1

Expression and structure-function characterisation of

herpesviral proteins

Sue-Li Dahlroth

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Doctoral thesis at Stockholm University

Department of Biochemistry and Biophysics

©Sue-Li Dahlroth, Stockholm 2008

ISBN 978-91-7155-755-1 pp 1-76

Printed in Sweden by Universitetsservice AB, Stockholm 2008

Distributor: Department of Biochemistry and Biophysics, Stockholm University

Papers I-III are reprinted with permission from the publisher.

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Abstract

Human viruses coexist with their hosts, sometimes silently and sometimes

by causing a vast range of symptoms. To fully understand these seemingly

simple particles, how they have evolved, their pathogenesis, to be able to

develop new drugs and potentially new vaccines and diagnostic tools we

need to study the individual viral proteins both functionally and structurally.

In order to determine and study a protein structure, large amounts of it is

needed. The easiest way to obtain a protein is to recombinantly overexpress

it in the well-studied bacterium Escherichia coli. However, this expression

host has one major disadvantage, overexpressed proteins might not be folded

or be insoluble. Within the field of structural genomics, protein production

has become one of the most challenging problems and the recombinant

overexpression of viral proteins has in particular proven to be very difficult.

The first part of the thesis concerns the recombinant overexpression of

troublesome proteins in E. coli. A new method has been developed to screen

for soluble overexpression in E. coli at the colony level, making it suitable

for screening large gene collections. This method was used to successfully

screen deletion libraries of troublesome mammalian proteins as well as

complete ORFeomes from five herpesviruses. As a result soluble expression

of previously insoluble mammalian proteins was obtained as well as crystals

of three proteins from two oncogenic human herpesviruses, all linked to

DNA synthesis of the viral genome. The second part of the work presented

here concerns the structural studies of three herpesviral proteins. SOX from

Kaposi’s sarcoma associated herpesvirus is involved in processing and

maturation of the viral genome. Recently SOX has also been implicated in

host shutoff at the mRNA level. With this structure, we propose a substrate

binding site and a likely exonucleolytic mechanism. The holoenzyme

ribonucleotide reductase is solely responsible for the production of

deoxyribonucleotides and regulates the nucleotide pool of the cell. The small

subunit, R2, has been solved from both Epstein Barr virus and Kaposi’s

sarcoma associated herpesvirus. Both structures show disordered secondary

structure elements in their apo-and mono metal forms, located close to the

iron binding sites in similarity to the p53 induced R2 indicating that these

two R2 proteins might play a similar and important role.

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Table of Contents

INTRODUCTION 8

HUMAN HERPESVIRUSES 11

HERPESVIRUS STRUCTURE 12 HERPESVIRUS SUBFAMILIES 13 ALPHA HERPESVIRUSES 14 BETA HERPESVIRUSES 15 GAMMA HERPESVIRUSES 15

STRUCTURAL GENOMICS 17

PROTEIN PRODUCTION 19

PHYSICAL PARAMETERS 20 FUSION PROTEINS AND PROTEIN TAGS 21 CONSTRUCT DESIGN 22

SCREENING FOR RECOMBINANT SOLUBLE OVEREXPRESSION

23

COLONY SCREENING METHODS 24 THE COFI BLOT (PAPER I AND III) 28

LIBRARY METHODS 31

DELETION LIBRARIES SCREENED WITH THE COFI BLOT (PAPER II) 32

EXPRESSION SCREENING COMPLETE GENOMES 36

STRUCTURAL GENOMICS AND HUMAN PATHOGENS 38

THE DAILY SCOOP (PAPER IV) 39 SCOOP AND OTHER HERPESVIRUS ORFEOMES 41

MOVING FURTHER DOWN THE PIPELINE 42

HOST SHUTOFF IN HHV 45

SOX 46 STRUCTURAL STUDIES OF THE SOX PROTEIN FROM KSHV (PAPER V) 48 SUBSTRATE BINDING 50 SOX AS AN RNASE? 51

NUCLEOTIDE SYNTHESIS 53

RIBONUCLEOTIDE REDUCTASE 53

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STRUCTURAL STUDIES OF THE R2 SUBUNIT OF THE RIBONUCLEOTIDE

REDUCTASE FROM EBV AND KSHV (MANUSCRIPT IN PREPARATION) 54

FUTURE PROSPECTS 57

ACKNOWLEDGEMENTS 58

REFERENCES 60

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List of papers

This thesis is based on the following papers, referred to in the text by their

roman numerals.

I. Cornvik, T., Dahlroth, S-L, Magnusdottir, A., Herman, M.D, Knaust,

R., Ekberg, M. and Nordlund P.

Colony-Filtration blot, A new screening method for soluble protein

expression in E. coli.

Nature Methods. 2005, 2(7):507-9.

II. Cornvik, T, Dahlroth, S-L., Magnusdottir, A., Flodin, S., Engvall, B.,

Lieu, V., Ekberg, M. and Nordlund, P.

An efficient and generic strategy for producing soluble human

proteins and domains in E.coli by screening construct libraries.

Proteins: Structure, Function and Bioinformatics. 2006, 1;65(2):266-

73.

III. Dahlroth, S-L., Nordlund, P. and Cornvik, T.

Colony filtration blot for screening soluble expression in Escherichia

coli.

Nature Protocols, 2006, 1(1):253-8.

IV. Dahlroth, S-L., Lieu., V, Haas, J. and Nordlund, P.

Screening Colonies of Pooled ORFeomes, SCOOP: A rapid and

efficient strategy for expression screening ORFeomes in E. coli.

Submitted

V. Dahlroth, S-L., Gurmu, D., Schmitzberger, F., Erlandsen, H. and

Nordlund, P.

Structure of the shutoff and exonuclease protein from the oncogenic

Kaposi’s sarcoma associated herpesvirus

Manuscript

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Abbreviations

2D-gel Two-dimensional gel

AE Alkaline exonuclease

AIDS Acquired immune deficiency syndrome

CAT Chloramphenicol acetyltransferase

CMV Cytomegalovirus

CoFi blot Colony filtration blot

EBV Epstein Barr virus

GFP Green fluorescent protein

HHV Human herpesvirus

HIV Human immunodeficiency virus

HSV-1, 2 Herpes simplex 1 and 2

IMAC Immobilised metal affinity chromatography

IPTG Isopropyl -D-1-thiogalactopyranoside

KS Kaposi’s sarcoma

KSHV Kaposi’s sarcoma associated herpes virus

MCD Multicentric Castleman’s disease

mCMV Murine Cytomegalovirus

MS Mass spectrometry

NMR Nuclear magnetic resonance

ORF Open reading frame

ORFeome The complete collection of ORFs from one organism

PEL Pleural effusion lymphoma

RNR Ribonucleotide reductase

SAD Single wavelength anomalous dispersion

SCOOP Screening colonies of ORFeome pools

SG Structural genomics

SOX Shut off and exonuclease

VZV Varicella zooster virus

WHO World health organisation

UNICEF United nations children’s fund

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a) b)

c) d)

e)

Introduction

Viruses are small biological entities, existing on the border of life as we

define it. It cannot survive on its own and behaves as a molecular parasite,

making use of its host’s cellular machinery to create infectious progeny.

They come in many different and diverse forms and have been around since

ancient times, evolving with its surroundings (1). Viruses are divided into

families, subfamilies, genus and strains and can vary in both shape and size.

The smallest known virus belongs to the Parvovirdae family with a size of

20-25 nm and the largest known virus is the Mimivirus of 400 nm (2). They

will carry their genetic material of only a few genes to hundreds as either

DNA or RNA, which is encapsulated in a protective layer of proteins

(capsid) and sometimes a membrane consisting of lipids and proteins.

Proteins in this outer shell will determine their mode of infection and which

hosts they infect, be it bacteria, plants, fungi, animals or humans (1, 2).

Figure 1

Pictures1 of different viruses taken with electron microscopy. a) Adenovirus (~90-100 nm) b)

Bacteriophages (~20-200 nm) c) Herpesvirus (~200 nm) d) Hepatitis C virus (50 nm) e) the

Ebola virus (~80 nm).

1 Pictures are part of the public domain and under no copyright restrictions. http://www.wikipedia.org

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Certain types of viruses that infect plants can destroy entire harvests of

certain crops causing enormous economic damage each year (3). In humans

viral infections can be life long or temporary and can cause symptoms that

can range from anything like a common cold, diarrhoea, the flu, chicken

pox, and measles to hepatitis, polio, cancer, AIDS (acquired immune

deficiency syndrome), encephalitis, Ebola hemorrhagic fever and so on.

Viruses can roughly be divided into DNA or RNA viruses depending on how

they carry their genetic material. For both classes, the genome can be double

stranded (ds) single stranded (ss), circular or linear. The life cycle of a virus

(Figure 2) can be divided into several stages, attachment to the target cell,

entry (by endocytosis, fusion or genetic injection), replication and shedding

(the process when new viral particles leave the cell). Shedding occurs either

through lysis, budding, apoptosis or exocytosis (2).

After host cell entry, DNA viruses must move its genome into the host cells

nucleus, the site for DNA replication and transcription. The RNA, from

RNA viruses, can either remain in the cell cytoplasm, which will then be the

scene for its life cycle, or it can convert its RNA into DNA that will move

into the nucleus and fuse with the hosts’ genome. These latter types of RNA

viruses, of which the best known is HIV (human immunodeficiency virus)

are called retroviruses and cause lifelong infections. An interesting fact is

that up to 8% of the human genome is believed to be remnants of retroviral

infections (4) and although we carry what is referred to as proviruses in our

genome, they do not as far as we know cause disease.

It is however not only retroviruses that can cause lifelong infections. Many

additional elements determine weather or not a viral infection will persist,

such as the target cell, the individual’s immune system and how the viral

genome is maintained and replicated once inside its host. For instance

herpesviruses are relatively large dsDNA viruses infecting a wide range of

host cells. Herpesviral infections are life long due to target cell type, genome

maintenance and a cunning strategy to evade the immune system (5).

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c)

d)

a) e)

Figure 2

The general life cycles of viruses from attachment to shedding. a) A DNA virus, that enters

through fusion. The DNA is exported to the nucleus where it is replicated and transcribed.

The mRNA is transported to the cytoplasm and translated into viral proteins. New virions are

shed through exocytosis. b) An RNA virus enters through endocytosis and is stripped. The

RNA is either c) translated directly into viral proteins and new virions are made or d) the

RNA is reversibly transcribed into DNA that enters the nucleus and fuses with the host

genome. This provirus is transcribed and translated into virions that are shed through budding.

e) The RNA genome is injected into the host cell and is translated into viral proteins, which

assemble into new infectious virions that are shed through host cell lysis.

Since viruses are the causative agents for numerous mild symptoms like

colds but also very brutal diseases like cervical cancer, Burkitt’s lymphoma,

liver cancer etc they are intensely studied. Their mode of infection,

pathogenesis and epidemiology as well as their molecular structure and

cellular interactions are of huge interest with the goal of developing

diagnostic tools, vaccines and antiviral drugs. As huge as the discovery and

development of penicillin and other antibiotics, as a treatment for bacterial

infections in the late 1920’s, is the discovery of vaccination (from the Latin

word vacca meaning cow) in the late 18th century. In 1796 Edward Jenner

used the cowpox virus to vaccinate humans, which resulted in protection

b)

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against the two smallpox viruses that can cause blister-like scarring in the

face, blindness and even death. Smallpox was officially declared eradicated

in 1979. Polio, caused by the poliovirus was for a very long time a dreaded

childhood disease that can cause paralysis, meningitis and even death.

