proteinsSTRUCTURE O FUNCTION O BIOINFORMATICS

Investigating the structural stability of theTup1-interaction domain of Ssn6: Evidencefor a conformational change on the complexMaria Palaiomylitou,1 Athanassios Tartas,1 Dimitrios Vlachakis,1 Dimitris Tzamarias,2 and

Metaxia Vlassi1*1 Institute of Biology, National Centre for Scientific Research ‘‘Demokritos’’, 15310 Ag. Paraskevi Attikis, Greece

2 Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, 71110 Heraklion, Greece

INTRODUCTION

Transcriptional repression of a wide variety of gene families in

Saccharomyces cerevisiae requires the function of two physically asso-

ciated proteins, Ssn6 and Tup1. The Ssn6-Tup1 complex consists of

one Ssn6 and four Tup1 molecules1 and is recruited to target pro-

moters by interacting with gene-specific DNA-binding proteins.2–5

Transcription regulators resembling Ssn6 and Tup1, both in amino

acid sequence and function, have also been found in worms, flies,

plants, and mammals, suggesting that transcriptional repression by

an Ssn6-Tup1-like system is evolutionary conserved.2,6

The N-terminal domain of the 966 amino acid Ssn6 protein contains

10 tandem tetratricopeptide repeats, known as TPRs (Fig. 1). The TPR

constitutes a degenerate recurrent sequence motif, the consensus

sequence of which is defined by a pattern of small and large hydropho-

bic amino acids.7,11 TPR mediated protein–protein interactions are

involved in a diverse spectrum of cellular functions.12–14 On the basis

of the functional diversity and importance of TPR containing proteins,

Blatch and Lassle14 proposed that the TPR motif may represent an an-

cient general protein–protein interaction module adopted by function-

ally different proteins and adapted for specific functions. The minimal

Tup1 interaction domain of Ssn6 includes its three N-terminal TPRs

(Fig. 1), whereas various combinations of TPR4 to TPR10 mediate

interactions with structurally distinct DNA-binding repressor proteins

specific for each gene family regulated by the Ssn6-Tup1 complex.15

The structure of Ssn6 is unknown. However, the crystal structures

of several natural16–26 as well as of designed TPR containing pro-

teins8 have been determined so far demonstrating that each TPR

motif is highly a-helical composed of a pair of antiparallel a-helices,termed helices A and B, as first determined by the crystal structure of

the three-TPR containing protein Pp5.16 The packing of helices

within and between adjacent TPR motifs is identical so that each

a-helix interacts with its two immediate a-helix neighbors8,16 in a

The Supplementary Material referred to in this article can be found online at http://www.

interscience.wiley.com/jpages/0887-3585/suppmat/

Grant sponsor: General Secretariat for Research and Technology of Greece, NCSR ‘‘Demoritos’’.

Maria Palaiomylitou and Athanassios Tartas contributed equally to this work.

*Correspondence to: Metaxia Vlassi, Institute of Biology, National Centre for Scientific Research

‘‘Demokritos’’, 15310 Ag. Paraskevi Attikis, Greece. E-mail: meta@bio.demokritos.gr

Received 30 December 2006; Revised 23 February 2007; Accepted 6 March 2007

Published online 16 July 2007 in Wiley InterScience (www.interscience.wiley.com).

DOI: 10.1002/prot.21489

ABSTRACT

Ssn6, a tetratricopeptide repeat (TPR) containing

protein, associates with the Tup1 repressor to

form a global transcriptional co-repressor com-

plex, which is conserved across species. The three

N-terminal TPR repeats of Ssn6, out of a total of

10, are involved in this particular interaction.

Our previously reported 3D-modeling and muta-

genesis data suggested that the structural integrity

of TPR1 and its correct positioning relatively to

TPR2 are crucial for Tup1 binding. In this study,

we first investigate the structural stability of the

Tup1 binding domain of Ssn6, in pure form,

through a combination of CD spectroscopy and

limited proteolysis mapping. The obtained data

were next combined with molecular dynamics sim-

ulations and disorder/order predictions. This com-

bined study revealed that, although competent to

fold, in the absence of Tup1, TPR1 is partially

unfolded with its helix B being highly dynamic

exposing an apolar surface to the solvent. Subse-

quent CD spectroscopy on this domain complexed

with a Tup1 fragment comprising its Ssn6 binding

region provided strong evidence for a conforma-

tional change consisting of acquisition of a-helical

structure with simultaneous stabilization of a

coiled-coil configuration upon complex formation.

We propose that this conformational change

occurs largely in the TPR1 of Ssn6 and is in

accord with the concept of folding coupled to

binding, proposed for other TPR domains. A

possible implication of the structural flexibility of

Ssn6 TPR1 in Tup1 recognition is discussed and a

novel mode of interaction is proposed for this par-

ticular TPR-mediated complex.

Proteins 2008; 70:72–82.VVC 2007 Wiley-Liss, Inc.

Key words: tetratricopeptide repeat; structural flexi-

bility; coupled folding and binding; circular dichro-

ism; limited proteolysis; molecular dynamics.

