Cell, Vol. 111, 565–576, November 15, 2002, Copyright 2002 by Cell Press

Structure of the N-WASP EVH1 Domain-WIPComplex: Insight into the Molecular Basisof Wiskott-Aldrich Syndrome

verrier et al., 2001). Similarly, a knockout mutation ofthe more ubiquitously expressed N-WASP gene resultsin embryonic lethality and defects in many but not allactin-based motility processes (Snapper et al., 2001).These proteins have also been hijacked by pathogens

Brian F. Volkman,1,4 Kenneth E. Prehoda,2,4,5

Jessica A. Scott,2,6 Francis C. Peterson,1

and Wendell A. Lim2,3

1Department of BiochemistryMedical College of Wisconsin

including shigella and vaccinia virus as required compo-Milwaukee, Wisconsin 53226nents for their own actin-based intracellular motility (Su-2 Department of Cellular and Molecularzuki et al., 1998; Moreau et al., 2000).Pharmacology

Recent studies have begun to uncover the molecularUniversity of California, San Franciscomechanism by which WASP/N-WASP regulates actinSan Francisco, California 94143polymerization. WASP and N-WASP have a highly mod-ular domain structure (Figure 1A). A �100 residueC-terminal “output” domain containing a verprolin-cofi-Summarylin-acidic motif (VCA) directly binds to and activates theactin-related protein (Arp)2/3 actin nucleating complexMissense mutants that cause the immune disorder(Machesky and Insall, 1998; Rohatgi et al., 1999),Wiskott-Aldrich Syndrome (WAS) map primarily to thewhereas the remaining regions of WASP/N-WASP,Enabled/VASP homology 1 (EVH1) domain of the actinwhich encompass multiple domains, are involved in tar-regulatory protein WASP. This domain has been impli-geting and regulating the activity of the output domaincated in both peptide and phospholipid binding. Wein response to upstream signaling inputs. An importantshow here that the N-WASP EVH1 domain does notgoal is to understand the function of the regulatory do-bind phosphatidyl inositol-(4,5)-bisphosphate, as pre-mains found in WASP/N-WASP in order to understand

viously reported, but does specifically bind a 25 resi-what signals regulate WASP and the mechanism of this

due motif from the WASP Interacting Protein (WIP).regulation. Several of the domains, including the basic

The NMR structure of the complex reveals a novelmotif (B), the GTPase binding domain (GBD), and the

recognition mechanism—the WIP ligand, which is farproline-rich region, have been found to participate di-

longer than canonical EVH1 ligands, wraps around the rectly or indirectly in autoinhibitory interactions that re-domain, contacting a narrow but extended surface. press or block the activity of the VCA output domainThis recognition mechanism provides a basis for un- (Miki et al., 1998; Kim et al., 2000; Prehoda et al., 2000).derstanding the effects of mutations that cause WAS. These autoinhibitory interactions are specifically re-

lieved by interaction with upstream activators includingIntroduction the phospholipid phosphatidyl inositol-(4,5)-bisphos-

phate (PIP2), the GTPase Cdc42, and Src homology 3Spatial and temporal regulation of the actin cytoskeleton (SH3) domain-containing proteins such as Nck (Higgsis essential for controlling cell shape and movement. and Pollard, 2000; Prehoda et al., 2000; Rohatgi et al.,The Wiskott-Aldrich Syndrome Protein (WASP) and its 2000, 2001; Carlier et al., 2000).homolog N-WASP are signal transduction proteins that One of the most important domains in WASP is the

Enabled/VASP Homology 1 domain (also known ashave emerged as central players that promote actinWASP Homology 1 or WH1 domain) located at the Npolymerization in response to upstream intracellular sig-terminus of the protein (Gertler et al., 1996; Symons etnals (Carlier et al., 1999; Snapper and Rosen, 1999;al., 1996; Callebaut et al., 1998; Renfranz and Beckerle,Higgs and Pollard, 2001; Millard and Machesky, 2001;2002). The functional importance of the WASP EVH1Pollard et al., 2000). Mutation of WASP, which is primar-domain is underscored by the fact that 28 of the 35ily expressed in hematopoetic cells, results in Wiskott-identified missense mutations that lead to the diseaseAldrich Syndrome (WAS), an X-linked recessive disorderWAS are concentrated within this domain (Derry et al.,characterized by immunodeficiency, eczema, and1995; El-Hakeh et al., 2002; Greer et al., 1996; Kolluri etthrombocytopenia (Derry et al., 1994; Kolluri et al., 1995;al., 1995; Zhu et al., 1997). In addition, this domain isVilla et al., 1995; Greer et al., 1996; Zhu et al., 1997).required for N-WASP-dependent intracellular motility ofThese symptoms are consistent with cytoskeletal de-vaccinia virus (Moreau et al., 2000).fects in hematopoetic cells (Zicha et al., 1998) and a

EVH1 domains are found in several other proteins,possible role of WASP/N-WASP in motility, thrombogen-including the cytoskeletal regulatory proteins Enabledesis, endocytosis, phagocytosis, and other actin-basedand VASP and the neuronal signaling protein Homerprocesses (Snapper et al., 1998; Snapper and Rosen,(Callebaut et al., 1998; Gertler et al., 1996). In these1999; Cannon et al., 2001; Coppolino et al., 2001; Le-cases, the EVH1 domain appears to serve as a modularprotein-peptide docking unit that can participate in pro-

3 Correspondence: wlim@itsa.ucsf.edu tein targeting. These previously characterized EVH1 do-4 These authors contributed equally to this work.