Vaccines against polio were developed by Jonas Salk in 1952 and Albert

Sabin in 1962. Since the start of a global vaccination effort in 1988 by

WHO, UNICEF and the Rotary Foundation, the number of reported cases

has dropped from hundreds of thousands to only thousands each year2.

Several continents are today declared as polio-free and a global eradication

has been proposed, although is still persists in some developing countries

(6). Even though many viral infections can be stopped by vaccinations there

are still many viruses, such as HIV, Hepatitis C, Dengue fever, for which

vaccines do not exist.

Human herpesviruses

An immense number of books and scientific articles have been written about

a wide range of topics concerning herpesviruses, from the overall virion

structure, the mode of transmission, prevalence in ethnic and social groups,

to the regulation of specific proteins in an infected cell. The main purpose of

this thesis is not to give an absolute introduction to herpesviruses, but just a

sneak peak and enough information about them for the reader to understand

the importance of this work as well as give a general grasp of the complex

relationship between these viruses and their hosts.

To date, hundreds of herpesviruses have been identified but only 8 are

known human pathogens and for the remainder of this thesis the focus will

be on the human herpesviruses and they will be referred to by their common

names or abbreviations (Table 1). All herpesviruses are large dsDNA viruses

that share a common overall structure and similar life cycle. Depending on

their mode of infection, the length of the life cycle and target cells, they are

further divided into subfamilies (2, 5). These subfamilies are further divided

into genera, although these will not be referred to or mentioned in this text.

2 http://www.who.int/en/

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Table 1

A list of the 8 human herpesviruses, their subfamily, formal names, common names and

abbreviations. In the text, they will henceforth be referred to either by their common name or

abbreviations in column 5, except for HHV-6 and HHV-7.

Subfamily Formal name Abbrev Common name Abbrev

Alpha herpesvirus Human

herpesvirus 1

HHV-1 Herpes simplex virus-1 HSV-1

Alpha herpesvirus Human

herpesvirus 2

HHV-2 Herpes simplex virus-2 HSV-2

Alpha herpesvirus Human

herpesvirus 3

HHV-3 Varicella-zoster virus VZV

Gamma herpesvirus Human

herpesvirus 4

HHV-4 Epstein-Barr virus EBV

Beta herpesvirus Human

herpesvirus 5

HHV-5 Human

cytomegalovirus

HCMV

Beta herpesvirus Human

herpesvirus 6

HHV-6 - -

Beta herpesvirus Human

herpesvirus 7

HHV-7 - -

Gamma herpesvirus Human

herpesvirus 8

HHV-8 Kaposi’s sarcoma-

associated herpesvirus

KSHV

Most of what we know today about herpesviruses has been based on studies

of the herpes simplex virus-1 (HSV-1) due to its early identification and easy

cultivation in cell cultures. Recently, however, significant progress has been

made in understanding the structure, biology and pathogenesis of the other

human herpesviruses.

Herpesvirus structure

As already mentioned, the herpesvirus family is a group of dsDNA viruses,

which are ~200 nm in diameter with a genome size of ~130-250 kbp (70-170

genes). What also unites herpesviruses is the common architecture of the

infectious particles (Figure 3). In the infectious virion, the genome is linear

and is wrapped around a core of proteins. This genome is contained within

an icosahedral shell, the capsid, which is made up of two types of oligomeric

proteins, hexon and penton capsomers. Surrounding the capsid is the poorly

characterized tegument, an amorphous mass consisting of various essential

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and non-essential proteins that are delivered to the cells at the very initial

stage if infection (5). Amino acid sequencing and MS analyses have been

carried out to determine the protein content of the tegument. Apart from

cellular proteins (that might be specifically or non-specifically packaged into

the virions) (7-9) the tegument contains more than 20 for HSV-1 and more

than 30 for HCMV virus-encoded proteins that aid in viral replication and

immune evasion (8, 10, 11). The tegument is surrounded by a membrane of

lipids and glycoproteins, (each of the herpesviruses encodes a set of 20-80

glycoproteins) used for target cell recognition, attachment and entry (5, 12,

13).

Figure 3

Schematic picture of the overall structure of a herpesvirus. The DNA core is surrounded by

the capsid, the tegument and a lipid envelope containing glycoproteins.

Herpesvirus subfamilies

All herpesviruses belong to one of three subfamilies, alpha, beta and gamma,

depending on their host range, length of reproductive cycle and target cells.

In addition to their overall structure, the three herpesvirus subfamilies share

the mode of infection and life cycle (5, 14). A herpesvirus life cycle has two

distinct phases, a latent and a lytic phase. After attachment to the target cell,

the lipid membrane is fused with the cellular membrane and the capsid and

tegument proteins are released into the cell. The capsid is dissolved and the

DNA is transported into the nucleus where is circularises into an episome.

The viral episome is replicated and maintained with the host genome. Only a

very small set of genes is expressed during the latent phase, and their

products block apoptotic pathways and aid in immune evasion and genome

maintenance. During the latent phase, the infected individual shows no

symptoms of infection. At a given signal, for instance a weakened immune

system due to a cold, the virus is reactivated and goes into lytic phase. The

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molecular signals that cause reactivation of herpesviruses are still not

entirely known, but HSV reactivation has been ascribed to physical damage,

ultraviolet light, hormones, or even fever. In the lytic phase, the virus will

take over host gene expression and shuts it down. It will then start

replication of its own genome and subsequently produce viral proteins. New

infectious virions will assemble and leave the host cell (5). A common

characteristic of herpesvirus infections is that they rarely pose any real

threats to a healthy person and although infection is life long a person can go

through life without even knowing that they are infected. The real problem

occurs in people with weakened immune systems where infection can lead to

organ failure and consequently death (15-18).

Alpha herpesviruses

HSV-1 is the prototype of the alpha herpesvirus family. The alpha

herpesviruses HSV-1/2 causes cold sores and genital herpes while VZV

causes chicken pox and shingles (14). Symptoms of infection will show in

epithelial cells such as skin and mucosa and these cells will consequently be

targeted by the immune system. However, the target destination is sensory

neurons in the brain. Infection starts at the mucosal surface, where the virus

will undergo lytic replication in the surrounding epithelial cells. After this it

enters a nearby sensory neuron, where it will establish a lifelong albeit latent

infection. The capsid travels up the axon on microtubules to the nucleus

where the genome enters the nucleus and circularises into an episome (19,

20). Upon reactivation, the virus will travel back down the axon to epithelial

cells, where further lytic spread will result in symptoms and a hopeful

transmission to new hosts. Although about 90 % of the general population is

infected with HSV it is only very rarely that this will cause any symptoms

other than cold sores (5). However, alpha herpesvirus infections can result in

encephalitis most commonly in children, the elderly, and people with

weakened immune systems (i.e. those with HIV/AIDS or cancer) although

this is very rare and only occasionally fatal (21).

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Beta herpesviruses

Beta herpesviruses, such as HCMV, HHV-6 and HHV-7 replicate more

slowly than alpha herpesviruses and establish latency in progenitor cells of

the bone marrow, monocytes and T-cells, which are all part of the immune

system (5).

The best characterised member in this subfamily is HCMV, which replicates

in a vast number of cells i.e. macrophages, dendritic cells, colonic and retinal

cells, endothelial cells (22). It has been estimated that >60% of the general

population carries CMV (5) and infection usually goes unnoticed in healthy

adults. It is only in immunocompromised individuals (like HIV patients,

organ transplant recipients as well as unborn babies) that serious conditions

such as pneumonia, encephalitis and retinitis arise (22, 23). HCMV infection

is considered the major cause of these conditions and the subsequent

mortality among immunosuppressed individuals (24, 25).

Gamma herpesviruses

There are two human herpesviruses that belong to the gamma herpesvirus

subfamily, EBV and KSHV. A key feature of these viruses is their capacity

to induce lymphoproliferation and cancers (5, 26). EBV’s major targets are

epithelial cells and B-cells where it also establishes latency. KSHV infects a

wide range of cells in vivo and in vitro for example endothelial cells, B-cells

epithelial cells and fibroblasts (27). After infection the linear genome

circularises into an episome, which tethers itself to the host chromosome by

a specific protein and replicates in concert with the hosts genome (5, 28, 29).

EBV causes infectious mononucleosis, better known as “kissing disease”

and it has been estimated that >90% of the worlds adult population carries

EBV3. EBV was the first human tumour virus discovered and is associated

with Burkitt’s lymphoma and nasopharyngeal carcinoma and to several other

malignancies for instance several types of Hodgkin’s lymphoma and gastric

carcinoma. In AIDS patients EBV causes a number of other lymphomas and

tumours (5, 30). In addition, there is a growing body of evidence, although

very controversial, that suggests a connection between EBV infection and

3 http://www.who.int/en/

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liver and breast cancer as well as certain types of auto-immune diseases like

multiple sclerosis, rheumatoid arthritis and diabetes (31, 32)

Kaposi’s sarcoma (KS) was first described in 1872 as a rare purplish-

pigmented sarcoma of the skin typically found in elderly men of Jewish and

Mediterranean descent. However, during the onset of AIDS in the 1980’s

there was a noticeable increase of KS and in 1994 the cause was identified as

a new human herpesvirus subsequently called Kaposi’s sarcoma associated

herpesvirus (KSHV) (33).

Besides causing KS, KSHV has been associated with some rare but lethal

lymphomas, pleural effusion lymphoma (PEL) and multicentric Castleman’s

disease (MCD) (5, 34). In Europe and the US <3% of the general population

are infected with KSHV. However, in some sub-Saharan countries where KS

was almost unknown before HIV, >50% are carriers (35) and in these

countries KS has become one of the most clinically described neoplasms. As

with EBV, KSHV has also been linked to other more controversial

conditions like sarcoidosis (5, 36, 37) and multiple myeloma (38, 39).

What really sets KSHV aside from the rest of the human herpesviruses is the

amount of cellular genes that is has copied throughout evolution, in

something called molecular mimicry or molecular piracy, where more than a

dozen cellular genes have been copied. Furthermore, several of these genes

have potential tumour related functions, so called oncogenes, meaning that

they can affect the cell cycle, apoptosis and other types of cell signalling

(40-42). EBV, on the other hand, encodes several highly evolved

transcriptions factors and signalling proteins that induce many of the same

cellular genes that KSHV has pirated into its own genome (5).

Herpesviruses have coevolved with their hosts to establish lifelong

infections in various cell types (43, 44). They are the major cause of several

minor syndromes and major malignancies in humans. Whether or not the

symptoms are manageable with today’s treatments or more severe is

determined by the individual’s immune system and genetic predispositions.

In immunocompromised patients, such as organ transplant recipients, cancer

patients, and AIDS patients a herpesviral infection can cause major

complications which could result in death.

To fully understand these viruses the individual proteins, such as virion

proteins and proteins from various stages of the viral life cycle, can be

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studied. However, some of these proteins can be hard to come by, since they

are not present or expressed in sufficient amounts in the virion particle or

target cells. The easiest way to obtain these proteins would therefore be to

recombinantly overexpress them. Recombinant proteins from herpesviruses

could help in creating vaccines, yield in high-resolution protein structures

that can help in understanding the viral life cycle, evolution, and

pathogenicity and might serve as potential drug targets.