72 PROTEINS VVC 2007 WILEY-LISS, INC.

three-stranded coiled-coil manner resulting in super-helical

architectures, the size and shape of which depends on the

number of tandem TPR motifs. A significant consequence

of the TPR superhelicity is the generation of a concave sur-

face formed by helices A of tandem TPRs and a convex sur-

face formed by both A and B helices.16 The concave surface

has been proposed to be involved in ligand binding16 and

this hypothesis was indeed confirmed experimentally in the

case of several Hsp-binding TPR proteins.17,22,24,27 The

crystal structure of the p67phox/Rac complex, however,

demonstrated that binding to the TPR concave groove is

not a general structural principle of ligand binding by TPR-

containing proteins, suggesting a versatility of TPR domains

in both target recognition and their general role as scaffolds

for protein assembly.18

Using the canonical TPR structure as template we have

previously modeled the 3D-structure of the TPR1-3

domain of Ssn6 involved in Tup1 binding. The model

combined with mutagenesis data indicated that the struc-

tural integrity of TPR1 and its correct positioning relatively

to TPR2 are essential for the Ssn6-Tup1 interaction.9 Con-

sistently, an independent mutagenesis work also attributed

a major role of the structural integrity of TPR1 in this par-

ticular interaction of Ssn6.10 In our previous study we

have also suggested that the Ssn6-Tup1 interaction may

have a hydrophobic character and in this respect it is rather

unique among known TPR-mediated interactions, making

its structural analysis of major interest.

The present study is a starting point of our efforts

towards an understanding of the structural basis of this

particular interaction of Ssn6. Towards this end, we

investigate the structural stability of the Tup1 interaction

domain of Ssn6, both in isolation and in complex with a

Tup1 fragment containing the Ssn6 interaction domain.

Circular dichroism (CD) spectroscopy, limited proteolysis

mapping, and molecular dynamics (MD) simulations

combined with disorder/order predictions show that, in

isolation, TPR1 is partially unfolded with its helix B

being highly dynamic exposing an apolar surface to the

solvent. Subsequent CD spectroscopy on an Ssn6-Tup1

complex provides strong evidence for a conformational

change involving acquisition of a-helical coiled structure.

We propose that this conformational change occurs

mainly in the TPR1 of Ssn6 and is in accord with the

idea of folding coupled to binding, proposed in other

TPR domains. A possible role of the structural flexibility

of TPR1 in Tup1 recognition is discussed. In addition, a

novel mode of association involving the convex TPR sur-

face and hydrophobic rather than polar residues is pro-

posed for this particular TPR-mediated interaction of

Ssn6 in contrast to modes of interaction observed so far

in other TPR-mediated complexes.

MATERIALS AND METHODS

Construction of plasmids

The minimal Tup1 interaction domain of S. cerevisiae

Ssn6 protein (Sc3T; residues 1–175) that includes its

three and a half N-terminal TPRs was cloned using the

pGEX-2T expression vector (Amersham Biosciences) as

described previously.15

A plasmid encoding the entire TPR domain of Ssn6

(Sc10T; residues 1–415) tagged to 6xHis was constructed

by inserting a PCR generated fragment between the NdeI

and BamHI sites of pET15b (Novagen) expression vector.

The PCR product was generated using the 50-TCATCCCGGGATGAATCCGGGCGGTGAAC-30 and 50-GTAAGGATCCTTACTATGGGTTTTCTAAC-30 forward and

reverse primers, respectively and was subsequently puri-

fied and digested with XmaI and BamHI. The NdeI site

of the vector was filled in with Klenow polymerase prior

to ligation with the PCR product.

A fragment of S. cerevisiae Tup1 protein (Tn108; resi-

dues 1–108) including the Ssn6 interaction domain5 was

cloned as a XmaI-AvaII blunt fragment at the XmaI-

EcoRI blunt sites of PGEX-2T expression vector. This par-

ticular molecule was chosen based on its stability and

solubility compared to shorter Tup1 protein fragments.

The sequence of the coding region of all constructs used

in this study was confirmed by nucleotide sequencing.

Protein expression and purification

Sc3T was expressed as a GST fusion protein in Esche-

richia coli. BL21(DE3) cells were grown at 378C in LB

Figure 1A: Schematic representation of the 10-TPR containing region of Ssn6 protein

(aa: 1–415). The two fragments used in this study (Sc3T and Sc10T) are

indicated. B: Sequence alignment of TPR repeats 1 to 3 involved in Tup1

binding. Predicted TPR a-helices are depicted as cylinders above the sequence.