mains all utilize a conserved aromatic binding surface5 Present address: Institute of Molecular Biology, University of Ore-to recognize specific, 6–10 residue proline-rich peptidegon, Eugene, Oregon 97403.motifs (Prehoda et al., 1999; Fedorov et al., 1999; Carl6 Present address: University of Wisconsin Medical School, Madison,

Wisconsin 53706. et al., 1999; Ball et al., 2000; Beneken et al., 2000; Barzik

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Figure 1. The N-Terminal Region of N-WASP Is an EVH1 Domain that Binds WIP via an Extended Peptide Ligand

(A) Proposed domain structure of N-WASP, as well as putative ligands for each domain.(B) Example PIP2 vesicle binding assays. S indicates supernatant, P indicates pellet. Bound protein is retained in the pellet. The PLC� PHdomain and the N-WASP EVH1 fragment were expressed as fusions to glutathione-S-transferase (GST). Remaining proteins were expressedas His6-tag fusions.(C) Example far-Western protein binding assays. Probe proteins were biotinylated by expressing them as fusions to the Promega Pinpointvector (see Experimental Procedures). Each protein was tested for binding to a GST-fusion to WASP Interacting Protein (WIP) fragmentcontaining residues 457–490 and to the ActA sequence DFPPPTDEEL, which is a ligand for the Mena EVH1 domain.(D) Summary of PIP2 and WIP (457–490) binding using various N-WASP constructs. None of the constructs bind PIP2, but all those that preciselyencompass the predicted EVH1 region can bind the WIP fragment.(E) Deletion mapping reveals that a 25 residue polypeptide from WIP (residues 461–485) is the minimal fragment required for binding to theN-WASP EVH1 domain (residues 26–147) in a far-Western gel overlay binding assay.

et al., 2001). Thus, it has been postulated that the WASP/ proline-rich sequences that may be putative EVH1 do-main binding motifs (Martinez-Quiles et al., 2001; Mo-N-WASP EVH1 domain may also serve a targeting

function. reau et al., 2000; Naqvi et al., 1998; Ramesh et al., 1997;Savoy et al., 2000). It has not been determined, however,Despite its importance in the pathology of WAS, the

precise molecular function of the WASP EVH1 domain if these motifs are sufficient to mediate the interactionbetween WASP and WIP. Furthermore, homology mod-has remained unclear (Insall and Machesky, 1999). Sev-

eral functions have been ascribed to the domain, includ- eling based on known EVH1 domain structures fails toexplain the behavior of several of the most commoning binding to the acidic phospholipid, phosphatidyl ino-

sitol (4,5) bisphosphate (PIP2) (Imai et al., 1999; Miki et WAS-causing mutations—specifically, the most signifi-cant mutational hotspot associated with severe WASal., 1996) and binding to the WASP interacting protein

(WIP) (Ramesh et al., 1997), both of which are known is located at a surface that is predicted to lie directlyopposite the canonical peptide binding surface (Be-regulators of N-WASP-mediated actin polymerization.

EVH1 domains share the same overall fold with Pleck- neken et al., 2000; Prehoda et al., 1999). These inconsis-tent findings have several possible explanations: (1) thestrin Homology (PH) domains (Fedorov et al., 1999; Pre-

hoda et al., 1999; Ball et al., 2000; Barzik et al., 2001; N-WASP/WASP EVH1 domain may differ significantly instructure from other EVH1 domains; (2) these domainsBeneken et al., 2000), many of which specifically bind

phosphoinositides (Rebecchi and Scarlata, 1998). Thus, may have multiple binding sites—one that interacts withpeptides and another that interacts with other ligandsit has been suggested that EVH1 domains may be bi-

functional, interacting with both peptides and phospho- such as PIP2; or (3) these domains may use a mechanismof peptide interaction that is significantly different frominositides (Prehoda et al., 1999).

WIP is an actin binding protein that contains several that of other EVH1 domains.

Structure of the N-WASP EVH1-WIP Complex567

Figure 2. N-WASP EVH1 Domain Sequence

(A) Constructs of the N-WASP EVH1 domain fused to the minimal WIP peptide. Fusions are linked via a (Gly-Ser-Gly-Ser-Gly) linker. Only thetop fusion yielded highly soluble protein amenable to structural analysis.(B) Structure-based alignment of the Rat N-WASP EVH1 domain with its close homologs WASP (H. sapiens) and Bee1p (S. cerevisiae). Alsoshown are sequences of other more distantly related, but structurally characterized EVH1 domains. Secondary structure elements in theN-WASP structure are indicated above the sequence. Sites that show a direct NOE contact with the WIP peptide are marked by filled triangles.Sites in WASP where missense mutation cause the disease WAS are circled (lavender, strong/moderate phenotype; green, mild phenotype).A conserved charged residue array in the WASP family EVH1 domains, described in Figure 5, are indicated in red (negative) or blue (positive).

Here, we have sought to clarify what ligands the generated several fragments encompassing the mouseN-WASP EVH1 domain and tested them for phospholipidN-WASP EVH1 domain can specifically bind and how

WAS-causing mutants may disrupt these interactions. binding. Phospholipid vesicle binding assays show thatnone of the N-WASP fragments are capable of bindingWe find that the N-WASP EVH1 domain and other EVH1

domains cannot bind PIP2 with significant affinity, con- PIP2 with detectable affinity using this assay (Figure 1B).In contrast, in the same assay, strong PIP2 binding istrary to a previous report. However, we find that the