Structural genomics

It is broadly accepted that the function of a proteins is dependant on the

amino acid sequence and how this chain of amino acids is folded in the

three-dimensional space. It is also widely accepted that the information for

the folding pattern of a protein resides in its linear DNA sequence. However,

this folding information is at present much too hidden from us and in order

to understand how a protein works at the molecular level we must study its

three-dimensional structure, preferably at high resolution.

In the wake of the genomics efforts, the effort to determine the complete

genomic sequence of all organisms (45-48), new emerging fields have risen

with aims to, on a-full-organism-scale determine patterns, trends, functions

and pathways among and within these genes and their corresponding gene

products, be it at the transcriptional, translational or degradation level. Huge

databases with massive amounts of information have become available for

searches, which in turn have yielded in vast amounts of new results and data.

Structural genomics (SG), the effort to structurally determine a large number

of proteins from one organism/genome, is such a field (49). In Table 2, the

approximate number of genes for some organisms and viruses are shown and

the number of unique protein structures for each of them4.

To date less than 5% of the human herpesviral proteins have been

structurally characterised and even less have been solved within the context

of SG (50, 51).

4 http://www.rcsb.org/pdb/home/home.do

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Table 2

Unique structures submitted to PDB for a few organisms/viruses also shown is the

approximate number of genes5.

Organism ~No of genes Unique structures in PDB

Homo sapiens 20,000 4080

Saccharomyces

cerevisiae

7,700 533

Escherichia coli 4,400 1403

HIV 9 32 (6)

EBV(HHV-4) 80 12

KSHV (HHV-8) 88 9

Within structural genomics projects there usually exists a common approach

to which all target proteins must succumb. This approach/strategy is usually

referred to as a pipeline and has the general outline of target selection,

cloning, overexpression, purification, crystallisation and structure

determination. Even though these steps are common, the execution of them

may vary, for instance how the targets are chosen (what criteria they are

based on) how the cloning is done (i.e. digestion followed by ligation,

recombination cloning or ligase independent cloning), which expression

system is used (i.e. bacteria, yeast, insect cells) (52-60). But the common

denominator to all these steps within SG initiatives is the aim to achieve as

high output of target structures as possible. To be able to work with as many

target proteins as possible and to increase success rates new methods within

all steps have been developed and evolved. In the early days of SG it became

evident that one of the major obstacles was, and still is, the production of

suitable protein samples of sufficient amount and purity (61). Since then,

statistics for all the steps in the SG pipeline from various worldwide SG

efforts have been gathered. SG initiatives have together cloned more than

100,000 targets from various organisms and produced suitable protein

samples for downstream processing for about 1/3 of the cloned targets7.

5 http://www.genomesonline.org 6 For HIV, the number of structures exceed the number of genes. The HIV genes are subject

to splicing, leaky scanning and frame shifting and products of translation are subjected to

protease activity hence creating more proteins than genes. In addition, structures from

different strains of HIV have been solved. 7 http://targetdb.pdb.org/statistics/targetstatistics.html

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Protein production

Since certain proteins are present only at very low levels and at certain time

points in a living organism, the most ethical and effective way of obtaining a

protein of interest is to recombinantly overexpress it. High-resolution

structures of biological macromolecules, such as proteins, can be determined

with several methods. The most convenient and efficient methods are X-ray

crystallography and NMR. For X-ray crystallography, the dominating

method, an absolute requirement is well diffracting protein crystals.

Numerous crystallisation trials and optimisations might be necessary to

produce a well diffracting crystal and therefore large amounts of pure and

soluble protein is needed.

The most widely used expression host to date for recombinant

overexpression is the well-studied bacterium E. coli. The major reason for

using E. coli is that with great ease and low cost, large amounts of biomass

can be generated (62-64). E. coli also possesses other advantages beneficial

for structural biologists. For instance, it will produce a very homogenous

protein sample since it lacks the machinery to create certain covalent

modifications like glycosylations.

In principal, E. coli has the same protein production machinery as any other

cell, although is differs in ways that might create problems. For instance,

when trying to overexpress a protein, the bacterium might form large

insoluble aggregates called inclusion bodies (62, 65, 66). Inclusion bodies

consist of unfolded or partly folded proteins and might take up a very large

volume of the cell (67, 68), although there have been situations where

correctly folded and active proteins have been found in inclusion bodies

(69). The already mentioned lack of covalent modifications, which might be

needed for proper protein folding and function, could be one reason for

inclusion body formation. E. coli may also lack certain important tRNAs

that could lead to halted translation as well as lacking certain folding

partners such as chaperones (67, 68, 70-73). Another problem when

overexpressing proteins is in vivo degradation by indigenous proteases or

that the target protein in itself is toxic to the bacterium (74). The

overexpression of eukaryotic proteins in E. coli is especially troublesome

(75), although even when trying to overexpress indigenous E. coli proteins,

in vivo proteolysis occurs as well as inclusion body formation (65, 66, 76).

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In contrast to prokaryotic proteins, eukaryotic proteins tend to be, on an

average, larger and consist of more domains that are connected by flexible

linker regions (77) and it has been shown that the size of a protein correlates

with the success rate of its soluble expression in E. coli (53). As already

mentioned, the official success rates from SG efforts for soluble production

of prokaryotic proteins, is 50% and 30% for eukaryotic proteins when a

basic SG pipeline is used (53, 60). However, in these statistics there is no

information of the success rates in regard to full-length protein expression or

protein size.

To increase the likelihood of obtaining soluble protein, when using E. coli,

there are generally two approaches: i) either change physical parameters of

the experiment (like the bacterial strains, culture conditions, promoters or

fusion partners or ii) change the properties of the target protein.

To ensure high success rates and low costs, the soluble recombinant

expression should be screened before proceeding with large-scale expression

and purification.

Physical parameters

To compensate for rare codons, tRNAs can be co-expressed in E. coli (78,

79) and numerous strains are commercially available that can co-express

these tRNAs. Typically these strains also lack certain proteases that could

lead to protein degradation (80). Even strains that should be more resistant to

toxic proteins and strains that provide the right oxidizing conditions,

permitting disulfide bonds to be formed, have been created (81-83). Another

parameter that can influence the recombinant expression of a target protein

is the culture conditions (62). The growth medium can be changed as well as

the growth temperature. Although very few systematic studies have been

reported (84, 85), it is still believed that the expression medium could

influence soluble expression. In regard to expression temperatures, more

support exists for its influence on the expression (62, 72) than for the

expression medium. For several proteins it has been shown that by

decreasing the temperature, target proteins could be rescued from the fate of

inclusion bodies (62, 85-87). How the solubility of a protein in a bacterium

correlates with a decrease in expression temperature is not fully understood

and might be due to a combination of factors involved in the transcription

21

and translation as well as folding of the protein. When the transcription and

translation machineries slow down, due to decreased temperature, the

protein might have time to fold in a proper way. The attractive forces,

between hydrophobic parts, that could lead to protein aggregation are

potentially weaker at low temperatures (88). It has also been shown that the

expression of several indigenous chaperones are induced at lower

temperatures (89).

Although new bacterial strains have been created and culture conditions are

varied, the problem of inclusion bodies and proteolysis still persists,

especially for mammalian proteins.

Fusion proteins and protein tags

Fusion proteins are often large soluble proteins that are subcloned upstream

or downstream of the target protein. It has been shown that by adding a large

soluble fusion protein, the folding propensity and therefore the solubility of

the target protein itself can be increased (75, 90-94). The most widely used

fusion proteins are glutathione S-transferase (GST), maltose binding protein

(MBP) and thioredoxin (TrxA). Fusion proteins do not only serve as

solubilising factors but can also aid in purification and detection of the target

protein. Although there are obvious benefits of adding a large soluble

protein, there are some clear limitations to it. For instance, the fusion protein

should preferably be removed before crystallisation trials either by laborious

recloning or digestions. Large fusion partners might also alter the solubility

of the target protein in a negative way and removal could therefore result in

an unpleasant surprise, such as protein aggregation and precipitation (94-98).

Instead of adding a large fusion protein for detection and purification, that

could potentially alter the solubility of the target protein, small peptide tags

can be used instead. The most commonly used peptide tag is the His-tag. A

stretch of six histidines is added, in the cloning step either upstream or

downstream of the target protein, which has the ability to bind divalent

cations, typically Ni2+ or Co2+. If these cations are immobilised on a gel

resin, the target protein can be caught and separated from non-histidine-

tagged proteins. This method, which we today refer to as IMAC

22

(immobilised metal affinity chromatography), was first described in the late

1980’s (99) and has since then revolutionised recombinant protein

purification. Commercially available antibodies and probes, conjugated with

horseradish peroxidase (HRP) or alkaline phosphatase (AP), directed

towards His-tags have since then been generated and therefore a His-tag can

be used for target protein detection based on immunochemicals.

Construct design

The second approach to increase the chances of obtaining recombinant

soluble expression is to change the characteristics of the target protein. As

already mentioned the expression of prokaryotic proteins can be problematic

and success rates for soluble expression of such a protein is approximately

~50 %. However, for eukaryotic proteins the success rates drop significantly

to ~30%8. This could be due to the larger size of these proteins, the number

of domains and flexible linker regions (which might be protease sensitive),

the requirement of specific chaperones for folding and the requirement for

post-translational modifications. A natural reaction to these problems has

been the attempts to clone and express the individual domains of the target

protein, which has been proven to be very useful (57, 75, 100-103). This

strategy is based on the theory that if the full-length protein fails to express

or crystallise, perhaps its individual domains might. Domains can be

predicted either experimentally, with limited proteolysis coupled with MS

analysis (101, 102), deuterium exchange MS (104) or with special computer

programs (105).

The latter strategy, designing new constructs partly with help of domain

predictions, has successfully been employed within SG-initiatives where

several expression constructs for one target protein are generated (Figure 4).

It has been shown that by using this approach the probability of generating

soluble protein increased two-fold (100). Since these types of domain

prediction programs have a fair degree of uncertainty, several expression

constructs have to be designed that start close to the predicted domains.

8 http://targetdb.pdb.org/statistics/targetstatistics.html

23

b)

Figure 4

Construct design. a) A schematic picture of a result from a domain prediction program of a

multi-domain protein. b) New expression constructs are designed to define domain borders in

hope of finding a better expressing construct.

The solubility of a protein can also be increased by making random or

focused mutations or deletions. This approach is called in vitro evolution and

will be described later in the text. Whether new expression constructs are

generated in a focused or random approach generate, all of them have to be

screened for soluble expression.

Screening for recombinant soluble overexpression

The aim of a screen, no matter how it is executed, is to rapidly reduce a large

number of clones/targets to a more easily handled number.

The traditional approach when screening for soluble expression is usually

done with liquid cultures in individual vials and more recently in a 96-well

format. 1 ml cultures are grown and induced in parallel, cells are harvested,

lysed and soluble material is separated from insoluble by centrifugation

and/or purification. The soluble fraction is usually analysed by SDS-PAGE

gels (53, 60, 85, 106-108). Robots can perform certain steps in this process

while others still have to be done manually.

A couple of years ago an effort at genome wide expression screening was

attempted. Some 10,167 ORFs from the nematode Caenorhabditis elegans

was screened for soluble expression in E. coli in a 96-well format. Soluble

expression could be detected for 1,356 ORFs corresponding to a success rate

of 13% (109). This number is very much lower than more recently reported

success rates for eukaryotic proteins and it was later shown that many ORFs

a)

24

were wrongly annotated, had mispredicted gene boundaries and were out of

frame (110). Although only one vector and one expression strain was used

and some steps were automated, the workload of this effort is likely to have

been very large.

