The crystal structure of human WD40 repeat-containing peptidylprolyl isomerase (PPWD1)

The crystal structure of human WD40 repeat-containingpeptidylprolyl isomerase (PPWD1)Tara L. Davis1,2, John R. Walker1, Hui Ouyang1, Farrell MacKenzie1, Christine Butler-Cole1,Elena M. Newman1, Elan Z. Eisenmesser3 and Sirano Dhe-Paganon1,2

1 Structural Genomics Consortium, Banting Institute, University of Toronto, Canada

2 Department of Physiology, University of Toronto, Canada

3 Department of Biochemistry and Molecular Genetics, University of Colorado, Denver and Health Sciences Center, Aurora, CO, USA

Cyclophilins (Cyps) are one of three subfamilies of the

peptidylprolyl isomerases (PPIases; E.C. 5.2.1.8),

together with the structurally unrelated FK506-binding

proteins and parvulins [1,2]. Biologists and clinicians

initially focused on the specific and high affinity of

PPIases for the immunosuppressants ciclosporin A,

FK506 and rapamycin [3–5]. These immunosuppressive

effects were eventually determined to be uncoupled

from the enzymatic function of the PPIases, which

involves the reversible cis–trans isomerization of Xaa–

Pro peptide bonds [2,6]. The evolutionary importance

of this fold and ⁄or function may be inferred from the

broad distribution of PPIases throughout Eukaryota,

Eubacteria, Archaea and, recently, a viral genome

[2,7,8]. However, there is very little direct evidence

to show that the enzymatic function encoded by the

PPIases is the only, or even the primary, physiological

role of these proteins in cells. There are a subset of

proteins whose association with Cyps is necessary for

correct folding and structural integrity: for instance,

maturation of steroid receptor complexes in conjunc-

tion with Hsp90 ⁄Hsc70 [9–11] and the function of Nin-

aA in Drosophila rhodopsin folding [12]. Cyp-catalyzed

isomerization can also play a part in host response

events, including the participation of host CypA in

binding the capsid protein Gag of HIV-1 during infec-

tion in humans and packaging into HIV virions, and

the association of a host CypB–viral polymerase com-

plex leading to viral replication during infection by

hepatitis C [13,14].

However, more recent studies of PPIases have

focused on their roles as signal transducers rather than

Keywords

crystal structure; cyclophilin; peptidyl-prolyl

isomerase; spliceosome; WD40

Correspondence

S. Dhe-Paganon, Structural Genomics

Consortium, Banting Institute, University of

Toronto, 100 College Street, Room 511,

Toronto, ON, Canada M5G 1L5

Fax: +1 416 946 0588

Tel: +1 416 946 3876

E-mail: [email protected]

(Received 18 January 2008, revised 3 March

2008, accepted 6 March 2008)

doi:10.1111/j.1742-4658.2008.06381.x

Cyclophilins comprise one of the three classes of peptidylprolyl isomerases

found in all eukaryotic and prokaryotic organisms, as well as viruses.

Many of the 17 annotated human cyclophilins contain the catalytic domain

in tandem with other domains, and many of the specific functions of a par-

ticular cyclophilin or its associated domains remain unknown. The struc-

ture of the isomerase domain from a spliceosome-associated cyclophilin,

PPWD1 (peptidylprolyl isomerase containing WD40 repeat), has been

solved to 1.65 A. In the crystal, the N-terminus of one isomerase domain is

bound in the active site of a neighboring isomerase molecule in a manner

analogous to substrate. NMR solution studies show that this sequence

binds to the active site of the cyclophilin, but cannot be turned over by the

enzyme. A pseudo-substrate immediately N-terminal to the cyclophilin

domain in PPWD1 could have wider implications for the function of this

cyclophilin in the spliceosome, where it is located in human cells.

Abbreviations

Cyp, cyclophilin; IC50, 50% inhibitory concentration; pNA, p-nitroaniline; PPIase, peptidylprolyl isomerase; PPWD1, peptidylprolyl isomerase

containing WD40 repeat; suc, succinyl.