Residues of the TPR signature7 are grey shaded. Amino acid preferences for each

TPR position8 are indicated below the aligned sequences. Hydrophobic residues

not-matching the TPR consensus character are black shaded. A hydrophobic

patch is boxed. Point mutation sites disrupting interaction with Tup19,10 are

indicated by open circles and asterisks (see discussion). Black and open arrows

denote recognized and predicted chymotrypsin sites, respectively. Chymotrypsin

recognition sites to Ala are not included for clarity reasons. C: Model of the

three-dimensional structure of the TPR1-3 region of Ssn6.9

Structural Stability of a Domain of Ssn6

DOI 10.1002/prot PROTEINS 73

medium containing ampicillin (100 lg/mL) to an OD600

of 0.7 and induced with 0.7 mM isopropyl-b-D-thiogalac-topyranoside (IPTG) at 258C for 3 h. The bacterial cell

pellet was lysed by sonication in lysis buffer A (20 mM

Tris-HCl pH 8.0, 50 mM NaCl, 20% (v/v) glycerol, 0.1%

(v/v) Triton X-100, 5 mM EDTA, and 1 mM DTT). The

lysate was centrifuged at 12,000g for 1 h at 48C. Proteaseinhibitors were included in all purification steps. The

GST-Sc3T fusion protein was purified by affinity chro-

matography on a Glutathione Sepharose 4B (GS-H)

(Amersham Biosciences) affinity column equilibrated

with lysis buffer A. After extensive washing of the col-

umn the bound protein was digested with thrombin pro-

tease (using a molar ratio of 1:500) for 2 h at 168C to

remove the GST moiety. The reaction was stopped by the

addition of benzamidine. The resulting purified protein

was subsequently loaded on a Sephacryl S200 (Amersham

Biosciences) gel-filtration column equilibrated with

20 mM Tris-HCl (pH 8.0) and 200 mM NaCl. Purified

protein not used immediately was stored at 2208C after

addition of 20% (v/v) glycerol.

Sc10T was expressed as a 6xHis-tagged protein using

E. coli BL21(DE3) cells. Cells were grown to an OD600 of

0.5 at 308C and induced with 1 mM IPTG at 208C for 16

h. Harvested cells were lysed by sonication in lysis buffer

B (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 3 mM imidaz-

ole, 0.5 mM DTT, 20% (v/v) glycerol, and 1% (v/v) NP-

40). After clarification, the His-tagged protein was puri-

fied to near homogeneity by affinity chromatography on

a Ni-nitrilotriacetic acid column (Qiagen) equilibrated

with buffer B and eluted with an imidazole gradient.

Tn108 was expressed as a GST-fused protein as

described above for Sc3T. The protein was purified to

near homogeneity using GS-H affinity chromatography

followed by removal of the GST moiety using thrombin

cleavage.

The Sc3T-Tn108 complex was produced in vitro by

incubating purified Tn108 and GH-S beads with pre-

bound GST-Sc3T. A fourfold excess of Tn108 over Sc3T

(in molar basis) was used to ensure complete saturation

of Sc3T. A control experiment using GH-S beads with

prebound GST was carried out, in order to test out for

any nonspecific binding of Tn108 on the matrix or on

GST. After incubation with Tn108 for 3 h at room tem-

perature, the GH-S beads were extensively washed to

remove any unbound Tn108 protein. Beads carrying the

bound GST-Sc3T-Tn108 complex were subsequently

incubated with thrombin to remove the GST moiety.

The resulting Sc3T-Tn108 complex was obtained by

centrifugation.

Molecular weight and Stokes radiusdetermination of Sc3T

The molecular weight and Stokes radius of Sc3T were

determined by gel-filtration chromatography. The chro-

matographic separation on the Sephacryl S200 column

was calibrated using the protein standards: bovine serum

albumin (66 kDa), ovalbumin (45 kDa), carbonic anhy-

drase (29 kDa), cytochrome c (12.4 kDa), and potassium

dicromate (approximately 200 Da). Blue dextran (� 2

MDa) was used to determine the void volume (V0) of

the column. The relative molecular weight of the elution

peak of Sc3T was then estimated using a standard curve

obtained by plotting the logarithm of the molecular

weight of the known proteins against Kav 5(Ve 2 V0)/

(Vt 2 V0), with Vt being the total bed volume}. Estima-

tion of the Stokes radius (RST) of Sc3T was obtained by a

modified Laurent and Killander method28,29 using a lin-

ear calibration plot of RST values versus (2log Kav).

Limited proteolysis and peptide mapping

Limited proteolysis mapping was performed on the

Sc10T fragment of Ssn6. Purified Sc10T protein, as eluted

from Ni-NTA column, was digested with a-chymotrypsinII

protease (Sigma–Aldrich) at a molar ratio of 1:200 (w/w,

protease:target protein). Chymotrypsin was selected for

its property to recognize exposed hydrophobic residues.

The reaction proceeded at room temperature for 2 h and

aliquots were collected at various times and analyzed by

SDS-PAGE. The gel was subsequently transferred onto a

polyvinylidene difluoride (PVDF) membrane. Protein

bands corresponding to proteolytically resistant fragments

were excised from the PVDF membrane and subjected to

N-terminal sequencing (Biocentrum Services, Krakow,

Poland).

Circular dichroism

Far-UV (180–260 nm) CD experiments were per-

formed on a JASCO J-715 spectropolarimeter equipped

with a Peltier element for temperature control. All scans

were performed using a 0.1 mm path-length quartz cell.