In our lab, we have previously developed a method that utilises filtration in

order to separate soluble material from insoluble called FiDo (filtration dot

blot) (111). Liquid cultures are grown and induced in a 96-well plate. A

small fraction of the culture is then transferred to another 96-well plate with

a low protein binding submicron filter in the bottom. The liquid media is

removed by vacuum and a bacterial pellet formed on top of the filter. The

pellet is either resuspended in a denaturing lysis buffer (solubilising all

proteins in the bacteria) or a native lysis buffer (only releasing the soluble

proteins). Vacuum is reapplied and the filtrate is collected in a collector

plate. The filtrate is then used to make dots on a membrane with a high

protein affinity, like nitrocellulose. The nitrocellulose is then blocked and

probed with an antibody or probe and developed like a Western blot.

The FiDo screen has also been modified to accommodate for an affinity

purification step to be able to determine the purifying ability of the target

protein (112).

In order to secure a high output of structures in an SG pipeline the best

strategy would be to generate multiple constructs subcloned with different

fusion proteins/tags, which would then be expressed in different strains and

at different temperatures. A quick calculation shows that working with 96

targets, creating 10 variants of each (based on domain predictions) cloned

with 2 different fusion proteins/tags and expressed in two different strains at

two different temperatures, would generate 7,680 different experiments and

although most of the work would be done in a 96-well format, it would be

quite labour intense. Therefore these types of combinatorial experiments are

currently not pursued within SG initiatives due to high costs and heavy

workload.

Colony screening methods

As already mentioned, solubility can be increased by adding a large fusion

protein. The solubility would then subsequently be screened based on the

physical characteristics of a soluble protein, such as its ability to be

25

separated from insoluble protein by centrifugation, filtration or purification

of liquid cultures.

However, solubility of a protein is intimately connected to its folding and

activity and could therefore be monitored by fusing the target protein to a

reporter protein, that when folded correctly would give rise to an easily

monitored phenotype. The theory relies on, that if the reporter protein is well

folded, hence soluble, the target protein should also be soluble and well

folded (Figure 5).

Figure 5

Schematic picture of a target protein (grey) fused to a reporter protein (black). a) If the target

protein is well folded and soluble, the reporter protein will fold and the phenotype can be

observed. b) If the target protein misfolds, the reporter protein will misfold and no phenotype

will be seen.

Solubility would not have to be screened in liquid cultures but instead at

colony level and would therefore lift the heavy burden of handling liquid

cultures since thousands of colonies, if they all carried different constructs,

could potentially be screened on one colony plate.

Waldo et al described in 1999 a method to monitor folding and therefore

solubility in colonies by fusing the target protein to GFP (green fluorescent

protein). Only bacterial colonies that fluoresce would have complete read-

through and express a well-folded and soluble target protein and vice versa

(113).

Another method relying on the same theory, presented by Maxwell et al, is

to fuse CAT (chloramphenicol acetyltransferase) to the protein of interest.

Only a bacterium that can grow in the presence of the antibiotic

chloramphenicol would express the target protein (114). Both these methods

are easy to use and allow thousands of colonies to be screened in one

experiment.

a) b)

26

a)

b)

Nevertheless, these types of methods have potential drawbacks. Firstly, the

reporter protein might affect the solubility of the target as already

mentioned. Secondly, false positives are seen for example when GFP is used

(115, 116), and thirdly, the reporter protein has to be removed before the

target protein can be used for structural studies.

In order to avoid the drawbacks of adding a large soluble protein, several

methods only relying on a small reporter peptide have been developed. In

practice, these methods rely on splitting a large reporter protein in two,

where none of the parts are active on their own. The target protein is fused to

a small peptide, corresponding to a vital part of the reporter protein. If the

target protein is soluble and well folded the phenotype should be detectable

when the “rest” of the reporter protein is added or co-expressed and vice

versa (Figure 6).

Figure 6

The theory behind a split reporter protein. The target (grey) is fused with a part of the reporter

protein (dark grey). If the target protein folds the tag will subsequently fold and when

combined with the rest of the reporter protein (black), the phenotype will show. If the target

protein is insoluble, the tag will be unfolded and no phenotype will be seen when adding the

rest of the reporter protein.

Wigley et al (117) used -galactosidase in this manner. -galactosidase was

split into a 52 amino acid -fragment, which was fused to different control

targets, and an -fragment corresponding to the rest of the protein. When the

control targets were expressed, colonies would turn blue or white depending

on the solubility. This method was effectively used to screen a hybrid gene

library of the human P450 for proper folding and solubility. Even though

this method seems to work well, especially when wanting to monitor slow

folding processes, a certain degree of false positives and negatives could be

27

observed. Additionally, in a review by G.S Waldo from 2003 it was claimed

that the -fragment could render proteins insoluble (118).

The developers of the previously described GFP-method have recently

developed it to better suit the demands of a smaller tag (119). Only part of

GFP, representing -strand 11, is fused to the target protein via a flexible

linker. This approach has successfully been used to screen mutation libraries

of proteins from Mycobacterium tuberculosis (115).

Both these methods have approached the problem, that reporter proteins can

remain active although it is fused to an insoluble target protein as well as

that they might alter protein solubility, by splitting the reporter protein in

two pieces. Both these methods have the advantage that they work in vivo

and in vitro making it easy to monitor solubility of the protein in a cell

lysate. A small complication is however, that in order for the rest of the

reporter protein to be present in the cell, two plasmids have to be used and

the protein has to be co-expressed.

A method that can directly monitor folding has been described by DeLisa et

al (120). It relies on that the twin-arginine translocation (Tat) pathway only

moves well-folded proteins across the inner membrane to the periplasmic

space of the E. coli cell (121). By fusing the target protein to a Tat

exporting-signal at the N-terminal and to -lactamase (which only confers

antibiotic resistance in the periplasm) at the C-terminal, the folding of the

target protein can be directly monitored. This method was tested on a

number of well-characterised proteins known to be soluble in the cytoplasm

(like GST, MBP, GFP, TrxA etc) as well as some cytoplasmic unstable

proteins and a good correlation could be seen. Potentially, -lactamase could

influence the folding in some unforeseen way and there were no reports on

any upper size-limit of the proteins that could be exported with the Tat-

pathway. In addition, before the target protein can be used the tag and/or the

-lactamase have to be removed either by recloning or digestions.

By fusing a gene to a C-terminal biotin acceptor peptide and therefore

enabling biotinylation in vivo by E. coli biotin ligase, BirA (122), detection

and affinity purification can be used based on the very strong binding of

biotin to the protein avidin (123).

Tarendeau et al (124) used this type of approach at the colony level.

Colonies carrying an expression construct with a C-terminal biotin acceptor

peptide (Avi-tag) are arrayed very closely, by a robot, on a nitrocellulose

28

membrane and induced for expression. The Avi-tag will be biotinylated in

vivo and after lysis, colonies expressing biotinylated proteins can be detected

on the nitrocellulose by probing with a fluorescent streptavidin conjugate. A

deletion library of almost 27,000 constructs (and with a seven-fold

oversampling!) of the influenza virus polymerase PB2 was successfully

screened in this manner and the approach was called ESPRIT (expression of

soluble proteins by incremental truncation). A major advantage is (at least in

theory) that since neither recloning nor digestions should be needed,

identified constructs can, if a plate replica were to be made, go directly in to

scale up experiments and subsequent affinity purifications based on biotins

affinity for avidin could be done. In addition no reports have so far surfaced

on any potential negative side effects by adding an Avi-tag, whether it

affects the solubility or if aggregated protein could potentially be

biotinylated and misinterpreted as soluble. Although this is a very powerful

method in its present implementation it still relies on expensive robotics,

which would not be part of standard laboratory equipment, and the use of

costly streptavidin-magnetic beads.

Several colony-based fusion protein or fusion tag screens have been

developed and evolved into well performing strategies. They are all elegant

methods that allow for a swift and easy way to identify targets that could be

suitable for large-scale protein production. Most importantly, however, is

that when it comes to colony-based screens they are best put to use when the

desire is to screen large collections of gene variants.

The CoFi blot (paper I and III)

We wanted to develop a method that works as a solubility screen at the

colony level but neither relies on a reporter protein that could potentially

affect the solubility of the target protein nor makes use of a tag that needs a

reporter protein to be co-expressed. Since we had very good experiences

with the previously described FiDo screen we adapted it in such a way so it

would allow us to screen soluble expression at the colony level.

In this method colonies are grown on a plate, called the master plate, and are

transferred to a submicron low protein-binding filter that can separate

inclusion bodies from soluble protein. Colonies on the master plate are

29

a) b)

c) d)

e) f)

g) h)

regrown and colonies on the filter are induced for expression by placing the

filter on a plate containing IPTG. After induction the filter is used to make a

filter sandwich (Figure 7). Upon lysis, soluble protein will diffuse through

the filter and attach to a high protein-binding membrane, such as

nitrocellulose. Detection is done by incubating the nitrocellulose with probes

or antibodies directed at the target protein and using standard

immunochemicals.

Figure 7

A schematic picture of the CoFi-blot method. a) Colonies are grown on a master plate, which

are b) transferred to a Durapore filter and c) expression is induced on a plate containing IPTG.

d) The filter with the colonies is then used to make a filter sandwich consisting of the

Durapore on top of a nitrocellulose and a Whatman paper with lysis buffer. e) Close up of

sandwich. f) Upon lysis, by repeated freeze thawing, soluble protein will diffuse through the

filter and bind to the nitrocellulose. g) The nitrocellulose is then blocked and incubated with

probes or antibodies directed at the target protein. h) The signals are detected by

chemiluminescence.

In our case we tend to use His-tag fusions, which efficiently can be probed

with Ni2+ conjugates as well as be used for purification. Colonies that give

rise to signals are picked from the master plate and can either go directly into

scale-up experiments or be further analysed. We chose to name this method

the CoFi blot (colony filtration blot).

30

In order to verify how well the CoFi blot works we decided to compare it to

the traditional method of growing liquid cultures and separating soluble

protein from insoluble by centrifugation.

32 eukaryotic and 24 E. coli proteins were subcloned in two different

expression vectors yielding either an N-terminal His- or FLAG-tag. Targets

were both grown and induced as colonies, which were subjected to the CoFi

blot, and as liquid cultures. The bacteria in the liquid cultures were

harvested, lysed and centrifuged. Dots were made on a nitrocellulose of both

total and soluble protein content and developed in the same way as for the

CoFi blot.

Figure 8

The CoFi blots performance was compared to traditional way of screening for soluble

expression, centrifugation. In the picture the results and correlation between the two methods

are shown for 32 eukaryotic proteins. Targets have been noted with (+), (-) or (0). (+) for

where the two methods are in agreement, (-) for disagreement and (0) for when there is no

total expression. Constructs with no total expression have been excluded from the statistics.

We have shown that the two methods are in 84% agreement with a fairly

good correlation between expression levels. We have also shown that the

CoFi blot is reproducible by re-screening clones in quadruplicates.

Differences between the two methods can be due to the different metabolic

states for a bacteria growing in a liquid culture as opposed to growing on a

solid support. Another factor that could explain the deviances is the affinity

of the filter for the proteins. Even though the filter is a low-protein binding

31

filter, some proteins might still stick and therefore the CoFi blot would not

work as a detection method for these particular proteins.

