The role of the turn in b-hairpin formation during WWdomain folding

TIM SHARPE,1,3 AMANDA L. JONSSON,2,3 TREVOR J. RUTHERFORD,1

VALERIE DAGGETT,2 AND ALAN R. FERSHT1

1MRC Centre for Protein Engineering and Cambridge University Chemical Laboratory, MRC Centre, CambridgeCB2 0QH, United Kingdom2Biomolecular Structure and Design Program, University of Washington, Seattle, Washington 98195-7610, USA

(RECEIVED May 17, 2007; FINAL REVISION July 16, 2007; ACCEPTED July 17, 2007)

Abstract

The folding of WW domains is rate limited by formation of a b-hairpin comprising residues fromstrands 1 and 2. Residues in the turn of this hairpin have reported F-values for folding close to 1 andhave been proposed to nucleate folding. High F-values do not necessarily imply that the energetics offormation are a driving force for initiating folding. We demonstrate by NMR studies and moleculardynamics simulations that the first turn of the hYAP, FBP28, and PIN1 WW domains is structurallydynamic and solvent exposed in the native and folding transition states. It is, therefore, unlikely that theformation of the b-turn per se provides the energetic driving force for hairpin folding. It is more likelythat the turn acts as an easily formed hinge that facilitates the formation of the hairpin; it is a nucleusas defined by the nucleationcondensation mechanism whereby a diffuse nucleus is stabilized byassociated interactions.

Keywords: protein structure/folding; NMR spectroscopy; computational analysis of protein structure

The transition states for folding of several b-sheetproteins have been characterized by F-value analysis(Grantcharova et al. 1998; Martinez and Serrano 1999;Cota et al. 2001; Fowler and Clarke 2001; Garcia-Miraet al. 2004). Recently, attention has focused on small,fast-folding b-sheet peptides that are amenable to simu-lation, in particular the WW domains. WW domains aresmall three-stranded b-sheets with a high aromatic con-tent including conserved tryptophan residues (Maciaset al. 2000). WW domains have some structural idiosyn-crasies: They lack a conventional solvent-inaccessiblehydrophobic core, they have abundant, largely solvent-

exposed aromatic residues, and they have relatively shortb-strands. Accordingly, WW domains may serve as amodel for b-hairpin formation (Petrovich et al. 2006).

The folding of several natural WW domain homologshas been studied (Crane et al. 2000; Ferguson et al. 2001a;Jager et al. 2001; Nguyen et al. 2003; Petrovich et al. 2006)as well as a rationally designed WW domain prototype(Macias et al. 2000; Ferguson et al. 2001a). F-values aremetrics for scoring experimentally the degree of formationof noncovalent interactions relative to the denatured andnative states on a scale of 01 (Matouschek et al. 1989;Fersht and Sato 2004). There are high F-values formutation of side chains in the first turn of the PIN1 WWdomain (Jager et al. 2001) and the FBP28 WW domain(Petrovich et al. 2006). Synthetic peptides with disruptedbackbone hydrogen bonding from substitution of specificamide bonds by flexible thio-ether bonds in hYAP (Fergusonet al. 2001b) or ester bonds in PIN1 (Deechongkit et al.2004) have similar patterns of F-values.

For PIN1, the high F-values in turn 1 were inter-preted as evidence of folding via a hydrophobic zipper

3These authors contributed equally to this work.Reprint requests to: Valerie Daggett, Biomolecular Structure and

Design Program, University of Washington, Seattle, WA 98195-7610,USA; e-mail: daggett@u.washington.edu; or Alan R. Fersht, e-mail:MRC Centre for Protein Engineering and Cambridge UniversityChemical Laboratory, MRC Centre, Hills Road, Cambridge CB20QH, UK; e-mail: arf25@cam.ac.uk; fax: 44-1223-336445.

Article published online ahead of print. Article and publication dateare at http://www.proteinscience.org/cgi/doi/10.1110/ps.073004907.