N-WASP EVH1 domain can specifically interact with a observed for two other known lipid binding modules:the Pleckstrin Homology (PH) domain from phospholi-25 residue peptide from WIP. We have determined the

structure of the EVH1-WIP peptide complex using NMR pase C-delta (PLC-�) and a fragment of N-WASP (resi-dues 178–274) that includes a highly basic region (Bspectroscopy, revealing that the domain adopts the

same overall fold as other EVH1 domains, but it utilizes domain) that has recently been shown to mediate PIP2

regulation in N-WASP (Prehoda et al., 2000; Rohatgi eta novel mechanism of peptide recognition. The WIPpeptide, which is more than twice as long as typical al., 2001). We also show that a canonical EVH1 domain,

from the protein Mena, is also not capable of bindingEVH1 ligands, wraps around the entire EVH1 domainlike a piece of string around a spool, contacting regions PIP2 in this assay. Thus, despite a high degree of struc-

tural homology with PH domains, EVH1 domains do noton the domain surface extending far beyond the canoni-cal EVH1 peptide binding site. The structure reveals a generally appear to share the function of high-affinity

phosphoinositide binding, at least at a level comparablenovel class of domain-mediated protein-protein interac-tions and suggests how WAS-causing mutations are to that observed for canonical PH domains (Kd �1 �M).likely to disrupt the WASP/WIP interaction.

The Minimal EVH1 Ligand Is a 25 ResiduePeptide from WIPResults and DiscussionIn contrast, in far-Western protein binding assays, wefound that N-WASP fragments encompassing the EVH1The EVH1 Domain of N-WASP Does Not Bind PIP2

It was previously reported that the N-terminal region of region were responsible for binding to a fragment fromWIP (residues 416–488) that had previously been shownN-WASP including the EVH1 domain was a PH or PH-

like domain capable of PIP2 binding (Miki et al., 1996). We to interact with N-WASP (Figure 1C). The fragment of

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N-WASP required for WIP binding correlates precisely Table 1. Structural Statistics for 20 WIP-EVH1 Structureswith the EVH1 domain boundaries (Figure 1D).

NOE Constraints NumberThe 72 residue WIP fragment contains many proline-rich putative EVH1 binding motifs, so we used deletion Long 803

Medium 278analysis to delineate the minimal recognition require-Short 455ments (Figure 1E). A 25 amino acid fragment of WIPIntraresidue 348(residues 461–485) is minimally required for recognition.Totala 1884

This motif is well conserved in certain isoforms of the WIP/EVH1 NOEsb 135recently identified WIP homolog CR16 (Ho et al., 2001)

Ramachandran Statisticscand the yeast homolog verprolin (Naqvi et al., 1998), andits presence appears to correlate with ability to bind Most favored 76.4%

Additionally allowed 20.7%N-WASP. The WIP motif encompasses a proline-richGenerously allowed 2.0%motif (DLPPPEP) similar to that recognized by the EVH1Disallowed 0.9%domain from Mena (DFPPPPT). In our assays, the WIPParameter Family Minimized Averagesequence, in fact, crossreacts with the Mena EVH1 do-

main, although the N-WASP EVH1 domain does not Target function (A2) 0.86 � 0.17 0.76crossreact with a 10 residue ligand for the Mena EVH1 Upper limit violationsdomain from the protein ActA (Niebuhr et al., 1997; Pre- Number � 0.1 A 10 � 3 4

Sum of violations (A) 5.1 � 0.7 3.8hoda et al., 1999).Maximum violation (A) 0.24 � 0.07 0.19The most striking feature of the minimal N-WASP

Torsion angle violationsEVH1 domain binding motif is that at 25 residues, it isNumber � 5� 0 � 0 0more than twice as long as the minimal 6–10 residue Sum of violations (�) 0.4 � 0.1 0.4

peptides sufficient for Mena, VASP, or Homer EVH1 rec- Maximum violation (�) 0.05 � 0.01 0.03ognition (Niebuhr et al., 1997; Prehoda et al., 1999; Ball Van der Waals violations

Number � 0.2 A 0 � 1 0et al., 2000; Barzik et al., 2001; Beneken et al., 2000),Sum of violations (A) 3.4 � 0.5 3.9suggesting that the binding energy is well-distributedMaximum violation (A) 0.17 � 0.07 0.16across this motif. Interestingly, the isolated N-WASP

EVH1 domain is highly insoluble and is poorly ex- Atomic Rmsdsd (A): Family of 20 Structures versus Meanpressed, precluding any high-resolution structural or

Backbone 0.50 � 0.06biochemical studies. However, we found that when we Heavy atom 0.94 � 0.08fused the 25 residue WIP motif to the N terminus of the

a Unique, nontrivial constraints derived from a total of 4305 NOEsEVH1 domain via a 5 residue linker, the protein nowobserved in four separate 2D and 3D NOESY spectra using the

expressed extremely well in bacteria and was highly CALIBA function of the DYANA program.soluble (Figure 2A). In contrast, however, a similar fusion b Constraints between residues of the WIP sequence and residuesof the WIP peptide to the C terminus did not yield soluble of the N-WASP EVH1 domain.

c Only residues observed by NMR, corresponding to WIP residuesprotein, thereby indicating a dependence on stereo-461–480 and N-WASP residues 34–137, were included in Ramachan-chemical orientation.dran analysis.d Disordered linker and loop residues were excluded. Rmsd calcula-Structure of the EVH1 Domain-WIP Peptidetions included WIP residues 462–468 and 474–479

Complex: A Novel “Wrapping”Mechanism of RecognitionThe unusual length of the WIP polypeptide required for

The overall fold of the N-WASP EVH1 domain is similarN-WASP EVH1 binding suggests a novel mechanism ofto that observed for the Mena, Homer, and VASP do-interaction that could, for example, depend on higher-mains (Fedorov et al., 1999; Prehoda et al., 1999; Be-order secondary or tertiary structure of the ligand. Toneken et al., 2000; Barzik et al., 2001). The structure-understand how the WIP polypeptide interacts with thebased alignment of several EVH1 domain sequences isN-WASP EVH1 domain, we determined the NMR struc-shown in Figure 2B.ture of the highly soluble fusion protein encompassing