The wavelength scans were made with the following

parameter settings: 0.2 nm step resolution, 20 nm scan

speed, 1 s response time, 1 s bandwidth, and 8 accumula-

tions. The CD signal from the buffer was subtracted

from the CD signals of the proteins. Conversion of the

observed ellipticities to molar ellipticities and deconvolu-

tion of the CD spectra were made using the CDNN

program.30,31 The expected CD spectrum of the

Sc3T-Tn108 complex was calculated by a summation of

the individual Sc3T and Tn108 CD spectra using the

equation: [y]theor 5 (NresSc3T 3 [y]Sc3T 1 NresTn108 3[y]Tn108)/NresSc3/Tn108, where [y]i and Nresi are the mean

residue ellipticity and residue number of protein i,

respectively. Thermal unfolding was recorded by moni-

toring changes in the CD signals at 222 nm correspond-

ing to the loss of the a-helical structure during thermal

denaturation with a scan rate of 908C/h. The melting

temperature (TM) for each protein fragment was taken as

M. Palaiomylitou/A. Tartas et al.

74 PROTEINS DOI 10.1002/prot

the temperature at which the fraction folded equals 0.5.

The protein concentrations used for the CD experiments

were 15–30 lM for Sc3T, 34 lM for Tn108, and 2.5 lMfor the Sc3T-Tn108 complex.

Sequence analysis for intrinsic disorder

Analysis of the Ssn6 amino acid sequence for intrinsic

disorder was performed using various disorder prediction

methods among which, the IUPred32 (http://iupred.

enzim.hu), RONN33 (http://www.strubi.ox.ac.ukRONN),

and PONDR1 VL-XT34,35 (http://www.pondr.com) pre-

dictors. The above predictors were chosen among avail-

able disorder predictors (reviewed in Ref. 36) because

they differ both methodologically and conceptually.

IUPRED is expected to recognize novel types of disorder

as it is not trained on disordered proteins. RONN can be

used to identify short regions of disorder with an

improved accuracy,33 whereas the VL-XT predictor of

PONDR1 may be used to predict short segments of

disorder that become ordered upon specific binding to a

biologically meaningful partner, indicating potential

binding sites.37,38

MD simulations

MD simulations were performed using MOE (The

Molecular Operating Environment) Version 2005.06 and

its built-in MD module using an NVT ensemble (N:

number of atoms, V: volume, T: temperature). A 3D-

model of the TPR1 to TPR3 region of Sc3T produced as

described previously9 was used as the initial structure.

The model was first optimized by energy minimization

using the MMFF94x molecular mechanics forcefield of

MOE. The protein was then solvated in a box of explicit

water molecules extending 8–12 A from the protein

atoms. Periodic boundaries were used in order to mini-

mize edge effects. The water structure was first equili-

brated for 100 ps keeping the whole protein fixed fol-

lowed by a 100 ps equilibration with fixed the protein

backbone. The equilibration phase was followed by a

2 ns MD simulation at 300 K with a time step for

integration of the potential function of 2 fs. A nonbonded

cutoff of at least 8 A was used.

Analysis of the MD simulation

The evaluation of the MD trajectory was done by ana-

lyzing the conformational changes over simulation time.

Coordinate files were extracted from the MD trajectory

every 0.5 ps for this purpose. The conformational analy-

sis focused on monitoring changes of the a-helical struc-ture during the simulation. Residues were assigned an a-helical conformation according to Kabsch and Sander.39

The percent simulation time during which a particular

residue followed the a-helical conformation was subse-

quently calculated. Moreover, a Ramachandran plot was

produced for all the MD structures in order to assess the

dynamics of the backbone conformational angles (u,w)during the simulation.

RESULTS

In isolation the Tup1-interaction domain ofSsn6 is partially unfolded

The minimal Tup1-interaction domain of Ssn615

(Sc3T; aa:1–175), including its three N-terminal TPR

repeats [Fig. 1(A)], was first expressed in recombinant

form and purified to homogeneity [Fig. 2(A)] in order to

study its structure in isolation. Sc3T eluted from the size

exclusion chromatography column at a volume equivalent

to a 23.2 kDa protein [Fig. 2(B)], which is close to its

deduced monomeric molecular mass (21.7 kDa). The sec-

ondary structure of this protein fragment was subse-

quently studied by CD. The far-UV CD spectrum of Sc3T

(Fig. 3, black line) revealed a predominately a-helicalstructure as indicated by the characteristic minima at 208

and 222 nm. However, deconvolution of the CD data gave

an estimate for the a-helical content of 34%, which is sig-

nificantly lower than the estimated one (�51%) based on

the expected TPR structure of this domain9 (see also

Fig. 1) possibly reflecting a partial unfolding of this Ssn6

fragment. Consistent with this possibility, the melting of

the a-helical structure of Sc3T extended from 20 to 908Cwith midpoint of the thermal denaturation (TM) of 588C(Fig. 3, Inset) as opposed to cooperative unfolding transi-

tions with high TM (838C) observed for ideal three-TPR

containing domains.8 This observation in conjunction

with the similarity of the thermal unfolding of Sc3T with

that of isolated a-helices in water,40 which also extends

Figure 2Purification and characterization of recombinant Sc3T. A: SDS-PAGE of purified

Sc3T stained with Coomassie Blue. Protein size markers are annotated. B:

Elution profile of Sc3T from the Sephacryl S200 gel-filtration column relative to

the protein standards: bovine serum albumin (BSA), ovalbumin (OVA),

carbonic anhydrase (CAH), and cytochrom c (CYT). Inset: Determination of the

Stokes radius (RST) using the same protein standards (BSA: RST 5 35.6 A,

OVA: RST 5 29.8 A, CAH: RST 5 23.5 A, CYT: RST 5 17.7 A). Sc3T is

indicated by an arrow.