Protein Science (2007), 16:22332239. Published by Cold Spring Harbor Laboratory Press. Copyright 2007 The Protein Society 2233

mechanism (Jager et al. 2001; Nguyen et al. 2003),propagating away from the structured turn, analogous toa single-sequence nucleation model of the GB1 hairpin(Munoz et al. 1998). All-atom molecular dynamics (MD)unfolding simulations, used in conjunction with F-valueanalysis to study the folding pathway of the FBP28 WWdomain, show that the first turn is present as a kink in thebackbone of the transition state, but the precise structureof the turn and its associated hydrogen bonds are notformed (Petrovich et al. 2006). Instead, the high F-valuesresult from side-chain interactions in the absence ofprecise native backbone structure. Here, we show thatthe first turn of WW domains is dynamic even in thenative state and discuss what this implies in terms ofmechanism of folding.

Results and Discussion

Mobility in the first turn

15N-1H backbone relaxation data were acquired forFBP28 and hYAPtm at 285 K and 288 K, respectively.We have also determined the structure of shortenedhYAPtm (A20R, L30Y, D34T) (Jiang et al. 2001; T.J.Rutherford and R. Canales, unpubl.) and there is goodagreement with the previous structures of hYAP (Pireset al. 2001). S2 order parameter values derived frommodel-free analysis had a very similar distribution inhYAPtm and FBP28 (Fig. 1). The first turn and the firstresidue of the second b-strand of both the hYAP andFBP28 WW domains have lower S2 values (0.680.74 forFBP28, 0.730.77 for hYAPtm) compared with the secondb-strand and the second turn, which form an islandof higher S2 values in the center of the b-sheet (;0.81for FBP28,;0.84 for hYAPtm). The lower S2 values reflectthe greater mobility of the NH bond vectors of the firstturn on the picosecondnanosecond timescale, comparedwith the second strand and second turn. We also observedreduced S2 values in the region of T37 (YAP)/S28 (FBP28)in the third b-strand. This region, and T22 (YAP)/T13(FBP28) in the first turn, make contacts with phosphopep-tide ligands in various WW domain ligand complexes(Macias et al. 2002). These residues have been identifiedas potential sites for regulatory phosphorylation of someWW domains (Shaw et al. 2005). It has been suggested thatregions with greater than average motion may triggerconformational changes necessary for ligand binding(Clore and Schwieters 2006).

Crystal structure B-factors (shown in Fig. 1), qualita-tive analysis of NMR structural restraints (Kowalski et al.2002), and NMR backbone dynamics studies (Jacobset al. 2003; Peng et al. 2007) show that the first loop inunbound PIN1 is dynamic on nanosecondpicosecondand millisecondmicrosecond timescales. Significant

changes in the conformation of turn 1 can be seen incrystal structures of PIN1 in ligand-bound (1F8A) andligand-free (1PIN) forms (Ranganathan et al. 1997;Verdecia et al. 2000). A recent comprehensive study ofPIN1 WW domain backbone dynamics (Peng et al. 2007)showed that the first loop rigidified upon binding tothe Cdc25 ligand (S2 values were increased by ;0.1for R17, S18, and G20 and by 0.7 for S19). Truncation ofthe loop (by deletion of S19) reduced both loop mobilityand binding affinity. Further recent loop modificationexperiments showed that shortening the loop, replacing itwith the loop from FBP28, and repositioning or deletingR17 abrogates ligand binding (Jager et al. 2006). Itappears that mobility in the first loop, observed forFBP28 and hYAPtm by NMR backbone dynamics andfor PIN1 by a variety of techniques, is a conserved featureof WW domains and that it has important effects uponligand binding.

S2 values calculated from low and room temperature(285 K and 298 K) MD simulations of the native state forFBP28, hYAPtm, and PIN1 show that the first turn ishighly mobile. Although the S2 values in the first turn arelower than those calculated from NMR measurements, thetrends are in good agreement with the NMR data fromFBP28 and hYAP and the crystallographic and NMR datafor PIN1 (Ranganathan et al. 1997; Peng et al. 2007). Thesimulations also display a dip in S2 values in the thirdb-strand, as observed by NMR for FBP28 and hYAPtm.

The flexibility of the backbone of the first turn was alsoreflected in the increased fluctuations of the a-carbonatoms about the average structure in the first turn com-pared with the rest of the protein (Fig. 2). In addition,when (f,c) space is divided into 5 3 5 bins, the resi-dues of the first turn of FBP28, hYAPtm, and PIN1 popu-lated 72%, 60%, and 39% of all bins, respectively (Table1). In each case, this represents the highest percentage of(f,c) space sampled for the structured region of eachprotein (i.e., excluding terminal tails if present).