The structure reveals, however, a novel mode ofthe interaction pair described above (Table 1).EVH1-mediated interaction—the WIP polypeptide existsStructural analysis of the EVH1-WIP complex utilizedin a largely extended conformation and wraps aroundthe single-chain construct described above. However,much of the domain, like a piece of string around ato confirm that the covalent linker did not affect theconcave spool (Figure 3A). Because of the extendedmode of WIP binding, we also produced a second analo-nature of the ligand, the surface of the EVH1 domaingous construct with a thrombin cleavage site in thethat is contacted for recognition is far larger (�900 A2 )linker. After purification, this construct could be cleavedthan the canonical binding surfaces utilized by the Menato completion and remained as a soluble complex. Noand Homer EVH1 domains to recognize their muchdifferences were observed in backbone triple-reso-shorter peptide ligands (�300 A2 ) (Figures 3B and 3C).nance or NOESY NMR spectra acquired on samples ofNonetheless, as a part of its interaction, the N-WASPeach version of the complex (cleaved or tethered), asideEVH1 domain does use the conserved canonical bindingfrom new residues introduced in the thrombin recogni-surface to recognize a highly proline-rich portion of itstion site (see Supplemental Figure S1 at http://www.cell.cognate motif (discussed below).com/cgi/content/full/111/4/565/DC1), illustrating that

The overall interaction of the N-WASP EVH1 domainthe presence of a covalent link has no effect on WIPpeptide binding mode. with this extended WIP polypeptide has unexpected

Structure of the N-WASP EVH1-WIP Complex569

Figure 3. Structure of the N-WASP/WIP Complex Determined by NMR and Comparison to Other EVH1 Domain-Mediated Complexes

(A) N-WASP EVH1 domain in complex with WIP residues 461–485. Note that residues 480–485 are required for binding but are poorly definedin the structure because of a lack of assignments and constraints. The likely path of those residues is indicated by the dotted line. The redarrow indicates the site of the most common severe WAS missense mutation, Arg76 [86]. This site falls at the opposite surface from thecanonical EVH1 binding surface.(B) Structure of the Mena EVH1 domain in complex with an 8 residue peptide from ActA.(C) Structure of the Homer EVH1 in complex with a 5 residue peptide from mGluR.(D) Structure of the RanBP EVH1 domain in complex with an extended, �25 residue polypeptide from Ran. Note that contacts between theEVH1 domain and other parts of Ran are made in the complex.Top and bottom images show two views rotated by 90� around a horizontal axis in the plane of the page. Note that in the N-WASP complex,the peptide ligand is recognized in the reverse N- to C-terminal orientation from other EVH1 complexes.

similarity to the interaction of the Ran Binding Protein to continue wrapping further around the N-WASP EVH1domain, along a continued path similar to that observed(RanBP) EVH1 domain with a fragment from the nuclear

import protein Ran (Figure 3D). In the crystal structure in the RanBP/Ran interaction.The extensive interaction interface in the N-WASP/of the Ran-RanBP complex, a �25 residue polypeptide

sequence from Ran wraps around the entire RanBP WIP interaction may explain the biochemical behavior ofthe domain—this exposed, largely hydrophobic bindingEVH1 domain, following the same path as that observed

for the WIP peptide when binding the N-WASP EVH1 surface may lead to the poor solubility of the unligandeddomain. Association of N-WASP with WIP or a relateddomain (Vetter et al., 1999). This interaction, however, is

only one of several extensive regions of contact between ligand may be obligatory for proper folding of the pro-tein—consistent with the observation that the N-WASP/the Ran and RanBP proteins, and it is unknown if this

wrapping interaction alone is sufficient for binding, as WIP interaction appears to be constitutive in vivo.is the case for the N-WASP/WIP interaction.

In the current NMR structure, the conformation of Common Elements of Proline-RichPeptide Bindingresidues 479–485 (C-terminal end of the WIP peptide)

are poorly defined because of a lack of resonance as- The most N-terminal portion of the WIP polypeptide,which contains the sequence LPPP, binds at the samesignments. The difficulty in assigning these residues

may be a result of several possibilities: first, the main conserved aromatic surface used by other EVH1 do-mains for proline peptide binding (Figure 4). As is ob-chain amide protons may have relatively high solvent

accessibility and exchange rates; second, the residues served in other EVH1-peptide complexes, this region ofthe peptide adopts a polyproline II (PPII) helical confor-may exist in multiple conformations that interconvert

within the intermediate exchange regime; and third, the mation, a left-handed helix with three residues per turn.Other proline-rich motif binding domains, including SH3region may be highly mobile. Dynamics of the polypep-

tide backbone were probed using the 15N-1H hetero- and WW domains, and profilin also recognize their li-gands in a PPII conformation. The PPII helix docksnuclear NOE. For residues 477–480, decreasing NOE

values are observed moving from N- to C-terminal, a against a complementary surface of largely aromaticside chains in a manner very similar to that observed intrend consistent with enhanced mobility for the adjacent

unobserved residues (data not shown). Nonetheless, ex- other EVH1 domains. A universal element of this recogni-tion is a central, conserved tryptophan residue (Trp 54),tension of the peptide C terminus beyond residue 480

is energetically important for binding (Figure 1E). We which forms a ridge over which the PPII helix packs,making favorable van der Waals contact. In addition,hypothesize that side chain atoms in this region of the

peptide make primary contacts with the EVH1 domain, the tryptophan indole nitrogen proton forms a hydrogenbond with a carbonyl oxygen of the PPII ligand. Less wellwhile the main chain atoms may be relatively mobile.