In summary, we have developed a colony expression screen, called the CoFi

blot, with good reproducibility and correlation to a more traditional

expression screen. The CoFi blot can be applied to any type of protein to

which antibodies have been generated or that contain a detectable tag, such

as a His-tag. Since it utilises standard molecular biology reagents and

equipment, it is a method suitable for any laboratory. Another advantage is

that the CoFi blot only utilises a small affinity tag for detection and not a

large fusion protein and therefore there is little risk that the tag will influence

the solubility/folding of the target protein. In addition, colonies containing

constructs that yield soluble protein, can be directly picked from the master

plate and be subjected to scale up experiments. One major advantage is that

the CoFi blot detects solubility after lysis and separation of cell debris,

meaning that the target protein has survived two additional steps required for

scale-up purification and is therefore, potentially, a better indicator of a

useful protein as compared to other methods where solubility is detected in

the cell before lysis. The CoFi blot also carries the same advantage of other

colony-based screens; the ability to screen thousands of colonies in a single

experiment.

Library methods

A successful strategy to make a protein soluble is by making mutations,

amino acid substitutions and deletions that could favour protein expression

and/or folding (115, 125). These types of alterations could be made in a

focused manner like changing the design of the expression construct based

on domain predictions (generating only a limited number of variants), or by

randomly generating thousands of variants (by error prone PCR, DNA

shuffling, truncations etc) creating a library. However, beneficial mutations

can be quite rare and one could potentially end up with a needle in a

haystack scenario. A colony screen is therefore an efficient mean to lift the

heavy burden of screening overexpression of such a library with traditional

methods.

32

As already mentioned, compared to prokaryotic proteins eukaryotic are often

larger and consist of more domains connected by flexible linker regions. As

discussed, a very common approach to increase a protein’s solubility is also

to change the expression construct by predicting domains. Domains are

predicted by computer programs, and expression constructs close to

predicted domain borders are cloned and tried for soluble expression. Even

though this approach has shown to be very successful, domain predictions

based on computer programs are not always accurate.

Domain borders can also be determined experimentally by limited

proteolysis coupled to MS, but this approach poses a kind of catch-22

situation since it relies on soluble expression from start. We wanted to

develop a strategy with which we could obtain or enhance soluble protein

expression and potentially map domain borders at the same time.

Deletion libraries screened with the CoFi blot (paper II)

We decided to produce deletion libraries of 21 human proteins that

previously, as determined in paper I, had been insoluble or did not express

protein at all to in an effort render them soluble and at the same time

investigate if we could experimentally map domain borders. Deletion

libraries were generated at DNA-level from the 5’ end of the target gene by a

method described by Henikoff et al (126) and is now available as a kit called

Erase-a-Base® (Promega). The target gene has to be cloned into a plasmid,

which is linearised and incubated with exonuclease III that removes

nucleotides from one strand of the DNA at a constant rate. By removing

timed aliquots a deletion library spanning the entire length of the gene can

be created.

In order for this method to work, two restriction sites have to be introduced

in front of the target gene to ensure unidirectional deletion. One to create a

5’-overhang that is susceptible to exonuclease III digestion and one that

generates a protecting 3’-overhang at the opposite end of the gene. The

remaining single strand is removed by S1 nuclease and the ends are flushed

by Klenow polymerase. The linear blunt-ended fragments are then re-ligated

and transformed into a cloning strain (Figure 9). Colonies are grown and

harvested and the library is extracted and transformed into an expression

33

strain. The library inside the expression strain is then screened for soluble

expression with the CoFi blot method.

Figure 9

Schematic picture of the Erase-a-base procedure. A plasmid containing appropriate

restrictions sites in front of the target gene is linearised. A deletion library, spanning the

entire gene, is generated by adding exonuclease III and removing timed aliquots. The

remaining single strand is removed by SI nuclease and blunt ends are generated by Klenow

polymerase. The plasmids are religated and a cloning strain is transformed.

21 human proteins were chosen, where 19 were successfully recloned into an

expression vector with appropriate restriction sites in front of the genes. For

detection and purification purposes we added a His-tag in front of the

restrictions sites. Deletion libraries were generated for all 19 genes and

positive colonies could be seen for 17 (Figure 10).

34

Figure 11

SDS-PAGE of constructs

identified with the CoFi blot

method a) The best expressing

constructs were purified by small

scale IMAC and the eluate was

run on SDS-PAGE. b) 6 different

constructs from two proteins

showing the difference in

construct length and their level

Figure 10

CoFi blots of 6 targets. Positive colonies can be seen as black spots on a, b, c, d, and f and

negative colonies are seen as very faint grey spots. Since this is a deletion library from the 5’-

end, only one third of the colonies can in theory be positive. e) Example of a completely

negative CoFi blot. Positive colonies were picked for analysis and identification purposes.

From each target, 24 positive colonies were picked and used to inoculate

small liquid cultures that were induced for expression. The soluble fraction

was purified by IMAC and the eluate was analysed as Dot blots. At this

stage 14 targets gave soluble expression and out of these, 11 could be seen

as a band on an SDS-PAGE gel (Figure 11). The remaining three proteins

with no soluble protein detected were either extracellular or membrane

associated.

35

Because we wanted to see if this approach could be used to experimentally

determine domain borders we ran two domain prediction programs, PFAM

and BIOZON9, and compared them with our translational start points. As can

be seen from (Figure 12), our well expressing constructs have translational

starts correlating well with predicted domain borders. Interestingly, for some

clones they even have a translational start within a predicted domain and

these constructs would therefore not have been identified using standard

domain predictions. It should also be noted that the two domain prediction

programs, determine domains based on different criteria, and therefore a

difference can be seen in their domain predictions, which shows the

importance in these type of experimental approaches compared to rational

construct design based on domain predictions.

Figure 12

Domains as predicted by PFAM and BIOZON and translational starts of constructs identified

using the CoFi blot method.

9 http://www.biozon.org/

36

Another very intriguing observation is that for most targets there are clones

that express soluble protein that is full length or close to full length. This

could be the result of favourable changes in these regions that affect the

transcription, translation and/or folding. This would be in concurrence with a

report that regions downstream of the initiation codon can act as

translational enhancers (127).

From the initial set used in paper I, where only 30% of the mammalian

proteins could be expressed we increased the success to 60%.

In retrospect we have developed an experimental method, by generating

random deletions to increase soluble expression and at the same time define

domain borders.

The CoFi blot is routinely used in our lab and has now also been adapted to

allow the screening of recombinant overexpression of integral membrane

proteins. It was verified by screening random mutation libraries of nine

integral membrane proteins (128) and has now been used to screen a very

large part of all human membrane proteins (Unpublished results).

Expression screening complete genomes

The main objective of an expression screen is to slim down the number of

samples from a larger set. It could be applied to a library of different variants

of the same gene, as earlier described, or a collection of genes representing

for example a protein family, a pathway or a whole genome. Particularly the

handling of an entire genome and the cloning into an expression vector could

become quite troublesome, and new strategies are needed to handle very

large gene collections for expression studies. A good start point, for such

studies are Gateway® adapted entry clones allowing for the easy subcloning

of a gene into an expression vector through recombination (Figure 13).

Today several ORFeomes (a collection of all ORFs from one

organism/genome) for a number of genomes are available as Gateway clones

(110, 129-132).

37

Figure 13

Schematic picture of Gateway® cloning from Invitrogen. A PCR product is generated flanked

by specific recombination sites that are complementary with a vector, pENTR. The PCR

product is moved through recombination into the pENTR creating an Entry clone with new

recombination sites. The Entry clone is incubated with an expression vector containing

recombination sites complementary to the new sites. An expression clone is created.

As earlier mentioned a genome wide expression screen was performed on

the C. elegans ORFeome v 1.1 with very low success rates. This screen was

done in the more traditional 96-well format. Even though all ORFs were

available as Gateway clones and some steps were automated, this work was

probably very labour intense. Another attempt at handling and screening a

part of the C .elegans ORFeome was reported by Gillette et al (133). This

strategy named POET (pooled ORFeome expression technology) was based

on pooling 688 C. elegans ORFs and in a single recombination reaction

transfer them into an expression vector with a His-tag. An expression strain

was subsequently transformed with this pooled ORFeome library. The

transformation reaction was then used to inoculate a larger volume to make a

large expression culture. This culture was induced for expression, harvested

and lysed. To separate soluble protein from insoluble, the lysate was purified

by IMAC. To analyse expression and separate the different overexpressed

proteins from each other, the eluate was run on a 2D-gel. Intense and deviant

spots were picked for MS analysis. 165 spots were picked and 50 were

identified as C. elegans proteins by MS. Out of these 50 proteins, 12 were

38

chosen for small-scale expression and 6 out of these for large-scale

expression.

This method acts as a rough sieve to identify strong expressers in a large

gene collection. It also makes the handling, from cloning to expression

screening simpler. However, individual DNA measurements and the pooling

and subpooling of equal amounts must have been tiresome. In addition, this

method might not be readily used in labs with more modest equipment,

referring to the access of 2D-gel electrophoresis or MS equipment. POET is

also haunted by some limiting factors, such as the step of converting a

transformation reaction into a large culture and the iso-electric focusing

range on the gel. In a culture containing several variants of a bacterial strain

there is always a risk that one or a few variants take over the entire culture

and several targets might be lost in this step. The iso-electric range of pH 4-7

will result in that only proteins with a pI within this range will be separated,

excluding a vast amount of potentially overexpressed proteins. In the end,

anything identified must be fished out from the original collection and

recloned for any downstream processes.

Structural genomics and human pathogens

In the beginning of this thesis, a general introduction was given to human

pathogens in the form of viruses. Although recombinant protein and protein

structures from viruses could aid in the development of vaccines and drugs,

relatively few protein structures of viral proteins have been solved10.

Especially from SG initiatives submission of viral protein structures in PDB

have been modest (53). This could partly be explained by that the focus,

within these initiatives, is on other types of proteins. In two articles (50, 51),

where the focus or part of the focus was on viral proteins, poor success rates

were reported. The main problem was low expression and low solubility in

E. coli. Success was predominantly achieved when each target was treated

individually or expressed in insect cells.

10 http://www.rcsb.org/pdb/home/home.do

39

The daily SCOOP (Paper IV)

We wanted to use the CoFi blot as a homing method to, within a genome,

identify easily expressed targets and targets in need of special care. We

designed a general strategy and applied it to the ORFeome of KSHV. KSHV

consist of approximately 90 ORFs, which were supplied to us as 113 entry

clones (132). ORFs coding for integral membrane proteins were cloned as

full-length constructs, as well as into separate non-transmembrane domains.

In similarity to POET, our approach also relied on pooling of Gateway®

adapted entry clones and a single recombination reaction. However, instead

of tedious DNA measurements we grew separate cultures of bacteria

containing the individual entry clones over night. OD600 measurements

showed similar density for all of them and equal amounts were pooled.

Plasmids from this pool were extracted and ORFs were moved, in one

recombination reaction, into an expression vector with an N-terminal His-

tag. A cloning strain was transformed with the reaction and plated. The

library was harvested and an expression strain was transformed and plated.