Hydrogen bonding in the first turn

Two backbone hydrogen bonds can form in turn 1 ofPIN1, FBP28, and hYAPtm. One of these hydrogen bondsinvolves the NH of the last residue in strand 1 (residue 13in FBP28 and residue 22 in hYAPtm, for example) and thei + 4 carbonyl group that corresponds to the first residuein strand 2 (residue 17 in FBP28 and residue 26 in hYAPtm).The second hydrogen bond is between the carbonylgroup of the last residue in strand 1 with the i + 3 NHgroup (residue 16 in FBP28 and 25 in hYAPtm). Weobserved limited amide protection for T22HN of hYAPtmand no protection for G25HN by NMR hydrogen ex-change experiments (T.J. Rutherford, unpubl. data). Sim-ilar results were obtained for a stabilized circularized

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2234 Protein Science, vol. 16

FBP28 (T. Sharpe, unpubl. data). No protection fromexchange was observed in the turn for wild-type FBP28and YQJ8 (Macias et al. 2000). Ninety-one percent of thetime the i! i + 4 hydrogen bond was intact in hYAPtm inthe MD simulations, while the value for the i ! i + 3hydrogen bond was 19%. The corresponding values were39% and 10% for FBP28. These data show that thehydrogen bonds in turn 1 are dynamic and undergo localexchange with solvent in the native state.

Interpretation of high F-valuesF-value analysis was designed to study the removal ofdefined interactions in a native structure (nondisruptivedeletions) and to infer the relative contributions of thoseinteractions in stabilizing transition and ground states(Fersht et al. 1987; Matouschek et al. 1989). A F-value of1 means that the energies of the native and transitionstates are perturbed equally by the mutation, and a F-value of 0 means that the transition and denatured states

Figure 1. (Top left and top middle panels) S2 values derived from NMR 1H-15N backbone relaxation data for FBP28 (285 K) and hYAPtm (288 K). (Top

right panel) Crystallographic B factors (temperature factors) were extracted from the structures of PIN1 in bound (1F8A, filled circles) and unbound (1PIN,

open circles) states. (Middle and bottom panels) S2 values for FBP28, hYAPtm, and PIN1 were calculated from the autocorrelation function of HN bond

vectors with a 250-ps or 2-ns window in two 5-ns molecular dynamics simulations at both 285 K and 298 K.

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are perturbed isoenergetically (Matouschek et al. 1989;Fersht et al. 1992; Fersht and Sato 2004). The most suit-able F-mutations, such as the paradigmatic mutation ofa buried hydrophobic residue to a smaller hydrophobicresidue and also Ala ! Gly scanning (Scott et al. 2007),delete specific interactions observed in the native state.For such mutations, one assumes that a residue with a F-value of 1 makes the same set of stabilizing interactionsin both the transition and native states, and also fractionalvalues of F are a good indication of the extent of bondformation. The equivalence of energetic and structuralperturbations results from the deletion of an equivalent setof interactions by mutation. But, F-analysis may also beapplied for extreme values of 0 and 1 for more drasticmutations and for mutations that do not change well-defined interactions (Matouschek et al. 1989; Fersht andSato 2004). In those examples, the F-values indicateenergetic equivalence with denatured and native states,respectively.

The mutations in the first turn of FBP28 are notgenerally the classical deletion of specific interactions(Petrovich et al. 2006). But, as they give F-values ofclose to 1, the turn is isoenergetic in the transition andnative states. Transition state structures identified fromunfolding simulations of FBP28 maintain a large fractionof contacts in the native state (average of 1.46 for residues1317) in the first turn while the native backboneconformation of the turn was not preserved. The averagefraction of contacts in the first turn correlates well with

experimental F-values in this region (Petrovich et al.2006). Thus, there are tertiary interactions in the absenceof fixed backbone structure in the transition state. Wehave shown by NMR and MD that the backbone structureis quite malleable in the native state, and MD shows thatit is also dynamic in the transition state.