Structural Stability of a Domain of Ssn6

DOI 10.1002/prot PROTEINS 75

from 20 to 1008C with TM of �588C, suggested that even

the formed a-helices are relatively weakly stabilized by ter-

tiary interactions in Sc3T.

To further investigate the partial unfolding hypothesis,

we performed far-UV CD experiments in the presence of

trifluoroethanol (TFE). TFE, at moderate concentrations,

induces a-helical structure in peptides and proteins with

propensity for a-helix formation.41 As shown in Figure

3, addition of TFE raised the a-helical content of Sc3T as

indicated by the deeper minima at 208 and 222 nm. At

TFE concentrations up to 20%, the increase of the ellip-

ticity minima was accompanied by a red shift of the par-

allel band at 208 nm and an inversion of the [y]222/[y]208ratio indicative of associating a-helices in a coiled-coil

manner.42,43 Notably, by using TFE concentrations

higher than 30%, the ellipticity minima were blue shifted

and the [y]222/[y]208 ratio changed back to less than 1.0

(Fig. 3), suggesting disruption of hydrophobic interac-

tions between individual helices, as previously reported

for other a-helical protein fragments at high TFE con-

centrations.44 These observations clearly indicated that

Sc3T, although competent to fold in to an a-helicalcoiled structure as expected for a canonical TPR domain,

is only partially folded.

The unfolded region of Ssn6 involves TPR1

The evidence for lack of proper structure prompted us

to investigate the conformational state of Sc3T by estimat-

ing its hydrodynamic radius (the Stokes radius) using gel-

filtration chromatography. By comparing the mobility of

Sc3T to those of a series of standard proteins on a

Sephacryl S-200 column the Stokes radius (RST) of Sc3T

was estimated to be 23.1 A [Fig. 2(B), Inset]. This value is

more close to the RST value expected for the native state

of Sc3T (22.16 A) as calculated using Uversky’s equa-

tions45 for a 21.7 kDa polypeptide. This finding indicated

that the partial unfolding of Sc3T is relatively confined.

To map the unstructured region of Ssn6 we used lim-

ited proteolysis, an approach widely used for mapping

lack of structure in proteins.46 The limited proteolysis

mapping was performed on a Ssn6 protein fragment

(Sc10T; aa: 1–415) containing the entire TPR domain

(TPR1 to TPR10) followed by 15 additional amino acids

corresponding to the ‘‘solvating helix’’ considered to be

essential for the solubility or stability of isolated TPR

domains.8,47 As shown in Figure 4, chymotrypsin

digested Sc10T rapidly, resulting in mainly two distinct

proteolytically resistant fragments. N-terminal amino

acid sequence analysis of the larger chymotryptic product

(fragment 1 in Fig. 4, apparent molecular weight of 45

kDa) revealed a mixture of two fragments starting by the

N-terminal sequences ‘‘DATLQFN’’ and ‘‘AYDATLQ’’

with 65 and 35% abundance, respectively. These chymo-

tryptic products were apparently generated upon cleavage

at the bulky hydrophobic residues Tyr69 (TPR1 position

24) and Met67 (TPR1 position 23), respectively [Fig.

1(B)], indicating that the anticipated TPR1 helix B is

Figure 3CD spectra of Sc3T. Far-UV CD spectra of 13.8 lM protein recorded at 208C in

50 mM phosphate buffer (pH 7.5) and in the absence (thin line) and presence

of 10% (-n-), 20% (-l-), 30% (-h-), and 50% (-*-) trifluoroethanol (TFE).

Inset: Thermal denaturation curve of Sc3T (at 30 lM protein concentration) as

monitored by CD spectroscopy at 222 nm. Thermal denaturation extends in a

broad temperature range (20–908C) indicative of noncooperative unfolding.

Figure 4Limited proteolysis mapping of Sc10T. Chymotrypsin digestion time course of

Sc10T (right lanes). For comparison, control Sc10T (left lanes) was also loaded

for the times indicated. Protein size markers are annotated. Labeled arrows

indicate the two proteolytically resistant fragments.

M. Palaiomylitou/A. Tartas et al.

76 PROTEINS DOI 10.1002/prot

unfolded exposing hydrophobic residues to the solvent.

The second chymotryptic product (fragment 2 in Fig. 4)

lacked the Sc3T fragment and therefore we did not pur-

sue it further, as it is not related to the Tup1 interaction

domain of Ssn6. The fact that potential chymotrypsin cut

sites existing within TPR2 and TPR3 sequences [Fig.

1(B)] were not actually recognized even in prolonged

incubation time points indicated that the unfolded region

of Sc3T involves only TPR1. This result along with the

estimated native-like state of Sc3T suggested that lack of

proper folding of the TPR region of Sc3T may be re-

stricted to TPR1.