4,000 colonies were subjected to the CoFi blot procedure. Positive colonies

were analysed based on their purifying propensity with small-scale

purification. Identification was done through regular Sanger sequencing. 23

unique constructs could be identified as expressing soluble protein with this

approach. 11 out of these 23 constructs were randomly chosen for large-

scale expression and purification.

In order to benchmark this method we compared it to the more traditional

handling and screening; individual subcloning, expression screening and

subsequent affinity purification all in a 96-well format. With the more

traditional setup 25 constructs could be identified as soluble expressers.

When comparing our new approach, now called SCOOP (screening colonies

of ORFeome pools), with the more traditional approach 23 and 25

constructs, respectively, were identified as yielding soluble protein in E. coli.

20 out of these 23 and 25 were the same for the two experiments. To

elucidate why 5 constructs were missed with SCOOP, their expression at the

colony level was investigated using the CoFi blot. Soluble expression at the

colony level could not be detected for 3/5. The additional 3 constructs that

were only identified with the CoFi blot were grown and induced for

expression in liquid cultures. The soluble fraction was affinity purified and

40

the eluate was run on SDS-PAGE gels and very faint bands could be seen on

the gel, indicating that these constructs were expressed at low levels.

It has long been known that a bacterium growing on solid support in a

colony behaves differently from bacteria growing in a liquid culture (134,

135). In this context we therefore propose that either the metabolic state of

the colony, compared to bacteria growing in liquid, does not allow for

soluble expression of some ORFs and vice versa, or that the CoFi blot

simply does not work for these proteins. In addition, when making pools of

this kind, some targets are inevitable lost along the way, perhaps accounting

for the additional two not identified by the CoFi blot. Success rates will

correlate to the overall coverage/normalisation of the library and although

we find several constructs more often than others, we must conclude that our

library is well dispersed since we cover almost all soluble expressers.

Although 113 constructs were screened by SCOOP, sequencing at a very late

stage revealed empty vectors, mutations leading to frame shifts and early

stop codons for 31 constructs. A TMHMM11 search of the remaining

constructs predicted 8 ORFs to code for transmembrane proteins with at

least 2 transmembrane regions. Of the 113 constructs used in the original

experiment only 74 could be expected to produce proteins of the expected

size.

In a matter of days we have been able to screen the ORFeome from a human

pathogen for soluble recombinant expression in E. coli using SCOOP

(Figure 14). Once again we have shown the general applicability and

advantages of the CoFi blot method, by which constructs identified can

directly be moved further down the SG pipeline. A normalised library is

created through pooling of bacterial cultures instead of at DNA-level, thus

avoiding tedious DNA measurements. Subcloning into an expression vector

is done in one recombination reaction and the subsequent screening is done

with the CoFi blot. Identification of positive constructs is done by

sequencing. From this very small set of targets, soluble expression in one

strain and at one temperature was roughly about 30%, which was also

verified by SCOOP. This number is a bit lower than the SG statistics of

37%, although this corresponds to proteins from all types of viruses and

11 http://www.cbs.dtu.dk/services/TMHMM-2.0/

41

there is no information about full-length expression nor expression

conditions (53).

Figure 14

Flow scheme of SCOOP.

SCOOP has also been applied to deletion libraries of the KSHV ORFeome.

Pools were generated based on restriction enzyme compatibility for the

Erase-a-base® protocol. Deletion libraries were generated and expression

was screened using the CoFi blot. Initial results show that several other

targets with widely different functions can be made to express in E. coli with

this approach. We have also screened the overexpression of all integral

membrane proteins from KSHV using the membrane protein CoFi-blot.

SCOOP and other herpesvirus ORFeomes

As mentioned, structure determination of viral proteins has been suffering, in

part, due to poor soluble expression in E. coli and therefore these types of

proteins have been considered as “difficult”. As a result only 525 unique

virus protein structures have been submitted to the PDB, a number that

includes proteins from all types of viruses, from SARS and HIV to

42

herpesviruses. Out of these 525 structures only 31 have been submitted by

SG initiatives, worldwide. SCOOP was applied to four additional

ORFeomes from herpesviruses, HSV-1, VZV, EBV and mCMV available as

Gateway® adapted entry clones. By using one expression strain and one

temperature we were able to identify 75 unique constructs as expressing

soluble protein. These constructs corresponded to proteins of various

functions like DNA metabolism, immune evasion and tegument proteins as

well as proteins of unknown function. For 65 there was no protein structure

in PDB. Large-scale expression, purification and crystallisation trials have

been attempted for all 65. So far 3 protein structures have been solved.

Moving further down the pipeline

One area that has been intensely studied for human herpesviruses is DNA

metabolism due to the high degree of conservation among the proteins

involved and the importance of DNA synthesis (5, 136). In fact the most

effective drug, available on the market today, targets the viral infection at the

level of DNA synthesis and therefore proteins involved in DNA metabolism

would be effective as antiviral drug targets. Acyclovir is an acyclic

nucleoside analogue used to treat alpha herpesvirus infections, which targets

the viral thymidine kinase that phosphorylates acyclovir to acyclovir-

monophosphate. Downstream phosphorylations, by cellular enzymes,

convert this monophosphate to a triphosphate that when incorporated in to

the growing DNA chain of the virus, by the viral DNA polymerase, causes

chain termination. Acyclovir and its related compounds represent a success

story in the general treatment of HSV, VZV and CMV infections (25, 137).

It can be administered as a lotion on symptomatic tissue, orally or

intravenously depending on the severity of infection. However, resistant

strains of these viruses have been identified, especially in HIV-infected

persons and almost all significant resistance has been seen in

immunocompromised patients (25, 137, 138). For these patients more

aggressive antiviral remedies have to be used.

In short, all herpesviruses encode a core set of six DNA synthesis enzymes

(Table 4); a DNA polymerase, a DNA polymerase processivity subunit,

three helicase primase subunits and a single strand DNA binding protein

43

(aka ICP8). In addition to their importance in DNA synthesis they also

mediate homologous recombination during viral replication (5, 139).

A second set of proteins with links to DNA synthesis, more or less

conserved in the herpesvirus family includes the thymidine kinase, alkaline

endo-exonuclease, ribonucleotide reductase, uracil N-glycosylase and a

dUTPase (Table 4). These proteins are considered non-essential for

replication in cultured cells but several appear essential for “normal”

behaviour of the virus in animal models (5).

Table 4

The proteins involved in DNA replication and synthesis, their abbreviations and their gene

names in all the human herpesviruses. White rows are proteins directly part of DNA synthesis

and grey rows are proteins with links to DNA synthesis.

Enzyme HSV-1/-2 VZV HCMV HHV6/7 EBV KSHV

DNA polymerase,

POL

UL30 ORF28 UL54 U38 BALF5 ORF9

DNA polymerase

processivity subunit,

PPS

UL42 ORF16

UL44 U27 BMRF1 ORF59

Helicase-primase

ATPase subunit, HP1

UL5 ORF55 UL105 U77 BBLF4 ORF44

Helicase-primase

RNA pol subunit B,

HP2

UL52 ORF6 UL70 U43 BSLF1 ORF56

Helicase-primase

subunit C, HP3

UL8 ORF52 UL102 U74 BBLF2/3 ORF40/41

Single strand DNA

binding protein, ICP8

UL29 ORF29 UL57 U41 BALF2 ORF6

Thymidine kinase, TK UL23 ORF36 - - BXLF1 ORF21

Alkaline exonuclease,

AE

UL12 ORF48 UL98 U70 BGLF5 ORf37

Deoxyuridine

triphosphate, dUTPase

UL50 ORF8 UL72 U45 BLLF3 ORF54

Uracil-DNA

glycosidase, UNG

UL2 ORF59 UL114 U81 BKRF3 ORF46

Ribonucleotide

reductase, large

subunit, R1

UL39 ORF19 UL45 U28 BORF2 ORF61

Ribonucleotide

reductase, small

subunit, R2

UL40 ORF18 - - BaRF1 ORF60

44

The alkaline endo-exonuclase (AE) is conserved in all herpes viruses and is

involved in the maturation and packaging of viral DNA (140, 141). In HSV

this protein interacts with ICP8 (142). These two proteins can also work

together to promote strand exchange similar to recombination events in

bacteriophage (143). Recently it was shown that the AE from both EBV

and KSHV is involved in host shutoff at the mRNA level, an activity that

has not been shown for the other HHV AEs (140, 144, 145).

The well-known allosterically regulated enzyme ribonucleotide reductase

(RNR) is solely responsible for the de novo synthesis of all four

deoxyribonucleotides. RNR is a heterotetramer of two subunits, R1 and R2

(146). All HHV carries homologues to R1 and R2 except beta herpesviruses,

which lack the R2 (5). Interestingly, the RNR of HSV is not negatively

regulated by high TTP pools as their cellular counterparts and seems

necessary for viral growth in “resting” cells (147, 148). There are reports

that this viral enzyme has an accessory function to protect HSV-1 infected

cells against cytokine induced apoptosis (149).

Uracil N-glycosylase functions as a repair enzyme that cleaves mutated U

residues from the DNA backbone (150, 151).

The virally encoded dUTPase breaks down dUTP, thus preventing its

incorporation into viral DNA(152, 153).

More understanding of proteins involved in DNA synthesis and replication,

their mode of action and interactions, amongst themselves but also with

cellular proteins, could aid in the development of new drugs targeting this

important step of the viral life cycle.

The fundamental mission of all viruses is to replicate, spread and persist in

the host environment to which they have become adapted. Viruses differ

with respect to the mechanisms by which they accomplish their objectives.

The difference is reflected not only in the basic mechanisms of viral entry,

synthesis of proteins, nucleic acid synthesis, virion assembly and egress but

also with respect to the basic strategies by which they counteract the

enormous recourses of the host cell and of the multicellular organism aiming

at total viral extinction. In principal, as a virus, you want no reports to the

immune system or to the apoptotic machinery of your presence and you will

take necessary precautions to achieve this. However, the immune system and

the apoptotic machinery are not so easily tricked with many back up systems

45

to fend off and terminate any invader. The infected cell, must in order to

pass immune system control, show several signs of being “normal”,

therefore certain mechanisms need to be upregulated while others need to be

blocked. Anything remotely suspicious will result in an attack by the

immune system. Immune evasion in the host is a major feature of

herpesviruses. To persist and replicate in a host cell the virus has to launch

several anti-host functions. For herpesviruses these functions tend to fall,

more or less, within four categories, i) selectively block synthesis of new

proteins, ii) block the function of preexisting host proteins activated after

infection, iii) selectively degrade cellular proteins and iv) block signalling to

the host immune system indicating that the cell is infected.

Host shutoff in HHV

Viruses i.e. polio, influenza, SARS-CoV etc frequently induce host shutoff

to avoid competition from host cell transcripts during the mass-production of

their own proteins and to prevent expression of factors involved in

stimulating an immune response (154).

One way to take over a cell is to down regulate cellular protein synthesis by

degrading RNA (155, 156). Not only are host proteins not synthesised due to

low mRNA levels but pre-existing polyribosomes are also degraded and

splicing is impaired. Host shutoff is a consequence of infection of alpha

herpesviruses and gamma herpesviruses and occurs a few hours after

infection (5, 157). One of the consequences of host shutoff is a block in the

synthesis of HLA class I and II molecules, revealed by reduced levels of

these antigen presenting complexes at the surface of cells in the EBV lytic

phase. This effect could lead to escape of T-cell recognition and elimination

(157).