Nucleation and the role of the first turn in folding

The nucleus in the nucleationcondensation mechanismis an unstable species that has to be stabilized by long-range and other interactions as the rest of the proteincondenses around it, and the nucleus is loosely definedas the region of the protein that is best formed in thetransition state (Itzhaki et al. 1995; Fersht 1997). The firstturn in the FBP28 WW domain is a nucleus for folding bythat definition, as it is nearly fully formed even though itis energetically weak and dynamic. The fractional F-values observed in PIN1 and FBP28 in the first andsecond strands are what are expected for a nucleationcondensation folding mechanism: The stabilizing inter-actions are in the process of being formed as the structurecondenses around the nucleus (Itzhaki et al. 1995). But, itdiffers from other nuclei in that it is solvent exposed andit is not stabilized by other residues condensing around it.Even though the turn is not rigid, it still responds tomutations that affect its energy: Any mutation thatdestabilizes the turn (entropically or enthalpically) willraise the free energy of formation of the transition statefor folding and vice versa.

The all-atom MD unfolding simulations of FBP28show that the backbone structure of the first turn isrelatively fluid in the transition state (Petrovich et al.2006). Consequently, mutations that allow the turn toexplore too great an area of Ramachandran space (forexample an X ! Gly mutation) or those that penalizeexploration of the necessary regions of Ramachandranspace (for example, a Gly ! Ala mutation or Gly dele-tion) (Jager et al. 2006), could frustrate protein foldingand destabilize the native state. Further insights can begained in silico. As the hairpin has a dynamic turn withrapid fluctuations in native state Ramachandran space,rather than use a model based on the attainment of native

Figure 2. (A) The structure of FBP28, hYAPtm, and PIN colored by

secondary structure: Strand 1 is red, strand 2 is green, and strand 3 is blue.

The thickness of the ribbon corresponds to the average root-mean-square

fluctuation of the a-carbon from the average structure (Ca-RMSF) from

the native state simulations at both 285 K and 298 K. This image was

generated using PyMOL. (B) Average Ca-RMSF of the native state

simulations at both 285 K and 298 K for FBP28, hYAPtm, and PIN1 in

pink, light blue, and orange, respectively. An offset was added to the

residue numbers of FBP28 and PIN1 so that the residue numbers in the

first turn overlapped with the residue numbers of hYAPtm.

Table 1. Percentage of 5 3 5 Ramachandran bins sampled inthe 285 K and 298 K native state simulations

FBP28 hYAPtm PIN

b1 22% 16% 19%

Turn 1 72% 60% 39%

b2 19% 12% 12%

Turn2 16% 15% 13%

b3 17% 16% 14%

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2236 Protein Science, vol. 16

(f,c) angles as the indicator of nativeness (Munoz et al.1997, 1998, 2006), we used unfolding simulations thatcan reproduce the dynamic behavior of the native statewith reasonable accuracy. For FBP28 such simulationsreveal that hydrogen bonds generally formed betweenstrands 1 and 2 before the backbone hydrogen bonds ofthe first turn, considering the unfolding simulation inreverse (Fig. 3; Petrovich et al. 2006). The increase inhydrophobic contacts between the residues of the firsttwo strands was preceded by, or concurrent with, thedecrease in the distance between the Ca atoms of residues13 and 17 that define turn 1 and the formation of thebackbone hydrogen bond between residues 13 and 17 ofturn 1 (Fig. 4). The detailed time course comparingacquisition of interstrand hydrophobic contacts and turnstructure indicates that folding proceeds through a looselystructured first turn and cross-strand hydrophobic con-tacts. However, this is where such labels become some-what arbitrarythe turn is critical to the process. Indeed,how can a b-hairpin form without a turn? The distinctionwe make is whether or not the precise structure of the turnis acquired before interactions across the sheet, such aswith a zipper. Instead, the simulations show a dynamic,loosely structured turn that serves both to bring the chainaround and to adapt as necessary for interstrand inter-actions to develop.

It is an intriguing possibility that the first turn of theWW domains may play an important role in misfoldingin addition to the roles in folding and binding outlinedabove and elsewhere. FBP28 forms amyloid fibers rapidlyunder physiological conditions (Ferguson et al. 2003).The amyloid fibers are formed from stacked nonnative b-hairpins. The turn between regions of b-space in thehairpin comprises the same residues that form the firstturn in the native state, but those residues have a non-native conformation in the fiber (Ferguson et al. 2006). Itmay be that the flexibility of the backbone in the firstturn, as observed in the native state, also facilitates theadoption of an alternative nonnative structure in theamyloid fiber. Thus, rather than being an optimized b-turn capable of driving folding, the first turn might bepassive in folding and well suited for the adoption of multiple conformations, including those involved in

ligand binding.