FEBS Journal 275 (2008) 2283–2295 ª 2008 The Authors Journal compilation ª 2008 FEBS 2283

as chaperones or protein foldases. Specific examples

include the participation of CypD in the mitochondrial

permeability transition, leading to necrotic cell death

[15], the isomerase-dependent regulation of ligand

specificity for the SH2 domain of the non-receptor

tyrosine kinase Itk [16,17], and the role of multiple

Cyps in regulating transcriptional and spliceosomal

events [18–20]. In these cases, the substrate proteins

are already folded and performing a subset of their

normal functions; an isomerase interacts with this sub-

strate conformation, thereby allowing for a new set of

molecular interactions to occur with the product con-

former [1,17]. The molecular switch function of Cyps

may well have different sequence determinants than

their isomerization function, but very few studies have

been performed to probe the sequence specificity of

binding versus catalysis of Cyps. Early evidence indi-

cated that CypA is able to catalyze the isomerization

of a wide array of Xaa-Pro sequences with nearly iden-

tical catalytic efficiency [21]. In contrast, phage display

and subsequent amino acid substitution analysis to

identify CypA binding specificity identified a clear

preference for sequences N- and C-terminal to the

Xaa-Pro target culminating in a consensus sequence of

FG*PXLp. This work indicates that preferential bind-

ing of target proteins may be dictated by a select sub-

set of amino acids distinct from sequences that are

substrates for catalysis [22]. The relevance of this find-

ing is dependent on studies showing an in vivo context

for peptides capable of binding to PPIases without

being used as isomerization substrates; to date, no

such peptide sequence has been described in the liter-

ature.

Further complicating the field of Cyp biology is the

fact that many of the 17 Cyps annotated in the human

genome have not been thoroughly studied in terms of

either enzymatic or other functional significance. For

some of these Cyps, their location inside the cell

provides the only clue as to their function. The large

subset of Cyps found to be stably associated with

spliceosomes provides an example of the issues

involved in studying the complexity of Cyp function.

Spliceosomes are multi-megadalton complexes contain-

ing hundreds of proteins and five essential small

nuclear RNAs, whose function is to excise out non-

coding regions (introns) of translated pre-mRNAs

[23,24]. Recent technological advances in the purifica-

tion of spliceosomal complexes, coupled with advances

in mass spectrophotometric methodology, have led to

a massive increase in the identification of proteins

found to be associated with spliceosomal complexes

[25–28]. At least 11 of the 17 human PPIases have

been found to be associated with intermediate splice-

osomal subcomplexes [26,29]. One of the spliceosomal

Cyps was identified using yeast two-hybrid screens for

known spliceosomal components; another was recog-

nized as a spliceosomal component based on sequence

homology to an SR repeat containing splicing factors

[20,30]. For two other spliceosomal Cyps, functional

and structural studies eventually identified their cog-

nate spliceosomal binding partners: PPIL1 binds to the

SNW domain-containing protein 1 (SKIP), and CypH

binds to the small ribonucleoprotein Prp4 [18,31–33].

Interestingly, in both of these cases, the interaction

with spliceosomal components was found on surfaces

not involving the isomerase active site and did not

affect cis–trans isomerase activity, indicating that the

function of the isomerase in the spliceosomal complex

may involve simultaneous active site and second-site

interactions. The binding partners for the other seven

spliceosomal Cyps are undetermined. Moreover, the

physiological function of the spliceosomal Cyps

remains unclear, which perhaps is not surprising, con-

sidering that so many proteins encoding the same

enzymatic function are found simultaneously in splice-

osomal complexes. It is theorized that this high level

of complexity and seeming duplication of effort in the

spliceosomal complexes are a function of the exquisite

sophistication of dynamic networks needed to properly

regulate and proofread the splicing process [23,34].

PPWD1 (peptidylprolyl isomerase containing WD40

repeat) was cloned in 1994 [35] and later purified as

part of the catalytically competent form of the spliceo-

some C complex [26]. This polypeptide encodes an

N-terminal WD40 repeat domain and a C-terminal

domain homologous to Cyps. As part of an attempt to

structurally characterize the spliceosomal Cyps, we

have determined the high-resolution X-ray crystal

structure of the isomerase domain of PPWD1 and

monitored its activity via both UV kinetics and NMR

solution experiments. In this structure, PPWD1 forms

distinct intermolecular interactions within the asym-

metric unit with an internal peptide containing a

Gly–Pro sequence. Interestingly, the Pro residue is

found in trans, an unusual circumstance for a substrate

peptide. Further experiments have shown that PPWD1

is indeed a functional isomerase against a standard

substrate sequence, but that, surprisingly, a peptide

containing the internal sequence is able to bind, but is

not catalyzed by the isomerase, suggesting that it is

not a substrate. Both the intermolecular interaction

and lack of enzymatic turnover were confirmed using

NMR solution studies of the PPWD1 protein. This

work represents the first structural and biochemical

characterization of a WD40 repeat-containing spliceos-

omal Cyp, and the first instance of a Pro-containing

Structure of spliceosomal cyclophilin PPWD1 T. L. Davis et al.

2284 FEBS Journal 275 (2008) 2283–2295 ª 2008 The Authors Journal compilation ª 2008 FEBS

sequence that is sufficient to bind specifically to the

active site of a Cyp, but is not a substrate for cis–trans

isomerization.