MD simulations show that TPR1 helix B ishighly dynamic

To further investigate the structural flexibility of Sc3T

we performed a 2 ns explicit solvent MD simulation on

the TPR domain of Sc3T at 300 K (native state). The

model of the TPR1-3 region of Sc3T corresponding to

the anticipated Tup1 bound form9 was used as the initial

structure. To measure the conformational transitions in

detail we followed the a-helical structure variation

during the simulation. Initial a-helices were assigned

according to the canonical TPR structure (Fig. 1). The

Ramachandran plot produced for the TPR helices in all

the MD structures illustrated that the TPR1 helix B and

to a lesser extend helix A was highly dynamic in terms of

coverage of the (u,w) space, while the remaining TPR

helices retained their structure during the simulation

with Ramachandran angles varying slightly around their

equilibrium positions [Fig. 5(A)]. Moreover, monitoring

of the a-helical structure showed that the TPR1 helix B

was disrupted for most of the simulation time with the

helix break involving residues of the hydrophobic patch

[Fig. 5(B)] in accord with the chymotrypsin digestion

results. Taken together, these observations showed that

the disorder may indeed be localized to TPR1, which

adopts a highly dynamic transient structure, resulting in

exposing hydrophobic residues to the solvent.

Sequence analysis for intrinsic disorder

To investigate whether the disorder of TPR1 is encoded

by its amino acid sequence we used various protein dis-

order predictors that calculate disorder tendencies

directly from the primary sequence (reviewed in Ref. 36).

In their majority these methods failed to predict the

observed disorder of TPR1 (data not shown). The calcu-

lated mean hydrophobicity of TPR1 was 0.49, which is

very close to the value of 0.48 � 0.03 derived by statisti-

cal analysis of folded proteins.48 These observations indi-

cated that order rather than disorder is encoded by the

amino acid sequence of TPR1, as expected for canonical

TPRs. However, closer inspection of the IUPRED and

RONN predictions revealed that TPR1 exhibited an ele-

vated disorder tendency compared to the remaining

TPRs [Fig. 6(A)]. RONN prediction in particular showed

a significantly higher disorder tendency below, yet close

to the disorder threshold for the region corresponding to

the hydrophobic patch of TPR1 helix B [Fig. 6(A)]. Such

RONN patterns often indicate true flexible regions.33

The PONDR1 VL-XT prediction [Fig. 6(B)] showed a

deep minimum of the disorder score (region marked by

grey bar) flanked by one strong and a relatively high dis-

order prediction. This particular disorder pattern is remi-

niscent of the so-called binding region prediction signa-

ture37 and especially of the a-MoRE pattern.38 MoREs

(Molecular Recognition Elements) define interaction-

Figure 5Analysis of the MD simulation. A: Ramachandran plot of all the 2 ns structures

for the regions corresponding to TPR helices. The color scale to the right is the

simulation time. The figure illustrates that TPR1 helix B, and to a lesser extent

helix A, is more dynamic in terms of coverage of the (u,w) space, while theremaining TPR helices retain their structure during the simulation. B: Percent of

a-helical conformations during the simulation plotted along the sequence. The

TPRs 1 to 3 are shown as grey bars on the top of the Figure. Gly62 (TPR

position 17) is indicated by an up triangle. Filled diamonds indicate

hydrophobic residues corresponding to the susceptible to chymotrypsin digestion

hydrophobic patch. TPR1 helix B is disrupted for most of the simulation time

and the helix break coincides with the observed chymotrypsin digestion site.

Structural Stability of a Domain of Ssn6

DOI 10.1002/prot PROTEINS 77

prone short segments of disorder that become ordered

upon specific binding to their biological partner.49 a-MoREs in particular define those MoREs that form a-helical structures upon binding. Interestingly, the chymo-

trypsin sensitive region (see above) overlaps with the pre-

dicted a-MoRE, suggesting that TPR1 may indeed be

part of a molecular recognition element that undergoes a

disorder-to-helix transition upon binding to a protein

partner. Given the requirement of the structural integrity

of TPR1.5 for the Ssn6-Tup1 interaction,9 we reasoned

that such a conformational transition might be possibly

induced by Tup1, the biological partner of Ssn6.

Tup1 interaction with Ssn6 is accompaniedby a conformational change

To test whether conformational changes occur upon

the interaction of Ssn6 with Tup1 we produced an Ssn6-

Tup1 protein complex in vitro [Fig. 7(A), see legend for

details] and studied its secondary structure by CD [Fig.

7(B)]. The complex was formed by Sc3T and a Tup1

derivative (Tn108; aa:1–108) comprising the Ssn6 inter-

action domain. The far UV CD spectrum of the Sc3T-

Tn108 complex [Fig. 7(B), filled squares] exhibited a

maximum at 195 nm and deep minima at 209 and 222

nm indicative of a highly a-helical structure.Towards the calculation of the expected CD spectrum

of the Sc3T-Tn108 complex, the individual far-UV CD

spectrum of Tn108 was also recorded [Fig. 7(B), open

circles]. Deconvolution of this spectrum gave a high a-helical content (64%) for Tn108. This observation com-

Figure 6Order/disorder predictions on various fragments of Ssn6 sequence comprising the

Tup1 interaction domain. A: Disorder tendencies predicted by the IUPred and

RONN disorder predictors. The threshold 0.5 distinguishing ordered from

disordered regions is marked by a dotted line. B: PONDR1 VL-XT predictions.