Materials and Methods

NMR backbone dynamics

15N-labeled FBP28 was expressed and purified as previouslydescribed (Ferguson et al. 2006). 15N-labeled hYAPtm wasprovided by Roberto Canales. All NMR data were acquired ona Bruker DRX500 using standard conditions for each protein:285 K, 20 mM sodium phosphate (pH 6.5), and 30 mM NaCl forFBP28 and 288 K, 50 mM sodium phosphate (pH 6), and 150mM NaCl for hYAPtm. All data were processed with nmrPipe

Figure 4. (A) A schematic of the first hairpin with side chains shown in

magenta, distance between the a-carbons that define the first turn

represented in green, and the hydrogen bond distance in cyan. (B) Number

of hydrophobic contacts between the residues of the first two b-strands

from a representative 373 K unfolding simulation of FBP28, with time

shown in reverse to represent the folding pathway. (C) Distance between

the a-carbons of residues 13 and 17 that define the first turn of WW

domain. (D) Hydrogen bond distance between residues 13 and 17 of the

first turn.

Figure 3. First 5 ns of a 373 K, low pH unfolding simulation of FBP28

(Petrovich et al. 2006) shown in reverse. Backbone hydrogen bonds are

shown as gray bars. Side chains of the two hydrophobic clusters are shown

and colored pink and light blue for cluster 1 and 2, respectively.

b-hairpin formation

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(Delaglio et al. 1995) and analyzed using Sparky (Goddard andKneller, 2004). Resonance assignments for FBP28 were madeusing an 1H-1H-15N TOCSY spectrum and the published assign-ments for FBP28 under identical conditions (Biomagresbank4714). For hYAPtm, the assignments were made using standardtriple resonance experiments.

NMR 1H-15N backbone relaxation data (R1, R2, and hetero-nuclear NOE) were acquired for FBP28 and hYAPtm usingstandard pulse sequences (Kay et al. 1992; Mandel et al. 1995).For R1 and R2 data, peak heights as a function of relaxationdelay time (161600 ms for R1 and 14350 ms for R2) werefitted to a single exponential function using Sparky. Hetero-nuclear NOE values (I/I0) were obtained by taking the ratio ofpeak heights in spectra obtained in the presence or absence ofpresaturation of amide proton resonances.

Model-free analysis

S2 values were calculated for FBP28 and hYAPtm using LipariSzabo model-free analysis (Lipari and Szabo 1982) performedin Tensor2 (Dosset et al. 2000). The errors for relaxation ratesand I/I0 (heteronuclear NOE) were conservatively estimated as5% of the average value for the structured region of the protein.The rotational correlation times of 4.1 ns for FBP28 and 4.4 nsfor hYAPtm were estimated using R2/R1 values from residueswith I/I0 > 0.55 and R2/R1 within 1.5 standard deviations of themean for the structured part of the molecule; the calculationassumed isotropic tumbling. Although FBP28 has been shownto form amyloid fibrils (Ferguson et al. 2003), these correlationtimes are inconsistent with oligomerization or self-aggregationand are consistent with data for a circularized FBP28 that doesnot form amyloid fibrils (T. Sharpe, unpubl. data). The datawere mostly fitted to the simplest models of internal motion(model 1 or 2), the S2 values reflecting trends clearly visible inthe raw R1 and I/I0 data. For FBP28 residues N22, L26, and E27had small chemical exchange contributions, with Rex between0.9 and 1.2 s1.

Molecular dynamics simulations

Two 20-ns simulations were performed at both 285 K and 298 Kin ilmm (Beck and Daggett 2004; Beck et al. 2006) using theLevitt et al. force field (Levitt et al. 1995; Armen et al. 2005)with a 10 A spherical cutoff for nonbonded terms. Eachsimulation included explicit F3C water molecules (Levitt et al.1997) at the appropriate density (0.9995 and 0.997 g/mL for285 K and 298 K, respectively) (Kell 1967). S2 values frommolecular dynamics simulations of FBP28, hYAPtm, and PIN1were calculated from the autocorrelation function of HN bondvectors (Wong and Daggett 1998) with a 250-ps or 5-ns windowat 285 K and 298 K.

Acknowledgments

We thank Dr. Neil Ferguson for suggesting these experimentsand for many helpful discussions and Dr. Mark Allen forassistance with NMR assignment.

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