Results

The crystal structure of the isomerase domain of

human PPWD1 (utilizing a construct encoding residues

473–646) was determined at 1.65 A resolution. The

final model of PPWD1 comprises three polypeptide

chains; each chain is disordered from residues 473–482,

such that the first interpretable density is for residue

Gln483. All other residues encoded by the PPWD1

construct are present in the final model. In addition,

the model contains a single glycerol molecule most

probably contributed from the cryoprotectant and 388

water molecules. With an architecture consisting of

eight antiparallel b-sheets forming a closed b-barrelwith two a-helices packed against the core (Fig. 1A),

the structure of PPWD1 is similar to the structure of

the canonical CypA as well as to CypH and PPIL1,

two other spliceosomal Cyps (Fig. 1B). The Ca atoms

of CypA align to the isomerase domain of PPWD1

with an rmsd of 1.34 A over 124 atoms, corresponding

well to the 60% sequence similarity between the two

isomerase domains. The main conformational differ-

ences lie in the a2–b8, b1–b2 and b4–b5 loops. The

b1–b2 loop (corresponding to residues Thr498–

Asp502) is shorter by five residues in PPWD1 com-

pared with CypA. This deletion also occurs in six of

the 17 currently annotated human Cyps, including

PPIL1–PPIL4 (Fig. 2), but the significance of the resul-

tant shorter b1–b2 loop is not understood.

PPWD1 crystallized with three protein molecules in

the asymmetric unit. Unexpectedly, there was an inter-

molecular interaction, propagated throughout the crys-

tal, in which the active sites of all three molecules in

the asymmetric unit were bound to seven residues

(QAEGP487KR) of an adjacent molecule (Fig. 1C).

The three polypeptide chains in the asymmetric unit

are conformationally identical, with rmsd values of 0.4

and 0.2 A over all atoms in 176 residues. In addition,

the N-terminal peptide is oriented in very similar fash-

ion across all three molecules, also with rmsd values of

0.4 and 0.2 A over all atoms for the first seven resi-

dues. Pro487 from one monomer is buried within the

active site of another, and is less than 3 A from the

side-chain of the conserved Arg535 (Arg55 in CypA)

(2.95 A for NH1 and 2.85 A for NH2). In addition,

Pro487 of the peptide is bound in a hydrophobic

pocket composed of Phe540, Phe593, Met541, Leu602

and His606, all of which are conserved between CypA

and PPWD1 (Figs 2 and 3). The Nd1 atom of the

conserved Trp602 (Trp121 in CypA) is coordinating

the backbone oxygen of Lys488 of the QAEGPKR

peptide (Fig. 3A), and Phe540 is coordinating Lys488

through a stacking interaction. The other interactions

between the side-chain of the QAEGPKR peptide in

the active site are centered about residue Glu485; in

addition to specific interactions between the backbone

nitrogen of Glu485 with the oxygen of Gly551, the

side-chain of Glu485 is nestled in a deep pocket of the

enzyme (Fig. 3B). An oxygen of the c-carboxyl groupforms hydrogen bonds with the backbone amide nitro-

gen of Asn582 and a water molecule buried in the

pocket; the other c-carboxyl oxygen is involved in a

network of water-mediated hydrogen bonds. Two resi-

dues in the PPWD1 active site, Gln543 and Gln591,

contribute to the complementary polar nature of this

pocket. The average B-factor for all three of the

QAEGPKR regions is 48 A2, and is comparable with

an all-atom B-factor of 45 A2 for all molecules in the

asymmetric unit. These observations indicate a stable

interaction, although PPWD1 runs as a monomer

using size exclusion chromatography, which suggests

that the interaction is either low affinity or has a high

off rate.

This mode of interaction and the trans conformation

of the peptide in the active site mimic the enzyme–sub-

strate interaction observed in the CypA–HIV-1 Gag

complex [36]. Pro487 is found to be bound to PPWD1

in the trans configuration, which is also the conformer

found in the complexes of the capsid protein Gag and

in some peptides derived from GAG protein with

CypA. For these complexes, the requirement for an

X-Gly-Pro (X „ Pro) sequence was proposed to

obtain stable binding of the trans isomer into the Cyp

active site; the PPWD1 structure confirms this observa-

tion and, indeed, any other amino acid at that position

would adopt /,w angles such that Cb would clash with

the catalytic Arg535 and destabilize the trans confor-

mation [36]. Although the backbone amide of the )2position has been shown to be involved in a configura-