A predicted a-MoRE (see text) is indicated by a grey bar. The black bar

indicates the sensitive to chymotrypsin digestion hydrophobic patch.

Figure 7In vitro production and characterization of the Sc3T-Tn108 complex. A:

Production of the complex. Lane 1: Glutathione Sepharose (GH-S) beads with

bound GST-Sc3T. Lane 2: purified Tn108. Lanes 3 and 4: GH-S beads with

prebound GST-ScB and flow-through, respectively, obtained by centrifugation

after interaction of the beads with Tn108 followed by extensive washing. Tn108

is present on the beads indicating that formation of the GST-Sc3T-Tn108

complex has been achieved. The presence of unbound Tn108 in the flow-through

indicates that the saturation of Sc3T is complete. Lane 5: Supernatant obtained

by centrifugation of the beads with bound GST-Sc3T-Tn108 following thrombin

digestion. Only the bands corresponding to Sc3T and Tn108 are present in the

supernatant, indicating a high purity of the resulting Sc3T-Tn108 complex.

Lanes 6 and 7: Flow-through and GH-S beads following interaction of Tn108

with beads with prebound GST. Tn108 is fully recovered on the flow-through

indicating that the protein does not interact with control beads or the GST. B:

CD spectra. Far-UV CD spectrum of the Sc3T-Tn108 complex recorded at 208Cin phosphate buffer (pH 7.5). The calculated spectrum of the complex (see

Methods) as well as the individual Sc3T (as in Fig. 3 without TFE) and Tn108

(in 50 mM phosphate buffer, pH 7.5 and 150 mM NaCl) far-UV CD spectra

used for its calculation are also shown for comparison. The difference between

the observed and expected CD spectra of the complex indicates that a

conformational change accompanies the Ssn6-Tup1 interaction.

M. Palaiomylitou/A. Tartas et al.

78 PROTEINS DOI 10.1002/prot

bined with the [y]222/[y]208 > 1 ratio [Fig. 7(B), open

circles] indicated that Tn108 adopts a predominant a-helical coiled-coil structure, consistent with previous

studies on a similar Tup1 fragment.50 This observation

provided additional evidence that in contrast to Sc3T, the

Tup1 fragment is completely folded in isolation.

Comparison of the far-UV CD spectrum of the Sc3T-

Tn108 complex with the spectrum produced by a summa-

tion of the individual Sc3T and Tn108 CD spectra revealed

major changes: deepening of the ellipticity minima of the

observed spectrum relatively to the expected accompanied

by a red shift of the parallel band (at 208 nm) and inversion

of the [y]222/[y]208 ratio [Fig. 7(B)]. This change of the CD

spectrum pattern reminiscent of the effects produced by

TFE (at moderate concentrations) provided strong evidence

that a conformational change involving acquisition of a-helical structure with simultaneous stabilization of an a-helical coiled-coil configuration occurs upon the Ssn6-

Tup1 interaction. The above result supports the idea of an

induced fit mechanism in agreement with both the TFE

experiments and the PONDR VL-XT predictions.

DISCUSSION

Ssn6 TPR1 structure flexibility, in isolation

In a previous study9 we suggested that the Ssn6-Tup1

interaction may have a hydrophobic character and in this

respect it is rather unique among known TPR-mediated

interactions. In the present study we initiated a structural

analysis of the Tup1 interaction domain of Ssn6 by using CD

spectroscopy. First, we found that, in isolation this domain

of Ssn6 is only partially folded, adopting a loose flexible

structure. Subsequent limited proteolysis experiments using

the entire TPR domain of Ssn6 showed that the unstructured

region involves TPR1. This observation is consistent with

hierarchical unfolding with faster exchange in the terminal

repeats than in the central ones observed in TPR51 and

other repeat (ankyrin) containing proteins.52,53 However,

the importance of the structural integrity of TPR1 for Tup1

binding9,10 raised the possibility that proper TPR1 structure

might be induced only upon binding to Tup1. Consistent

with this, there is now increasing experimental evidence for

structural flexibility of repeat domains in the absence of their

ligand.52,54,55 Cliff et al.55 provided evidence for a confor-

mational change upon ligand binding for the TPR domain

of the Pp5 protein and proposed that folding coupled to

binding might be a common mechanism of ligand recogni-

tion by TPR domains although this idea has been debated.56

The disorder of TPR1 may be necessary forthe recognition of Ssn6 by intrinsichydrophobic coiled-coil residues of Tup1

Intrinsic disorder has been now recognized to many

protein domains and has been proposed to be essential

for biological function57–59 and especially for molecular

recognition.60 A signature of a probable intrinsically dis-

ordered region is the presence of low sequence complex-

ity coupled with an amino acid composition bias, charac-

terized by a low content of bulky hydrophobic amino

acids.34,61 However, our chymotrypsin digestion experi-

ments in conjunction with MD simulations showed that

the disordered domain of TPR1 includes a region rich in

hydrophobic residues corresponding to its anticipated he-

lix B. PONDR VL-XT disorder/order predictions identi-

fied this region as being part of a potential molecular

recognition element that may undergo disorder-to-order

transition upon binding to a specific biological partner.