As shown in Figure 3A, these residues are most likely conserved but largely hydrophobic residues comprise

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Figure 4. The N-WASP EVH1 Surface Usedto Recognize the Proline-Rich Element in theWIP Ligand Is Similar to that Used by OtherEVH1 Domains

(A) Close-up view of region of N-WASP EVH1domain that contacts residues 481–487 of theWIP ligand. The ligand adopts a polyprolineII (PPII) helical conformation that docks overTrp 54. The carbonyl oxygen between the api-cal ligand prolines also makes a hydrogenbond to the Trp nitrogen. Pockets sur-rounding Trp 54 are made by residues 46 and104 or 48 and 106.(B) Corresponding residues in the Homer andMena EVH1 domains are also used in recogni-tion of the proline-rich peptide ligand. In thesestructures, however, the PPII helix binds inthe opposite N- to C-terminal orientation.

residue binding pockets that flank the central tryp- Buried Mutations Linked to Wiskott-AldrichSyndrometophan.Because of the extremely high homology betweenWASP and N-WASP (Figure 2), this EVH1 domain struc-

Two Orientations for EVH1-Peptide Binding ture and its novel mode of peptide binding providesOne of the most striking features of this structure is that the first opportunity to accurately interpret missensethe WIP polypeptide docks on the EVH1 domain in an mutations that result in the disease WAS. WAS is anN- to C-terminal orientation that is the reverse of that X-linked recessive disorder that is characterized byobserved in all previous EVH1-peptide complexes (Fig- thrombocytopenia (platelet deficiency), eczema, and re-ures 3 and 4B). Thus, the EVH1 domain, like other pro- currence of bacterial and viral infections. A mild formline-rich binding modules (including SH3 and WW do- of the disease is known as X-linked thrombocytopeniamains) and profilin, can bind PPII ligands in two possible (XLT). WAS/XLT-causing missense mutations occur atorientations. This orientational flexibility, first explored �30 residues within the WASP EVH1 domain. For thein depth for SH3 domains, is due largely to the 2-fold discussion below we will describe sites of interactionrotational pseudosymmetry of the PPII structure, both using the residue numbering for N-WASP. However, forin terms of shape and hydrogen bonding moieties (Lim clarity and comparison, the corresponding residue num-et al., 1994; Feng et al. 1994). This rotational symmetry ber from WASP will also be given in square brackets.allows the relatively isoenergetic docking of the PPII Several of the common WAS-causing mutations maphelix in two orientations against a single compatible to the hydrophobic core of the EVH1 domain, especiallysurface. It is interesting to note that in the RanBP EVH1 at residues that are absolutely conserved betweenstructure, the extended peptide from Ran binds in the WASP and N-WASP (Table 2A, Figure 5A). Mutations atopposite orientation from that observed in the current buried positions often destabilize a protein and indi-

rectly decrease function. However, it is noteworthy thatstructure.

Structure of the N-WASP EVH1-WIP Complex571

Table 2. WAS Missense Mutations Localized to the EVH1 Domain and Their Role in the N-WASP-WIP Complex

Type of Position in N-WASP DiseaseMutation [WASP numbering] Substitution Severity Description of Position Probable Effect

(A) BURIED Trp87 [97] Cys strong core destabilization/conform. changeHis105 [115] Tyr strong core; H bond to Tyr97 destabilization/conform. changePhe118 [128] Ser strong core; beneath pept. bind. surf. destabilization/conform. changeAla124 [134] Thr strong core destabilization/conform. changeTyr73 [83] Cys mild core; beneath pept. bind. surf. destabilization/conform. changea

Tyr97 [107] Cys mild core; H bond to His 105 destabilization/conform. changea

(B) SURFACE Leu31 [39] Pro strong near peptide C term. disrupt WIP interactionCys63 [73] Arg strong near peptide N term. disrupt WIP interactionVal65 [75] Met strong near peptide C term. disrupt WIP interactionSer72 [82] Phe strong near peptide C term. (NOE) disrupt WIP interactionArg76 [86] Cys, His, Leu, Pro strong near peptide C term. disrupt WIP interactiona

Arg101 [111] Pro strong near peptide C term. (NOE) disrupt WIP interactionAla115 [Gly125] Arg strong near peptide N term. (NOE) disrupt WIP interactionGlu123 [133] Lys strong away from peptide structural salt bridge?Thr37 [45] Met mild (XLT) near peptide C term. disrupt WIP interactionGln44 [52] His mild (XLT) near peptide N term. disrupt WIP interactionLys33 [Arg41] Gly mild near peptide C term. disrupt WIP interactionSer39 [47] Asp mild near peptide C term. disrupt WIP interactionSer40 [48] Ile mild near peptide C term. disrupt WIP interactionAla48 [56] Val mild near peptide N term. (NOE) disrupt WIP interactionPhe74 [84] Leu mild near peptide C term. disrupt WIP interactionGln89 [99] Arg mild near peptide C term. (NOE) disrupt WIP interactionGly109 [119] Glu mild near peptide N term. (NOE) disrupt WIP interactionGlu121 [131] Lys mild salt bridge with Lys125 destabilization/conform. changeArg128 [138] Pro strong H bond to Ser99 destabilization/conform. change

Data compiled from WASP database (http://www.tmd.ac.jp/med/ped/WASPbase.html)“NOE” indicates observed NOE contact between WIP peptide and this residue.a Mutation experimentally observed to disrupt WASP-WIP interaction by yeast two-hybrid assay (Stewart et al., 1999).