tion-dependent hydrogen bond with the b4–b5 loop,

the function of the side-chain at this position has not

been exhaustively studied. As opposed to the mainly

hydrophobic residues found in the context of HIV-1

capsid variants (containing Ala, Val, or Met at posi-

tion )2), PPWD1 contains Glu at this position

(Glu485), which is pointing into a region of charge

complementarity in the PPWD1 active site that would

not be well accommodated by hydrophobic side-

chains. The relevance of finding a peptide bound in

trans in the active site of isomerases is ambiguous; in

the case of the HIV capsid, it has been proposed that

an X-Gly-Pro sequence represents a poor substrate for

T. L. Davis et al. Structure of spliceosomal cyclophilin PPWD1


catalysis, explaining the capture of the trans conformer

in the crystal [36]. However, as the HIV capsid protein

is indeed an efficient substrate for isomerization by

CypA [37], it cannot yet be stated definitively that

Gly-Pro sequences are the minimal determinant for

binding versus catalysis for Cyps, or what role

sequence variation amongst the Cyps may play. In

summary, the interactions seen between the

QAEGPKR peptide and PPWD1 active site in the cur-

rent structure might be a product of an inability of the

Fig. 1. Structure of PPWD1 and comparison with other Cyp structures. (A) The structure of the isomerase domain of PPWD1 is shown in

ribbon format. The N-terminus and secondary structural elements are labeled. (B) Structural alignment of PPWD1 (magenta) with Ca stick

representations of the canonical CypA (2RMA) in forest green and the spliceosomal CypH (1QOI) in light green. Unless otherwise men-

tioned, all figures were generated using PyMOL (http://pymol.sourceforge.net/). (C) The arrangement of molecules in the asymmetric unit in

the PPWD1 crystal. N- and C-termini are labeled. The N-terminal peptide is bound in the active site of an adjacent protein. The homotypic

interaction between the N-terminal peptide and the active site of a neighboring molecule could be a crystal contact; that is, it may be an arti-

fact of the crystallization condition. Unfortunately, crystallographic methods cannot distinguish between crystal contacts and specific pro-

tein–protein interactions; however, the results presented here indicate that the homotypic interaction modeled in the crystal structure is

supported by solution methods.



Fig. 2. PPWD1 domain structure and sequence alignment of selected human isomerase domains. Top: a graphical overview of the PPWD1

gene. The internal peptide sequence QAEGP487KR is highlighted. Bottom: an alignment of selected human isomerase domains; output is

from MULTALIN [52]. Residues conserved at 90% or more are indicated by capital letters, and between 50% and 90% by lower case letters;

(I ⁄ V) conserved positions are indicated by !; (N ⁄ D ⁄ E) conserved positions are indicated by #. All Cyps shown in this alignment (with the

exception of CypA) have been experimentally described as being associated with spliceosomes, as referenced in the text. Green boxes high-

light the conserved positions in the active site of CypA [2]; blue boxes outline the positions of the spliceosomal binding site on CypH [32];

pink boxes outline the main spliceosomal interaction regions on PPIL1 [31]. Asterisks (*) and crosses (+) above the alignment indicate the

positions of the S1¢ and S2¢-S3¢ subsites as described in [38].



enzyme to catalyze the isomerization of this particular

sequence; alternatively, the trans conformer of the sub-

strate Gly–Pro sequence might simply have a higher

affinity for the PPWD1 binding pocket. Further analy-

sis is therefore necessary to attempt to distinguish

between these two possibilities.

To determine whether PPWD1 is catalytically com-

petent to isomerize Cyp substrates, we conducted bio-

chemical and biophysical assays. Using a colorimetric

coupled assay against succinyl-Ala-Ala-Pro-Phe-p-nit-

roaniline (suc-AAPF-pNA), a well-characterized CypA

substrate, we found that the isomerase domain of

PPWD1 (using the crystallographic construct compris-

ing residues 473–646) is indeed an active isomerase

with a catalytic efficiency of 1.5 lm)1Æs)1, similar to

the values obtained in previous studies (Fig. 4A)

[21,38]. This is not surprising as the active site residues

of PPWD1 are nearly completely conserved when com-

pared with CypA (Fig. 2). Furthermore, we found that

ciclosporin A binds tightly to PPWD1 with a 50%

inhibitory concentration (IC50) between 1 and 2 nm, a

value similar to that for CypA (Fig. 4B). These enzy-

matic characteristics are not significantly different for

a truncated PPWD1 construct without the N-terminal

sequence (encoding residues 493–646), implying that

this region does not interfere with the active site at the

pico- to micromolar concentrations of protein used in

the assay. As a result of the technical limitations of the

enzyme coupled assay described above, we may not

have detected enzymatic activity against low-affinity

substrates in the millimolar KD range, nor could we

detect binding without catalysis. To address this issue,

we conducted direct NMR measurements, as described

previously [39]. PPWD1 (residues 473–646) was active

on the standard Suc-AAPF-pNA substrate, as indi-

cated by a collapse of the peaks contributed by the cis

and trans conformers (caused by enzyme-catalyzed

isomerization that is rapid compared with the chemical

shift differences between the cis and trans resonances)