In alpha herpesviruses, mRNA degradation is a result of the tegument

protein vhs (virion host shutoff) protein encoded by UL41 (158). However,

although vhs functions as an RNase it cannot succeed in complete host

shutoff by itself and collaborates with ICP27, inhibiting splicing of mRNA

(5, 159). Intriguingly, it appears that some mRNAs are more resistant to

degradation than others (160). Vhs homologues are found in all alpha

herpesviruses but not in beta-or gamma herpesviruses (157).

46

Host shutoff at the mRNA level has also been observed in gamma

herpesviruses. However, this occurrence has been ascribed to another

protein, the already mentioned alkaline exonucleases (AEs) of these viruses

(140, 144, 145). A screen of KSHV genes for ability of their corresponding

gene products to, in vivo, diminish expression of a GFP reporter protein lead

to the identification of the gene product of ORF37 as a host shutoff factor.

Since this protein functions both as an exo- and endonuclease as well as a

shutoff protein it was named SOX (shutoff and exonuclease) (140).

SOX

SOX is conserved in all herpesviruses as an alkaline exonuclease involved in

DNA maturation and packaging, but it is only the gamma herpesvirus

homologues that display a shutoff activity in vivo. SOX expression initiates

8 hours after lytic reactivation, which precisely coincides with the beginning

of host shutoff (161). Unlike vhs, SOX is not a tegument protein (162) and

the co-expression with GFP resulted in loss of GFP fluorescence, which was

the result of the GFP mRNA degradation. This discovery was further

verified by siRNA knockdowns (140, 144).

In vitro studies of the herpesviral AEs exhibit a 5’-3’ and a 3’-5’ (10-fold

weaker) dsDNA exonuclease activity and an endonuclease activity (163)

They work best at an alkaline pH and have an absolute requirement for Mg2+

(although Mn2+ can work, equally well for HSV-1/2 AE at some

concentrations) (164-166). For the HSV null mutant in gene UL12, growth is

severely restricted but is still able to replicate and encapsidate considerable

amounts of viral DNA. Capsids, as a result from this mutant, are impaired in

the egress from the nucleus to the cytoplasm (167). In addition the HSV AE,

UL12, exhibits strand exchange activity, in vitro, and acts together with

ICP8 as a recombinase (143, 168). In contrast to UL12, which only localises

to the nucleus, SOX localises both to the nucleus and cytoplasm (144).

Complex sequence alignments showed that all herpesviral AEs belong to the

PD-(D/E)XK superfamily, which comprises endonucleases (i.e. EcoRI,

EcoRV BamHI etc), nicking enzymes and one exonuclease (169-172).

Typically, sequence similarity between these proteins is so low that it is only

upon structure determination that they are assigned to this superfamily.

Although low sequence similarity, it has been proposed that all these

47

proteins share a common core of a 4-5-stranded -sheet that brings together

the charged residues in the conserved motif PDX10-30(D/E)XK that is

involved in Mg2+ binding and catalysis (169).

When aligning all herpesviral AEs, seven conserved linear motifs can be

identified (173). Both random and focused mutations identified single and

double function mutants. Single function mutants lacked either DNase or

shutoff activity in vitro or in vivo respectively, whereas double function

mutants had neither. When linearly mapping these mutations it was revealed

that mutations that abolished shutoff activity located outside of these

conserved motifs (except one, which is not conserved) whereas double

function mutations and single function mutations affecting only DNase

activity located within the conserved linear motifs (Table 5, Figure 15)

(144).

Table 5

Summary of locations of mutations that affect SOX activity (140, 144)

Mutation DNase activity Shutoff activity Conserved motif Conserved

residue in HHV

AEs

T24I Yes No No No

A61T Yes No No No

Q129H No Yes I Yes

P176S Yes No No No

G217A No No II Yes

D221E No No II Yes

V369I Yes No VI No

D474N Yes No No No

Y477Stop Yes No No No

Figure 15

Schematic figure of the seven (I-VII) conserved motifs of SOX and the mutations affecting

the activity. Black box indicates a double function mutation. Grey box indicates single

function mutations abolishing DNase activity and white boxes indicate single function

mutations abolishing shutoff activity.

48

As a result it was proposed that SOX is a bifunctional protein that exhibits

DNase activity in the nucleus and shutoff activity in the cytoplasm, inducing

host shutoff. Because mutations that abolished shutoff activity located

outside of conserved linear motifs, it was proposed that this activity had

been acquired later in evolution (140). However, since the shutoff activity

only could be shown in vivo, it was suggested that SOX might need an

additional cellular or viral-binding partner to induce this action.

The EBV homologue, BGLF5, has been shown to possess the same features

as SOX by similar characterisations (145, 166, 174, 175). With an amino

acid sequence identity of 42% it has now been shown that both these

proteins down regulate expression of HLA molecules, which impairs the

recognition of infected cells, by T-cells and that this is not a feature of the

other herpesviral AEs (175).

Structural studies of the SOX protein from KSHV (Paper V)

One of the proteins identified as positive with SCOOP was the KSHV SOX

protein. Catalytically active SOX, both native and selenomethionine

labelled, was expressed with an N-terminal His-tag in E. coli BL21[DE3]

and purified to homogeneity with our generic two-step purification scheme

by IMAC and gel filtration. Well diffracting crystals were obtained through

vapour diffusion. Native crystals diffracted to 1.85 Å and phases were

obtained by SAD.

SOX consists of 487 residues with a molecular mass of 55 kDa. It consists of

two domains giving it a rough bowl shape. The two domains, the N-terminal

domain and the core domain are connected by a partly unmodelled region

called the bridge. The N-terminal domain consists of -helices and the core

domain consist of a 5 stranded -sheet flanked by several helices in

similarity to the rest of the members in the PD(D/E)-XK superfamily. The

two domains form a crevice where the bottom is made up of a U-shaped

loop, called the U-loop, stretching from core domain into the N-terminal

domain (Figure 16). This crevice is positively charged, consists of highly

conserved amino acids in all herpesviral AEs as well as the residues in the

already mentioned conserved sequence motif of the PD(D/E)-XK

superfamily.

49

U-loop

Figure 16

Overall model of SOX. The N-terminal domain in light and the core domain in dark. Three sulphates can be seen in the density and have therefore been modelled. Density for what we believe is a magnesium ion could be seen in the cleft and has therefore been modelled. The bridge connecting the N-terminal domain with the core domain is indicated by a dotted line

Densities can be seen for what we believe are three sulphates, where two are

coordinated by totally or semi-conserved residues proposed to be binding the

site for the substrate backbone, and one is involved in crystal contacts. A

magnesium ion has also been modelled in the crevice. This magnesium ion

is partly coordinated by residues in the PD(D/E)XK motif. A DALI search

revealed a surprisingly similar fold to the only known exonuclease within

the superfamily, the -exonuclease (Figure 17) (176).

Figure 17 Superposition of a monomer from the -exonuclease (dark) on SOX (light). -exonuclease is also a two-domain protein. The two domains in -exonuclease are connected by a completely modelled bridge. The partly modelled bridge in SOX is an ordered -helix and the U-loop has small -sheet in the -exonuclease. The bridge in -exonuclease is completely modelled and a sulphate in SOX is bound in the same location as a phosphate in -exonuclease. In addition, the magnesium ion in SOX is located in the same position as a manganese ion in -exonuclease (not shown).

50

Substrate binding

In a superposition of SOX and the -exonuclease, we can see that one of the

sulphates is located and coordinated in the same way as a phosphate in the -

exonuclease structure. The magnesium ion in SOX is also located and

coordinated as a manganese ion in the -exonuclease structure as well as

other divalent ions in other proteins belonging to the same superfamily.

Since this area in SOX is highly conserved and both the tertiary structure as

well as the aligning of ligands with the -exonuclease, we propose that this

is the site for substrate binding. However, although great similarities are

observed in both the overall fold of these two enzymes, dissimilarities can be

seen. For instance, SOX is larger than -exonuclease and where SOX has

partly unstructured and unmodelled regions (such as the bridge and the U-

loop) -exonuclease has an ordered secondary structure. We believe that the

-exonuclease structure represents what for SOX would be a substrate bound

state, where conformational changes lead to ordered secondary structures in

the bridge and the U-loop. Another major difference between these two

proteins is their quaternary structure, where the -exonuclease is active as a

trimer (176), which could explain its processivity. No prior reports or our

results indicate that SOX functions as anything but a monomer (166).

The second modelled sulphate in SOX, located at the beginning of the

crevice, is coordinated by totally or semi-conserved residues, which would

indicate that this is a true substrate backbone binding site. To elucidate

whether this sulphate could represent part of the substrate backbone, we

modelled a 4bp oligo by aligning the phosphates in the backbone to the two

sulphates in the model. As can be seen (Figure 18) this oligo fits nicely in

this highly conserved crevice and aligns very well with our sulphates.

The second phosphate in the oligo backbone places itself very close to the

modelled magnesium ion. It is possible that the phosphodiester bond is

cleaved by a nucleophilic attack by an activated water molecule close to the

magnesium, which stabilises negative charges in this area, cleaving off one

nucleotide at a time. Since the substrate of SOX can be both double stranded

and single stranded SOX must have a type of unwinding activity. We

therefore believe that SOX separates the strands of the dsDNA and the single

stranded substrate destined for digestion is guided into the active site by the

bridge and is stabilised by binding to conserved and partly conserved

51

aromatic residues in and around the crevice. The backbone is cleaved and

the free nucleotide is released on the opposite side of the “tunnel” created

under the bridge. The remaining free single strand of the substrate is directed

away from the protein surface.

Figure 18

Conserved residues in SOX as determined by sequence alignments with all other HHV AEs.

Totally conserved residues are depicted in red and least conserved residues are light blue. A

4bp oligo was modelled in the crevice based on the location of the two sulphates.

SOX as an RNase?

An intriguing question is whether SOX can mediate host shut off through an

intrinsic RNase activity. As already mentioned, functional characterisations

of SOX in vivo, show that it affects the total mRNA content of the host cell

and although host shutoff is observed in alpha herpesviruses, the other

herpesviral AE’s cannot induce this type of degradation.

Figure 19

Mapping mutations that affect the activity of SOX. Red deletes overall activity. Orange

deletes DNase activity and pink the shutoff activity.

52

In addition, single function mutations abolishing the shutoff activity of SOX

locate outside of conserved linear sequence motifs found in all herpesviral

AE’s. It has therefore been proposed that the host shutoff trait of SOX, and

BGLF5 from EBV, has been acquired later in evolution. It has also been

proposed that SOX might need an additional cellular binding partner or that

it does not have any intrinsic RNase activity and only activates some sort of

RNA decay pathway (140, 144).

With the structure in hand we can now map known mutations, which affect

the shutoff function. As can be seen (Figure 19), mutations that abolish

shutoff function are spread all over the protein, with predominance in the N-

terminal domain. The mutations locate both on the exposed surface and in

buried regions although they are mostly found close to the surface.

Since SOX has a nuclease type active site, with an extended pocket that

appear well suited for binding nucleic acids, it could be argued that both the

DNase and a potential shutoff RNase activity are carried out in the same

active site, since no other obvious binding or active site can be seen in the

structure. As support for this theory, both double-function mutations

(G217A, D221E), located in the U-loop, as well as one shutoff mutation

(P176S), located in the end of the unmodelled bridge are either directly

involved in metal binding or reside very close to the substrate binding and

active site. As in most mutational studies carried out in vivo, it cannot be

ruled out that these mutations only affect the folding and stability of SOX,

cancelling out activity all together. Even so, these mutations emphasize the

importance of this region. However, since RNase activity cannot be detected

for SOX in vitro, additional proteins are likely to be required enabling the

nuclease activity for mRNA degradation. Possibly this can be viral or host

factors mediating the interaction of SOX with conserved elements of

mRNA.