many of these buried mutations cluster into a single tation of Trp54 has been shown to disrupt proper local-packing unit (Trp87 [97], Tyr97 [107], His105 [115], ization to vaccinia virus (Moreau et al., 2000). In fact,Phe118 [128], and Ala124 [134]) that includes a buried the most dramatic hotspot of disease mutations is onhydrogen bond between residues Tyr97 [107] and the face of the EVH1 domain directly opposite the pro-His105 [115]. Even fairly subtle mutations at these posi- line-rich peptide binding surface (Beneken et al., 2000).tions (i.e., Tyr97 [107]-Cys, His105 [115]-Tyr, Ala124 For example, the most frequent mutated residue re-[134]-Thr), which maintain hydrophobicity and thus sulting in a severe disease phenotype is Arg76 [86].might not be expected to be very destabilizing, result Every possible single base change missense mutationin the disease. Interestingly, this packing unit lies directly (Cys, His, Leu, Pro) has been found in the WAS patientbeneath the proline-rich peptide binding surface. In fact, population (Table 2B). Mutations are also found at otherone of these residues, His105 [115], is the covalent surface residues that flank Arg76 [86], including Leu31neighbor of two residues that directly contact this seg- [39], Thr37 [45], Ser40 [Thr48], Cys63 [73], and Phe74ment of the WIP ligand (Phe104 [114], Thr106 [116]). [84]. Given the distance of this mutational hotspot fromThus, it seems possible that subtle mutations in this the canonical peptide binding site identified in pre-packing unit perturb the precise stereochemistry of the viously characterized EVH1 domain complexes, it hasproline-rich peptide binding surface and thereby impair been difficult to reconcile how these mutations in WASPWIP binding. This hypothesis is consistent with several might disrupt EVH1 domain interactions and functionprevious findings: first, the Tyr97 [107]-Cys and Ala124 (Beneken et al., 2000).[134]-Thr mutations were found to impair interaction In the context of this new structure, however, we nowwith WIP in an in vitro yeast two-hybrid binding assay know that the WIP ligand wraps around the N-WASP(Stewart et al., 1999); and second, studies of patient EVH1 domain, extending from the canonical proline-richsamples reveal no significant decrease of endogenous peptide-docking site all the way to mutational hotspotexpression levels of the WASP protein in a patient bear- surrounding Arg76 [86]. Contacts made in this regioning a mutation at Tyr97 [107]. are therefore likely to be critical for WIP binding. Unfortu-

nately, because of a lack of assignments, we do notknow the precise conformation of the last seven resi-Surface Mutations Linked to Wiskott-Aldrichdues of the WIP polypetide and exactly how these resi-Syndromedues interact with the hotspot centered around Arg76Approximately 20 of the reported WAS-causing muta-[86]. However, these seven residues are required fortions map to the EVH1 domain surface (Table 2B, Figurebinding and therefore must be involved in energetically5B). Oddly, there are no naturally occurring mutations

at the proline-rich peptide binding surface (although mu- important interactions.

Cell572

Figure 5. Structural Basis of Mutations thatCause the Disorder WAS

(A) Residues in the core of the N-WASP EVH1domain that when mutated lead to WAS areshown in space-filling depiction. Severe/moderate mutations are colored in lavender;mild mutation are colored in green. Residuenumbering is that of N-WASP, with the corre-sponding numbering from WASP given insquare brackets. Peptide is colored orange,with the postulated path of the peptide C ter-minus indicated by small orange spheres.(B) Front face of N-WASP EVH1 domain indi-cating surface mutations associated withWAS. Residues are colored as above.(C). Back face of N-WASP EVH1 domain, indi-cating surface mutations associated withWAS. Molecular has been rotated 180� fromthe view in (B).(D) Transparent surface view identical to thatin (C) reveals a conserved network of chargedresidues, centered around Arg76 [86] (seeFigure 2 for conservation).(E) Alignment of WIP homologs in the WASPbinding peptide reveals two regions of highconservation, including the C-terminalPSKxxR/K motif that is likely to dock at thesurface shown in (D). Charged residues arecolored in red (negative) or blue (positive).(F) Cartoon of how the extended WIP peptideand its homologs may wrap around theN-WASP EVH1 domain to contact two distantbut energetically important binding surfaces.

Based on the evidence given below, we postulate that conserved in all WIP homologs, including mouse WIP,human CR16, and yeast verprolin—all of which havethe C-terminal end of the WIP peptide binds at this

mutational hotspot and participates in an energetically been shown to interact with WASP or WASP homologs(Figure 5E). This cluster of basic residues is within thecritical network of electrostatic interactions. First, the

proposed binding path for the remaining portions of conserved motif: P-S-K-x-x-R/K-x(1-3)-R. In summary, wetherefore propose a model in which the conserved basicthe peptide correlates exactly with the binding groove

utilized by the RanBP EVH1 domain to recognize one motif found at the C terminus of the WIP ligand specifi-cally interacts with a complementary network of con-terminus of the Ran peptide (see Figure 3D). Second,

mutation of the central Arg76 [86] to His has been shown served acidic residues that are properly positioned bythe central Arg76 [86] residue (Figure 5F). Such an inter-to impair interaction with WIP in an in vitro yeast two-

hybrid assay (Stewart et al., 1999). Third, Arg76 [86] is action may have a fairly high degree of mobility, consis-tent with the difficulty in observing assignable reso-the central residue in a conserved electrostatic network

presented on this surface of the EVH1 domain (Figure nances, despite its apparent energetic importance.Overall, this structure is therefore consistent with a5D). Surrounding Arg76 [86] are a ring of acidic residues,