(Fig. 4C, red peaks). PPWD1 also bound to a syntheti-

cally derived QAEGPKR peptide, as shown by the

small shifts for resonances on addition of the enzyme,

especially for the +2 Arg resonances, which correlate

well with the Arg interactions seen in the crystal struc-

ture. However, PPWD1 did not catalyze the isomeriza-

tion of this peptide sequence, as it does for the model

peptide substrate AAPF (Fig. 4C, black peaks;

Fig. 4D). Finally, NMR-based experiments were

undertaken to validate the intermolecular association

of PPWD1 in the context of the protein construct used

to obtain the crystal structure. 1H,15N-HSQC measure-

ments were conducted on the PPWD1 construct which

crystallized (residues 473–646), as well as an N-termi-

nally truncated construct (residues 493–646). As

expected, the overall spectra of these two constructs

were similar. However, it is clear that the line widths

of most resonances in the spectrum of the longer con-

struct are broader than those in the shorter construct

Fig. 3. Details of QAEGP487KR peptide interaction. (A) Cartoon model of PPWD1 and stick representation of the amino terminal peptide.

Active site residues are shown in stick format and water molecules are shown as spheres. Note the water molecules in a hydrogen bonding

network with Glu486 of the N-terminal peptide. (B) Electrostatic surface representation (+10 kTÆe)1, red; )10 kTÆe)1, blue) of PPWD1, gener-

ated by the APBS software package [53], showing the side-chains of Pro487 and Glu486 buried in deep pockets formed by the PPWD1 active

site.



(Fig. 5). The simplest interpretation of this line broad-

ening is that it is a result of a weak interaction

between protein molecules, and it is quite possible that

the interaction seen in solution using NMR is caused

by the specific interactions captured in the crystal

structure. Taken together, these data suggest that the

intermolecular interaction trapped in the crystal struc-

ture can indeed be recapitulated using solution meth-

ods, and that this intermolecular interaction is a

consequence of binding, but not efficient catalysis, of

the QAEGPKR sequence by PPWD1.

Discussion

The QAEGPKR peptide is capable of binding the

PPI domain of PPWD1 without being an efficient

substrate for cis–trans isomerization. It is clear from

earlier work that there is very little selectivity for

substrates at the )1 or +1 position in the Cyp

active site, with the caveat that Gly–Pro may pro-

mote the shift of equilibrium binding in the active

site to the trans over the cis conformer (although

with very little difference in catalytic efficiency)

Fig. 4. PPWD1 is an active isomerase and binds to QAEGPK, but does not catalyze its isomerization. (A) Increasing amounts of PPWD1 accel-

erate the isomerization of suc-AAPF-pNA in a standard colorimetric assay. The bottom curve (squares) shows the unaccelerated cleavage of

peptide relative to the catalyzed reaction (top curve, circles). (B) Ciclosporin A inhibits PPWD1 activity against suc-AAPF-pNA. (C) Binding and

catalysis of isomerization for the standard substrate suc-AAPF-pNA. Black and red spectra of the Ala residues in the peptide are obtained from

the uncatalyzed and catalyzed reaction, respectively. Acceleration of the cis–trans isomerization of the peptide results in the collapse of these

resonances into a single set of peaks. (D) Binding, but not isomerization, of the N-terminal peptide of PPWD1. A synthetic peptide correspond-

ing to the seven residues seen in the crystal structure (QAEGPKR) was added to the PPWD1 protein (residues 473–646) to assess binding as

in (C). Notice that the peak shift observed for Arg resonances is more pronounced than that for Lys (labeled with R and K, respectively), indicat-

ing a change in the chemical environment for this residue; this is confirmed by the model of the crystallographic data, which shows Arg489

pointing into the active site of the isomerase, whereas Lys488 points into the solvent. There are chemical shifts on addition of the enzyme, but

no collapse of cis and trans peaks, suggesting that PPWD1 binds the N-terminal peptide but does not catalyze isomerization.