What would support the hypothesis of an additional cellular binding partner

for shutoff activity is the predominance of mutations affecting shutoff in the

N-terminal domain, especially in parts that are not present in the -

exonuclease. Perhaps this domain, which also carries a more overall positive

charge than the rest of the protein, will play a role in cellular protein binding.

In this scenario, whether or not it is SOX that carries out the RNA

degradation or the potential binding partner cannot be determined presently.

53

Nucleotide synthesis

For every living cell, cell division is a highly regulated process and even the

slightest stray could lead to serious consequences, where cancer is a classic

sign. In a multicellular organism, there has to be a strict balance between

cells that rest, divide and die. The cell cycle is a series of events that lead to

cell division and is under strict control of key enzymes called cyclins and

cyclin dependent kinases. The cell cycle is roughly divided into four phases;

G1-, S-, G2- and M-phase. During G1, cells synthesise RNA and proteins

that are needed for S-phase, which is characterised by DNA synthesis and

chromosome replication. In the G2-phase, proteins needed for cell division

are synthesised, which is followed by actual cell division during the M-

phase. An additional phase exists for cells, called G0, this phase is entered by

cells in G1 and is the state of cells that are non-dividing or are fully

differentiated. The actual time period a cell is present in G0 varies with cell

type some, i.e. neurons, never leave. The passage from one phase to another

is under rigorous regulation and has certain check points that need to be

cleared in order for safe cell division. Deoxyribonucleotides are the building

blocks of DNA and the synthesis of deoxyribonucleotides peaks during S-

phase. The enzyme responsible for the production of deoxyribonucleotides is

the extremely well studied and strictly regulated enzyme ribonucleotide

reductase (146). However, this enzyme is also important for the synthesis of

deoxyribonucleotides in resting cells, as a response to DNA damage. Both

alpha and gamma herpesviruses encode their own RNR while beta

herpesviruses only encode one of the RNR subunits (R1) (5).

Ribonucleotide reductase

RNR is an enzyme that can be found in all kingdoms of life as well as in

viruses. As already mentioned, RNR performs the chemically difficult

reduction of all four ribonucleotides to their corresponding

deoxyribonucleotides in a reaction involving a free radical (177). The

allosterically regulated RNR is the only enzyme that can perform this

reaction and it therefore controls the entire dNTP pool in the cell.

54

Fig 20

The reaction catalysed by RNR.

RNR’s have been divided based on their radical cofactors into three classes,

I, II and III, where class I further is divided into subclasses Ia and Ib. A

strictly balanced supply of deoxyribonucleotides is essential for accurate

DNA replication and repair (146). RNR could therefore be a potential drug

target for the treatment of different types of cancer and viral infections (178-

182).

RNR is a heterotetramer consisting of two homodimers, R1 and R2 and has

an absolute requirement for iron in the form of a di-iron site located in the

R2 subunit. A free radical is generated, by reductive cleavage of molecular

oxygen at the di-iron site of the R2 subunit, which is transported to the R1

subunit to activate the nucleotide substrate for catalysis (177).

RNR activity is under strict transcriptional and postranscriptional control

and in replicating mammalian cells enzyme activity is regulated by control

of R2 protein stability (146, 177). However, to produce

deoxyribonucleotides as a response to DNA damage in resting cells, the

expression of another cellular R2 is induced, which lies under the control of

the transcription factor and tumour suppressor p53, hence it has been named

p53R2 (183, 184). p53R2 also appears to play a role in providing

deoxyribonucleotides for mitochondrial DNA synthesis (188)

Structural studies of the R2 subunit of the ribonucleotide

reductase from EBV and KSHV (manuscript in preparation)

The EBV genome encodes for a class I RNR R2 subunit with low amino

acid sequence identity to bacterial and eukaryotic R2s. This R2, encoded by

the BaRF1 gene was identified as positive by SCOOP of the EBV ORFeome

and is different to R2s of other organisms in that it has a glutamate (Glu61)

in the position of a metal-coordinating aspartate common to most R2s. The

structure of EBV R2 was solved in its apo, mono metal and di-metal form

55

(Figure 21) at 1.9, 1.6 and 2.0 Å respectively and their radical generating

abilities were investigated using electron paramagnetic resonance (EPR).

The monomer of the EBV R2 is an -helix bundle in similarity to other R2s,

but with several interesting differences in the region around the metal site

(Figure 22). In one of the monomers in the apo and monometal structures,

residues 62-73 of -helix B are disordered and Glu61 is either disordered

or oriented away from the metal binding site. Additionally, there is no

traceable density for residues 120-143 in 2 and D indicating that these

elements too are disordered. These stretches seem to order themselves only

when the di-ferrous metal site is fully formed, where Glu61 participates in

metal coordination. This large conformational flexibility upon iron site

formation has not previously been described for any R2 subunits. Recently,

however, disorder in this particular region is found in three R2 structures

also recently available in PDB; of H. sapiens p53R2 (2VUX) in Bacillus

halodurans (2RCC), Plasmodium vivax (2O1Z) and S. cerevisiae (2RCC).

It is likely that the extra flexibility of helix B affects the rate of iron

binding and therefore the timing of the radical generation reaction. EPR

confirms that EBV R2 is slower in generating the radical and that the radical

site is more accessible to radical scavengers. These observed deviances in

the different metal bound forms of EBV R2, become all the more interesting

when put into a wider virus-host cell context. p53R2 also displays this

disordered region. In nonproliferating cells, DNA damage induces

expression of the p53R2 and also the R1 protein. Together they form an

active RNR complex supplying the resting cell with dNTPs for DNA repair

(183, 186). Since the host cell already encodes for its own RNR it is

therefore believed that the viral RNR supplies nucleotides in a resting host

cell (187).

The mobility of these regions in both EBV R2 and p53R2 may be a relevant

adaptation to particular chemical environments in a resting cell, and may

have a functional role in modulating iron and oxygen access, respectively to

the active site.

56

a) b)

c)

Figure 21

Model of the EBV R2 homodimer in its di-metal form. The subunits are coloured in light blue

and dark blue. Iron atoms are coloured in orange.

We have also solved the apo structure of KSHV R2 encoded by ORF60.

Initial results show that the apo form of KSHV R2 also has the unstructured

stretch in the -helix B, which additionally supports the hypothesised and

specific role of gamma herpesvirus R2s and p53R2. However, the chemical

basis for this role still remains to be elucidated.

Figure 22

Cartoon model of the a) apo-EBV R2 b)

mono metal and c) di-metal EBV R2. The

secondary structure elements described in the

text, which are disordered for one of the

monomers in the apo and monometal forms,

are coloured in cyan (helix B) and light

pink (helix 2 and D).

57

Future prospects

Needless to say, viral biology is an interesting field. To thoroughly

understand its evolution, pathogenesis, to develop new drugs and potentially

new vaccines and diagnostic assays etc we need to study the individual viral

proteins both functionally and structurally. Unfortunately, the recombinant

overexpression of viral proteins has so far proven to be very difficult and

therefore very few viral proteins have been structurally determined. We have

developed methods and strategies to speed up the structural genomics

pipeline and increase the throughput. Recombinant viral proteins are being

routinely pushed through our pipeline and protein structures are coming out

as a result. Three structures have so far been solved from proteins involved

in DNA metabolism, which is an area of both scientific as well as medical

interest.

However, to really comprehend a virus’ interaction with its host, structural

studies of protein complexes are necessary and this will in the future be

pursued. The CoFi blot and SCOOP are routinely used in our lab, screening

gene collections of various kinds. The CoFi blot is evolving and we are

finding new applications for it.

58

Acknowledgements When wanting to write the acknowledgements I realised that what I appreciate the most, with

all the people that have surrounded me during this journey, is their fantastic patience and their

never fading trust and support. Many people have been part and I would especially like to

thank a few of them.

Pär, för ditt förtoende. Inte bara när att du antog mig som student utan också för din

övertygelse om att mitt projekt skulle fungera. För att du lät mig åka ända till Kina. Du har

skapat en inspirerande miljö och jag har lärt mig mycket, både om forskning och mig själv.

Stefan Nordlund, för att du alltid lyssnar tålmodigt på en students predikament.

Alla på DBB, speciellt Anki, Kicki, Lotta och Ann, som hjälpt mig många gånger med

administrativa saker.

Past and present members of PN group especially: Martin A, Vickan, Susanne F, Helena,

Susanne VdB, Deb, Tove, Karl-Magnus, Agnes, Martin M, Martin Hä, Amin, Benita,

Anders T, Jessica A, Andreas, Herwig, Albert, Ken, Lina-Beth, Alberto, Marie, Pelle,

Hanna, Henrik, Anna, Florian, Elisabeth and Marina.

Monica, för alla visdomsord om livet.

Said, för ditt fantastiska tålamod och för att du tar hand om labbet!!

Ulrika, U som i ullig, L för labrador!!!! För att du är en fantastisk vän.

Audur, för sjukt roliga stunder både i och utanför labbet! Och all hjälp!!!!

Lola, for always sharing ranting experiences and listening to my ranting.

Heidi, för prat och stöd.

Martin, Stina, Pål och Emma, för upplyftande diskussioner som berör alla möjliga ämnen!

Daniel M-M, för att du alltid kan få mig att skratta.

Karin, för vänskap på labbet och utanför.

Daniel G, för din humor.

Damian, för att du tålmodigt lyssnar och hjälper mig med mina dataproblem.

Maria D, för alla leenden.

People at Beida, especially Xiao Dong, Erik, Nan Jie, Xiaoyan, Koh and Dr Gao! Xie xie.

Sunny “shi pa” and family, your generosity and curiosity overwhelm me. I am certain we

will meet again!

Mina Kina-kompisar, Anders K, Kim, Tove, Anna, Sara, Anders L, Ola och Maria!!!!

Stockholms biovetenskapliga forskarskola, med Eva och Camilla i spetsen.

Mina fosko-kompisar, speciellt Anton, Anna och Linda. Med era historier inser jag att jag

inte är själv!

59

Mia och Linda för att ni är helt utanför labblivet, men alltid är på min sida!

Jenny, för att du alltid är redo att luncha.

Crista and Jill, for your never seizing friendship although you are so far away.

Jasmine and Stephanie, for great times and really being there for me!

Familjerna Nolvi, Jansson/Umana, Tronström och Sundesson.

My entire family in Singapore, who means the world to me, see you soon!

Familjen Cornvik, för att ni alltid lyssnar på mig och mitt eviga tjatande om allt och inget.

Anna-Karin, det finns inga ord som beskriver vår vänskap. Du är världens bästa och ingen

kan ersätta dig!

Kim, världens bästa bror, som alltid ser till att ta ner mig på jorden och som alltid ställer upp

för mig.

Mamma och Pappa, för att ni helt utan tvekan står vid mig. Ni ger mig perspektiv och

försöker alltid förstå vad jag håller på med, både på labbet och med mitt liv.

Tobias, för den fantastiska person du är. För det du betytt, betyder och alltid kommer att

betyda för mig. Du gör mig komplett i alla avseenden. DOJ, DOJ!

60

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