Asp67 [77], Glu88 [98], and Glu90 [100] (Figure 5D), all simple model for the cause of WAS: the major defect inthese diverse WAS missense mutations is likely to beof which are conserved from human WASP/N-WASP to

the yeast ortholog, Bee1p (see Figure 2). In the current disruption of the interaction with WIP. Disruption of thisinteraction may prevent proper subcellular targeting ofstructure, Arg76 [86] is very well ordered for an arginine

residue—several interresidue NOEs are observed for the WASP. The reason that mutations are found at siteswidely spread over the EVH1 domain surface, well be-Arg side chain, and its epsilon NH proton exchanges

very slowly. This observation is consistent with Arg76 yond the canonical EVH1 peptide binding surface, isbecause this EVH1 domain utilizes a novel mode of inter-[86] playing a central role in coordinating and orienting

this otherwise repulsive network of surrounding acidic action—the WIP ligand wraps around almost two-thirdsof the entire circumference of the EVH1 domain, con-residues. Fourth, the C terminus of the WIP peptide

contains a cluster of basic residues that is very well tacting many distant surfaces.

Structure of the N-WASP EVH1-WIP Complex573

cated and centrifuged lysate was used directly as probe, after esti-Conclusionsmating fusion protein concentration by SDS-PAGE. Washing andThis structure of the N-WASP EVH1 domain in complexchemiluminescence detection was performed as described (Pre-with a peptide ligand from WIP reveals a novel mecha-hoda et al., 1999).

nism of peptide recognition by EVH1 domains and high-lights the versatility of EVH1 domains as a recognition

Protein Labeling and Purificationplatform. While the majority of EVH1 domains and otherThe WIP-EVH1 sequence (residues 461–485 of human WIP and resi-similar protein-protein modules bind short 6–12 residuedues 26–147 of rat N-WASP, linked by the intervening sequence

motifs, the N-WASP EVH1 domain binds a single long GSGSG) was ligated into the pBH4 expression vector, which con-25 residue motif. Comparison of the WIP ligand and its tains a His6-tag followed by a Tobacco Etch Virus (TEV) proteasehomologs from different species suggest that there are cleavage site. For uniform labeling with 15N and 13C, BL21(DE3) cells

freshly transformed with the WIP-EVH1 expression plasmid weretwo conserved submotifs that are recognized: thegrown in M9 minimal media containing [13C]glucose (2 g/l) andN-terminal PPP submotif, similar to other EVH1 ligands,[15N]ammonium chloride (1 g/l) as the respective sole carbon andand the C-terminal PSKxxR submotif. These two sub-nitrogen sources. Expression was induced by the addition of IPTG

motifs interact on opposite faces of the EVH1 domain, to a final concentration of 1 mM. After expression for 3 hr, the cellsleading to this very extensive wrapping mechanism of were harvested by centrifugation, resuspended in Lysis Buffer (50recognition (Figures 5E and 5F). The extensive binding mM PO4, 300 mM NaCl, 10 mM imidazole [pH 8.0]), disrupted by

sonication, and centrifuged, and the soluble fraction bound in batchsurface employed provides an explanation for the spa-to Ni-NTA agarose. After extensive washing with lysis buffer, boundtially dispersed set of WAS-causing mutants that mapprotein was eluted with lysis buffer � 300 mM imidazole. The His6-to the EVH1 domain.tag was removed by incubation with TEV protease for 2 hr at room

This interaction represents a novel or hybrid class of temperature. Cleaved WIP-EVH1 protein was further purified on aprotein-protein interactions. Most protein-protein inter- ResourceS (Pharmacia) column at pH 7.5 with a 1–500 mM NaClactions are divided into two major classes: (1) interac- gradient. Pure protein was pooled and concentrated to �1 mM.

Protein size was confirmed by mass spectroscopy before and aftertions that involve the docking of two large folded proteinNMR data collection.surfaces, and (2) interactions that involve the docking

A cleavable version of the linked WIP-EVH1 protein was generatedof a short, largely linear peptide epitope on a proteinas described above, but with an intervening linker sequence

surface (Stanfield and Wilson, 1995). Like the second GGLVPRGSGG (underlined sequence is thrombin cleavage site).class of interactions, the N-WASP EVH1-WIP peptide 13C/15N-labeled protein was prepared as described above andinteraction involves recognition of a linear epitope. How- cleaved at a concentration of 0.5 mM by adding 50 nM of bovine

thrombin (Sigma). Cleavage was complete in 1 hr as monitored byever, because the epitope is extremely long, the contactSDS-PAGE.area involved is as large as that observed in the first

class of interactions. It will be interesting to see if otherEVH1 domains also have the ability to recognize longer NMR Spectroscopy

NMR experiments were carried out on Bruker DRX600 spectrome-peptides with higher specificity by utilizing more of theirters, either at the Medical College of Wisconsin (with triple-reso-surface binding potential. It is also possible that in somenance Z-axis gradient CryoProbe�) or at the National Magnetic Res-cases EVH1 domains may be able to use their extensiveonance Facility at Madison (with conventional probe). Samples

binding surfaces to simultaneously interact with two or contained �1 mM WIP-EVH1 protein in 90% H2O/10% D2O with 20more ligands, recognizing short, proline-rich peptides mM sodium phosphate (pH 7.0), 20 mM sodium chloride, 1 mMwith the canonical binding surface and recognizing other dithiothreitol, and 0.05% sodium azide. Resonance assignments

and distance constraints were derived from the following experi-yet unidentified ligands at remaining portions of the do-ments: 3D 15N NOESY-HSQC (Talluri and Wagner, 1996), 3D 15N SEmain surface.TOCSY-HSQC (Zhang et al., 1994), 3D HCCH-TOCSY (Kay et al.,1993), 3D SE CCONH (Grzesiek et al., 1993), 3D SE HNCA (Grzesiek