[21,22,38]. Therefore, it is reasonable to look N-ter-

minal and C-terminal to these positions to find

potential binding specificity in Cyps. Previous studies

have delineated binding determinants for Cyps con-

tained outside of the )1 and +1 positions using dif-

ferent methodologies. For instance, by comparing

binding affinities with the ability to inhibit PPIase

activity for peptide-based inhibitors, it was found

that the active site of CypA could be separated into

two distinct subsites (called S1¢ and S2¢–S3¢), pre-

sumably indicating binding determinants on the pro-

tein distal to the )1 position [38]. In addition, a

phage display experiment, which independently con-

firmed the preference for binding of Gly–Pro at the

central position, described a binding preference for

Phe at the )2 position and various amino acids at

+3 and +4 for CypA [22]. These studies indicated

that there might be separate sequence determinants

for binding that are stricter than those for substrate

turnover, and that these determinants are somehow

dictated by residues outside the minimal active sur-

face of the Cyps. The structure of PPWD1 indicates

that the isomerase domain may bind tightly to a

subset of protein targets with a polar side-chain at

the )2 position, without losing the ability to be an

efficient isomerase for substrates containing alterna-

tive residues at this position (A ⁄G-P, for instance),

as opposed to the hydrophobic preference of CypA

at this position. Although residues that form the

skeletal S2 pocket are identical between CypA and

PPWD1, those adjacent to it, particularly in the b4–b5 loop (including residues Gly551–Gly553, Glu555

and Gly560–Glu561) are different between CypA and

PPWD1, and may influence the side-chain specificity

at the )2 position. Indeed, this region of the active site,

including the b5–b6 loop (residues Ala583–Thr587), is

the most divergent when comparing all Cyp active sur-

faces, and we therefore predict that these regions will

dictate the greatest differences in specificity at the )2position. In addition, using this structural rationale,

there is some preference for a bulky side-chain at the

+2 position because of potential stacking interactions

with Trp601 and Phe540. Our current data cannot

address positions C-terminal to +2, as our

QAEGPKR peptide is constrained by its attachment

to a neighboring Cyp molecule.

Fig. 5. PPWD1 protein shows association in solution. Two overlain 1H,15N-HSQC spectra of PPWD1 proteins are shown. The 473–646 con-

struct (black) contains the amino terminal sequence QAEGPKR, but 493–646 (red) does not. The 473–646 construct is the same protein that

led to the crystal model. Experiments were conducted in parallel and under conditions of identical buffer and protein concentration.



As mentioned earlier, many of the residues that

make up the distal subsites on PPWD1 are conserved

between the spliceosomal Cyps and CypA (Fig. 2).

Two exceptions are Ala583, which is variously changed

to Ser, Arg or Asn in spliceosomal Cyps, and Trp601

which is largely conserved amongst the non-spliceoso-

mal Cyps, but is variably changed to His, glutamate,

or Tyr in the spliceosomal subclass of Cyps (Fig. 2).

Interestingly Ala583 is part of the S1¢ subsite, and

Trp601 and Phe540 are part of the S2¢–S3¢ subsite

described previously [38]. It is reasonable to propose

that the ability of PPWD1 to bind to sequence deter-

minants that are not substrates for isomerization may

be quite relevant to the larger biological function of

Cyps if it is found to be a more general phenomenon.

The intermolecular interaction seen in the crystal

structure of PPWD1 may imply a new function within

the spliceosome, where it possibly plays a role in

spliceosomal assembly or activity. Homotypic interac-

tions are also observed in the crystal structure of

another spliceosomal Cyp, CypH (SnuCyp-20), where

isomerase domains interact with each other, again

through an extended loop region N-terminal to the

isomerase domain. PPWD1 and CypH have no

sequence similarity in this region and crystallize under

different conditions and with different spacegroups

[32]. It is interesting in the context of this dissimilarity

to observe that the two spliceosomal Cyps exhibit a

similar type of intermolecular association in structural

studies. Although the solution properties of the homo-

typic CypH interaction were not studied, the crystal

structure of CypH bound to a peptide derived from

the spliceosome shows that it binds a surface directly

opposite to the isomerase active site (Fig. 2), suggest-

ing that the homotypic interaction seen in the structure

would not be impaired by spliceosomal association

[32,33]. The overall sequence similarity between the

isomerase domain of CypH and the isomerase domain

of PPWD1 is reasonably high (55% over approxi-

mately 140 residues), but many of the residues that

form the spliceosomal binding site of CypH are not

conserved in PPWD1 (blue boxes, Fig. 2). In the case

of CypJ (PPIL3), another spliceosomal Cyp whose

interaction surface with the spliceosomal protein SKIP

has been probed using NMR, the interaction with the

spliceosomal component was again found to be distinct

from the active site (pink boxes, Fig. 2) [31]. Again,

the spliceosomal interacting region of CypJ is not well

conserved amongst spliceosomal Cyps, including

PPWD1. Although the spliceosomal target or interact-

ing region of PPWD1 has not been isolated, it is rea-

sonable to believe, based on the cases of CypH and

CypJ, that this region may lie well outside the active

site residues, and that these surfaces may be variable

in terms of sequence amongst the spliceosomal Cyps in

order to target isomerase binding to distinct spliceoso-

mal substituents. It may be that the bifunctional isom-

erase domains of spliceosomal Cyps may be directed

towards internal sequences in order to regulate the

activity of these enzymes or to serve as a signal trans-

duction element in addition to the isomerase function.