The apolar character of this region supports our previous

suggestion that the Ssn6-Tup1 binding may be mediated

by hydrophobic interactions.9 In further support of

this idea, hydrophobic cluster analysis62 on the Tup1

sequence revealed that Leu62, mutation of which (L62R)

disrupts the interaction with Ssn6,63 is part of a heptad

repeat occupying intrinsic hydrophobic position d (Sup-

plementary Fig. S1). Notably, the L62R mutation does

not affect the coiled-coil structure of Tup1 or its ability

to form tetramers50,63 implying an important role of

hydrophobic heptad positions of Tup1 around Leu62 in

Ssn6 interaction rather than in self association.

The observation that Leu62 occupies an intrinsic

hydrophobic coiled-coil position combined with the non-

native structure of TPR1 raised the possibility that the

structural flexibility of this Ssn6 region may be necessary

for exposing an apolar surface to the solvent in order to

be recognized by Tup1 hydrophobic coiled-coil residues.

Such recognition mechanism has been revealed for the

first time in the case of a coiled-coil chaperone and has

been proposed to extend to other coiled-coil proteins

that interact with molecules exposing an apolar sur-

face.64 Consistent with this postulation, the aminoacid

sequence of the TPR1 helix B does not conform to the

consensus at various positions (black shaded in Fig. 1).

Namely, position 17 although occupied by bulky hydro-

phobic residues in canonical TPRs,8 is occupied by a Gly

in TPR1 [Fig. 1(B)] probably for facilitating the struc-

tural flexibility of this region and the exposure of hydro-

phobic residues to the solvent. Moreover, the enrichment

of the TPR1 helix B in nonconsensus alanine residues

[Fig. 1(B)] results in a relatively low complexity of this

region probably adding to its ability to exhibit the pre-

sumably necessary structural plasticity. Finally, the struc-

tural flexibility of TPR1 may be facilitated further by the

flexible, low complexity (poly-Gln) N-terminus (Fig. 1).

Possible involvement of the convex TPRsurface of Ssn6 in Tup1 binding

The present study suggested a direct role of TPR1 he-

lix B in Tup1 binding. In further support to this postu-

lation, two disruptive mutations of Tup1 interaction

Structural Stability of a Domain of Ssn6

DOI 10.1002/prot PROTEINS 79

concern Ala68,9 which is located at TPR1 helix B [indi-

cated with asterisks in Fig. 1(B)]. This site corresponds

to an external TPR position (position 23)8 and therefore

a mutation at this position is anticipated to affect Tup1

binding directly than the structural integrity of Ssn6.

Involvement of the TPR1 helix B in turn suggests for

the first time involvement of the convex TPR surface

(Fig. 8) in binding of this particular partner of Ssn6

consistent with the notion that interaction through the

concave surface is not a common feature of TPR-medi-

ated interactions.18 Dedication of the convex surface to

Tup1 binding may leave the concave TPR surface avail-

able for other partners of Ssn6 that interact with this

region, as is for example a2.65

Coupled folding and binding

More importantly and consistent with the idea of

coupled folding and binding, we provided evidence that

the formation of the Ssn6-Tup1 (Sc3T-Tn108) complex is

accompanied by a conformational change involving ac-

quisition of a-helical structure and stabilization of a

more compact a-helical coil structure. The increased hel-

icity detected upon complex formation combined with

the observation that similar Tup1 fragments comprising

its Ssn6 interaction domain are fully folded in isolation

(Ref. 50 and this study) suggested that the a-helix forma-

tion in the complex occurs largely within Ssn6. This ob-

servation strongly supports the idea of coupled folding

and binding in Ssn6 suggesting that such a mechanism may

indeed be more common in TPR domains as proposed by

Cliff et al.55 Moreover, coupled folding and binding is now

becoming recognized as a relatively common feature in

diverse regulatory proteins.58,60 Disorder-to-helix transi-

tions, in particular, have been predicted to be quite frequent

in various protein interaction networks.66

CONCLUSIONS

The structural flexibility of the TPR domain of Ssn6

we observed in this study may be crucial for its function.

We propose that TPR1 needs to display some flexibility

in isolation in order to be recognized by Tup1 among

the wide range of biological partners available to Ssn6.

This structural versatility may provide the means for an

easy turnover of the repression mechanism by the Ssn6-

Tup1 complex.

Moreover, the induced fit mechanism we observed

provides additional support of the idea of coupled fold-

ing and binding being a more common feature of TPR

mediated interactions.

ACKNOWLEDGMENTS

M.P and A.T acknowledge support from NCSR

‘‘Demokritos’’ Fellowship Programs. The use of the facili-

ties of the Centre for Crystallographic Studies of Macro-

molecules of NCSR ‘‘Demokritos’’ is acknowledged as

well as N. Gounalaki for plasmid construction. PONDR

was provided by Molecular Kinetics, 6201 La Pas Trail,

Suite 160, Indianapolis, IN 46268 (main@molecularkine-

tics.com). The MOE software was available from Chemi-

cal Computing Group Inc., 1010 Sherbrooke Street

West, Suite 910, Montreal, Canada H3A 2R7, http://www.

chemcomp.com.

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