Experimental Procedures and Bax, 1992; Kay et al., 1994), 3D SE HNCOCA (Grzesiek and Bax,1992), 3D SE HNCO (Grzesiek and Bax, 1992; Muhandiram and Kay,

Lipid Vesicle Binding Assays 1994), 3D 13C SE NOESY-HSQC (one each for the aromatic andCosedimentation lipid vesicle binding assays were performed as aliphatic 13C regions) (Kay et al., 1993), and 13C constant-time SEdescribed by Kavran et al. (1998). Specifically, 3 �M protein and HSQC (Santoro and King, 1992). NOESY mixing times were 80 ms.vesicles (�20 �M PIP2) were mixed in 20 mM HEPES (pH 7.0), 100 Isotropic mixing periods were 22 ms (CCONH) and 60 ms (15NmM NaCl, 1 mM DTT (total assay volume 85 �l), incubated at 25�C TOCSY-HSQC). Heteronuclear NOEs were measured from an inter-for 5 min, and centrifuged at 100,000 � g for 15 min. The vesicles leaved pair of 2D 15N-1H gradient sensitivity enhanced correlationcomprised PC:PS:PIP2 at ratios of 48:48:2. Apparent concentrations spectra (Farrow et al., 1994) recorded with and without a 3.5 s protonof PIP2 were calculated as 50% of total. The vesicle pellet was saturation period using a total recycle delay of 5.5 s. Data werewashed with 1000 �l of buffer three times and suspended in a processed using NMRPipe (Delaglio et al., 1995) and analyzed usingvolume of buffer equal to the supernatant. Bound (pellet) and un- XEASY (Bartels et al., 1995). Chemical shifts were referenced tobound proteins (supernatant) were identified by SDS-PAGE. This 2,2-dimethylsilapentane-5-sulfonic acid (DSS) directly for 1H andassay can detect binding of the PLC-� PH domain, which has an indirectly for 15N and 13C (Markley et al., 1998). The CSI programaffinity for PIP2 of Kd � 2 �M. was used for determining the secondary structure from the chemical

shift index (Wishart and Sykes, 1994; Wishart et al., 1992). For thethrombin-cleaved protein, resonance assignments and NOE pat-Far-Western Protein Binding Assays

Assays were performed as described (Prehoda et al., 1999). Putative terns were determined for the cleaved WIP-EVH1 complex using 3DSE HNCA, 3D CCONH, 3D 15N NOESY-HSQC, and 3D 13C NOESY-EVH1 peptide ligands were generated as fusions to glutathione

S-transferase (GST), electrophoresed on a 10%–20% SDS-PAGE HSQC spectra, under the same conditions. No significant chemicalshift differences were observed apart from residues of the linkergel, and transferred to nitrocellulose by electroblotting, and the blot

was blocked for 1 hr at 4�C in Superblock (Pierce). Biotinylated that differ in the two constructs (see Supplemental Figure S1 athttp://www.cell.com/cgi/content/full/111/4/565/DC1), and bothEVH1 domains were produced as fusions to a naturally biotinylated

E. coli protein using the PIN-POINT system (Promega). The soni- samples displayed the same pattern of WIP-EVH1 NOEs.

Cell574

Structure Calculation and Analysis Derry, J.M., Ochs, H.D., and Francke, U. (1994). Isolation of a novelgene mutated in Wiskott-Aldrich syndrome. Cell 78, 635–644.Structures were iteratively refined using the simulated annealing

protocol (10,000 steps total) of the torsion angle dynamics program Derry, J.M., Kerns, J.A., Weinberg, K.I., Ochs, H.D., Volpini, V., Esti-DYANA (Guntert et al., 1997). NOE peak intensities were converted vill, X., Walker, A.P., and Francke, U. (1995). WASP gene mutationsinto upper distance bounds with the CALIBA function of DYANA. A in Wiskott-Aldrich syndrome and X-linked thrombocytopenia. Hum.total of 143 φ and dihedral angle constraints were generated from Mol. Genet. 4, 1127–1135.1H, 13C, 13C�, 13C’, and 15N secondary shifts using TALOS (Cornilescu

El-Hakeh, J., Rosenzweig, S., Oleastro, M., Basack, N., Berozdnik,et al., 1999). Of the final 50 structures calculated, the 20 conformers

L., Molina, F., Rivas, E.M., Zelazko, M., and Danielian, S. (2002).with the lowest target function values were selected for analysis.

Wiskott-Aldrich syndrome in Argentina: 17 unique, including nineThe mean structure, calculated from this ensemble of 20 structures

novel, mutations. Hum. Mutat. 19, 186–187.in MOLMOL (Koradi et al., 1996), was minimized in DYANA using

Farrow, N.A., Muhandiram, R., Singer, A.U., Pascal, S.M., Kay, C.M.,8000 steps of conjugate gradient minimization. Coordinates (mini-Gish, G., Shoelson, S.E., Pawson, T., Forman-Kay, J.D., and Kay,mized average structure and final 20 structures) have been depos-L.E. (1994). Backbone dynamics of a free and a phosphopeptide-ited in the RCSB database (accession code 1MKE). Residues in thecomplexed src homology 2 domain studeid by 15N NMR relaxation.PDB file are numbered consecutively with the following residueBiochemistry 33, 5984–6003.number translation: PDB(1–25) � WIP(461–485); PDB(26–30) �

Fedorov, A.A., Fedorov, E., Gertler, F., and Almo, S.C. (1999). Struc-GSGSG linker; PDB(31–144) � N-WASP(26–139) (note: residuesture of EVH1, a novel proline-rich ligand-binding module involved140–147 of N-WASP were not observed by NMR and are not includedin cytoskeletal dynamics and neural function. Nat. Struct. Biol. 6,in the PDB file).661–665.

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