Indeed, it may be that isomerization must be prevented

in order for spliceosomal Cyps to perform these addi-

tional functions, and perhaps the homotypic interac-

tions seen in the spliceosomal Cyps are indicative of

peptide sequences that are binding determinants, but

not efficient substrates, as in the case of PPWD1.

Experimental procedures

Cloning, expression and purification

Full-length cDNA encoding human PPWD1 was obtained

from the Mammalian Gene Collection (BC015385). Con-

structs based around the predicted isomerase domain

boundaries were cloned into pET28a-LIC and transformed

into BL21 Gold (DE3) cells (Stratagene, La Jolla, CA,

USA). Cultures were grown in Terrific Broth medium

at 37 �C to D � 6, and induced at 15 �C with the addition

of 50 lm isopropyl thio-b-d-galactoside. Pellets were resus-

pended in 20 mL of lysis buffer (50 mm Tris, pH 8.0,

500 mm NaCl, 1 mm phenylmethanesulfonyl fluoride and

0.1 mL general protease inhibitor; Sigma P2714, St Louis,

MO, USA) and lysed by sonication; lysates were then cen-

trifuged for 20 min at 69 673 g. The supernatant was

loaded onto nickel nitrilotriacetic acid resin (Qiagen, Valen-

cia, CA, USA), washed with five column volumes of lysis

buffer and five column volumes of low imidazole buffer

(lysis buffer + 10 mm imidazole, pH 8), and eluted in

10 mL of elution buffer (lysis buffer + 250 mm imidazole,

pH 8, and 10% glycerol). One unit of thrombin (Sigma)

per milligram of protein was added to remove the tag

overnight at 4 �C. For gel filtration, an XK 16 · 65 column

(GE Healthcare, Piscataway, NJ, USA) packed with

HiLoad Superdex 200 resin was pre-equilibrated with gel

filtration buffer (lysis buffer + 5 mm b-mercaptoethanol

and 1 mm EDTA). Peak fractions were pooled and

concentrated using Amicon concentrators (10 000 molecular

mass cut-off; Millipore, Danvers, MA, USA). The protein

was used at 15 mgÆmL)1 for crystallization studies.

Crystallization, data collection and structure

solution

A construct of the PPWD1 isomerase domain containing

residues 473–646 crystallized in 1.7 m NH4SO4, 0.1 m

sodium cacodylate, pH 5.7, and 0.2 m sodium acetate.



Crystals were harvested into mother liquor with 15% glyc-

erol and frozen in liquid nitrogen. Diffraction data were

collected on an FR-E SuperBright Cu rotating anode ⁄Raxis

IV++ detector (Rigaku Americas, The Woodlands, TX,

USA), and integrated and scaled using the hkl2000 pro-

gram package [40,41]. For structure solution and refine-

ment, the program phaser [42] was used as part of the

ccp4 suite [43] to find the molecular replacement solution

in the resolution range between 20 and 2.8 A, using PDB

ID 1XO7 as a search model. Following phaser, a round of

maximum-likelihood refinement and phase extension to

1.65 A was carried out with refmac5 [43,44], and the

phases were then input into arp ⁄warp [45] for automatic

model building and iterative refinement. The models were

completed using the graphics program o [46], and further

rounds of refinement using refmac5 resulted in an R-factor

of 19.9% (Rfree = 24.5%) for data from 44.32 to 1.65 A.

The model has excellent stereochemistry as judged by

procheck [47], with no residues in disallowed or unfavor-

able regions of Ramachandran space. The final model of

PPWD1 comprises three polypeptide chains corresponding

to the three molecules in the asymmetric unit; each chain is

disordered from residues 473–482, such that each model

reflects residues Gln483–Lys646. Data collection and refine-

ment statistics are provided in Table 1. Coordinates and

structure factors have been deposited in the Protein Data

Bank (PDB) with ID 2A2N.

PPIase colorimetric activity

PPIase activity was measured using a standard protease-cou-

pled assay [3,48] adapted to a 96-well format. The reaction

mixture contained 64 pm to 1 lm of PPIase and 200 nm chy-

motrypsin (Sigma) in reaction buffer (35 mm Hepes, pH 7.8,

368 mm trifluoroethanol, 50 mm NaCl2, 10 mm LiCl2, 5 mm

b-mercaptoethanol). The reaction was performed at 25 �Cusing 33–400 lm suc-AAPF-pNA (Bachem Americas, King

of Prussia, PA, USA), and read at 390 nm on a BioTek Syn-

ergy 2 plate reader using flat-bottomed well plates (Costar

3695). Initial velocities were plotted against the substrate

peptide concentrations to compare the uncatalyzed chymo-

trypsin rate with the isomerase-catalyzed reaction.

NMR experiments

Cells were inoculated into 20 mL of modified M9 medium

containing (15NH4)2SO4, trace elements and Kao & Mich-

ayluk vitamin solution (Sigma). Growth and induction were

performed as above, except that cells were induced at

D > � 3 with 100 lm isopropyl thio-b-d-galactoside. Pro-tein was purified as above. 15N-labeled proteins at 1 mm in

20 mm Hepes, pH 7.5, 100 mm NaCl, 10 mm dithiothreitol

and 10% D2O were pre-centrifuged at 35 000 g for 10 min,

and then subjected to NMR using a cryoprobed Bruker

AV500 spectrometer (Bruker, Milton, Canada). All spectra

were recorded at 25 �C. For 1H,15N-HSQC experiments, a

pulse sequence with ‘flip-back’ water suppression was used.

Typically, sweep widths of 8000 and 2000 Hz were used for

the F2 and F1 dimension, respectively. The data were pro-

cessed with Topspin [49] or NMRpipe [50] software.

All samples aimed at assessing PPWD1 binding and cataly-

sis of peptides were diluted to 300 lL with 5% D2O and

placed into a Shigemi microcell (Allison Park, PA, USA) in

50 mm Hepes, pH 7, 500 mm NaCl and 1 mm dithiothreitol.

Samples contained 3 mm peptide and either 0.5 mm suc-

AAPF-pNA (Bachem) or TQAEGPKR (Sigma-Genosys),

with and without 1 mm PPWD1-Cyp. Spectra were collected

at 10 �C on a Varian 600 or 900 MHz spectrometer (Palo

Alto, CA, USA). Spectra were acquired using standard Var-

ian BioPack sequences, processed using NMRpipe software

[50] and visualized using ccpn software [51].

Acknowledgements

Some of the NMR instrumentation used in the current

study belongs to the Ontario Center for Structural

Table 1. Data collection, phasing and refinement statistics [atomic

coordinates were deposited in the Protein Data Bank (PDB) (http://

www.rcsb.org): 2A2N].

Data set PPWD1

Space group C 1 2 1

Unit cell (A)

Unit cell (deg)

139.658, 39.893, 115.638

90.00, 122.33, 90.00

Beamline Rigaku FR-E

Wavelength (A) 1.54178

Resolution (A) 23.20–1.65

Unique reflections 59 800

Data redundancy (-fold) 3.4 (2.6)

Completeness (%)a 91.4 (63.1)

I ⁄ sigI 34.86 (2.0)

Rsymb 0.036

Refinement

Resolution (A) 23.20–1.65

Reflections used 56 773

All atoms {solvent} 4259 {338}

Rwork ⁄ Rfreec 0.199 ⁄ 0.245

rmsd bond length (A) 0.015

rmsd bond angle (deg) 1.368

Figure of merit 0.815

Average B-factor (A2) 46.22

Ramachandran plot

Favored (%) 94.46

Allowed (%) 5.54

Disallowed (%) 0

a Highest resolution shell is shown in parentheses. b Rsym = 100 ·sum(|I ) <I>|) ⁄ sum(<I>), where I is the observed intensity and <I>

is the average intensity from multiple observations of symmetry-

related reflections. c Rfree value was calculated with 5% of the

data.



Proteomics. The Structural Genomics Consortium is a

registered charity (number 1097737) that receives funds

from the Canadian Institutes for Health Research,

the Canadian Foundation for Innovation, Genome

Canada through the Ontario Genomics Institute, Glaxo-

SmithKline, Karolinska Institutet, the Knut and Alice

Wallenberg Foundation, the Ontario Innovation Trust,

the Ontario Ministry for Research and Innovation,

Merck & Co., Inc., the Novartis Research Foundation,

the Swedish Agency for Innovation Systems, the

Swedish Foundation for Strategic Research and the

Wellcome Trust.

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The crystal structure of human WD40 repeat-containing peptidylprolyl isomerase (PPWD1)

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Transcript of The crystal structure of human WD40 repeat-containing peptidylprolyl isomerase (PPWD1)