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Originally published In Press as doi:10.1074/jbc.M609902200 on January 16, 2007

J. Biol. Chem., Vol. 282, Issue 11, 8446-8453, March 16, 2007
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Multiple WASP-interacting Protein Recognition Motifs Are Required for a Functional Interaction with N-WASP*

Francis C. Peterson{ddagger}, Qing Deng{ddagger}, Markus Zettl§, Kenneth E. Prehoda, Wendell A. Lim||, Michael Way**, and Brian F. Volkman{ddagger}1

From the {ddagger}Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, §Laboratory of Molecular Biology, Medical Research Council, Cambridge CB2 2QH, United Kingdom, Department of Chemistry, University of Oregon, Eugene, Oregon 97403-1229, ||Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143-2240, and **Cell Motility Laboratory, Cancer Research UK, London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom

Received for publication, October 23, 2006 , and in revised form, December 27, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The WASP-interacting protein (WIP) targets WASP/WAVE proteins through a constitutive interaction with an amino-terminal enabled/VASP homology (EVH1) domain. Parallel investigations had previously identified two distinct N-WASP binding motifs corresponding to WIP residues 451-461 and 461-485, and we determined the structure of a complex between WIP-(461-485) and the N-WASP EVH1 domain (Volkman, B. F., Prehoda, K. E., Scott, J. A., Peterson, F. C., and Lim, W. A. (2002) Cell 111, 565-576). The present results show that, when combined, the WIP-(451-485) sequence wraps further around the EVH1 domain, extending the interface observed previously. Specific contacts with three WIP epitopes corresponded to regions of high sequence conservation in the verprolin family. A central polyproline motif occupied the canonical binding site but in a reversed orientation relative to other EVH1 complexes. This interaction was augmented in the amino- and carboxyl-terminal directions by additional hydrophobic contacts involving WIP residues 454-459 and 475-478, respectively. Disruption of any of the three WIP epitopes reduced N-WASP binding in cells, demonstrating a functional requirement for the entire binding domain, which is significantly longer than the polyproline motifs recognized by other EVH1 domains.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mutations in the WASP gene give rise to X-linked thrombocytopenia and the Wiskott-Aldrich syndrome (WAS)2 (1), a platelet and immune deficiency first identified in 1937. The WAS protein (WASP), expressed in cells of hematopoietic lineage, and its ubiquitously expressed neuronal homolog (N-WASP) encode multidomain proteins that stimulate actin polymerization in a phospholipid- and GTPase-dependent manner (2-4). WASP also associates with WASP-interacting protein (WIP) via an enabled/VASP homology-1 (EVH1) domain at the WASP amino terminus; the majority of identified WAS mutations are located within this domain (5). The WIP-WASP interaction is required for T cell activation (6, 7) and in vitro chemotaxis of T cells (8) and monocytes (9) in response to the chemokine CXCL12. Macrophages from patients with WAS also display impaired chemotaxis (10-12). Thus, WAS mutations may produce a general defect in leukocyte chemotaxis and T cell-mediated immune responses (13).

WIP, WIP-related (WIRE/WICH), and CR16 comprise the mammalian verprolin family and function as regulators of the actin cytoskeleton (14). The verprolins are proline-rich proteins that bind profilin and various Src homology 3 domain-containing proteins, and contain a conserved WASP binding domain (WBD) near the carboxyl terminus. N-WASP exists in cells predominantly as an inactive complex with WIP or CR16 (6, 15). WASP/N-WASP is stabilized by its constitutive association with WIP (8, 16), and PKC{theta}-mediated phosphorylation of a serine near the WIP carboxyl terminus weakens that interaction (6). Disease-causing mutations in the EVH1 domain are thus thought to disrupt the complex with WIP and perhaps promote WASP degradation (16).

The conserved binding surface of EVH1 domains includes an invariant tryptophan side chain that recognizes a proline-rich sequence in the target protein (17, 18), although variations in the length, sequence, and binding orientation have been observed in structures of EVH1·target complexes. For example, our previous structural analysis (19) demonstrated that the N-WASP EVH1 domain binds a short proline-rich sequence (462LPPPEP467) at the canonical binding surface but only in the context of a longer sequence (WIP residues 461-485) that includes ~20 residues carboxyl-terminal to the polyproline motif. Moreover, upon determination of the WIP·EVH1 structure we found the orientation of the WIP polypeptide was reversed in comparison to all other EVH1 domain complexes (17, 20, 21). In a parallel study, Zettl and Way (22) also showed that whereas substitution of the conserved EVH1 tryptophan abrogated binding, the polyproline motif alone was insufficient for WIP/N-WASP interaction. However, they also found that a 10-residue segment of WIP (451ESRFYFHPISD461) immediately amino-terminal to the LPPPEP sequence was by itself sufficient to bind the EVH1 domain. This interaction depended on conserved aromatic side chains (Phe454 and Phe456) but required no proline-rich sequence whatsoever. A subsequent analysis by Aspenstrøm (23) indicated that the analogous aromatic motif and proline-rich region of WIRE are both required for the interaction with the EVH1 domain of WASP. Results from different groups thus have suggested that elements both within and adjacent to the WIP-(461-485) sequence are required for functional WIP-WASP interactions in vivo.


Figure 1
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  		FIGURE 1.
An extended WIP sequence interacts with the N-WASP EVH1 domain. A, additional WIP residues in the exWIP·EVH1 complex (gold contours) perturb signals from the EVH1 domain in the 1H-15N HSQC spectrum of WIP·EVH1 (blue contours). B, EVH1 domain 1H/15N chemical shift differences of >1 ppm (dashed line) are shown in cyan, green, and gold. C, WIP·EVH1 structure (19) (Protein Data Bank code 1MKE) with WIP residues shown in blue and perturbed residues of the EVH1 domain highlighted as described in B. D, EVH1 shift perturbations are adjacent to the WIP fragment amino terminus (D461), the point of attachment for residues 451-460 in the exWIP·EVH1 complex.

 
To clarify these apparently discordant results, we determined the structure of a complex between the EVH1 domain of N-WASP and an extended WIP sequence that encompasses both of the previously defined interaction domains. Our results show that WIP wraps around the EVH1 domain and interacts with three distinct surfaces. As anticipated, residues at the amino-terminal end of the WIP fragment make specific contacts with the EVH1 domain. Binding measurements in cells demonstrated the essential role for multiple WIP epitopes conserved in the verprolin family.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Protein Production—Residues 461-485, 451-485, or 451-493 from human WIP were linked to the EVH1 domain (residues 26-147) from rat N-WASP by the sequence GGLVPRGSGG and expressed with an amino-terminal His6 affinity tag followed by a tobacco etch virus protease cleavage site. WIP·EVH1 fusion constructs were produced in Escherichia coli as described previously (19).

NMR Spectroscopy—NMR samples were prepared in 20 mM sodium phosphate, pH 7.0, 20 mM sodium chloride, 2 mM dithiothreitol, 0.05% sodium azide, and 10% 2H2O. NMR data were acquired at 30 °C on a Bruker 600 MHz spectrometer with a triple resonance CryoProbe and processed using NMRPipe (24). Backbone 1H, 15N, and 13C assignments for exWIP·EVH1 were transferred from WIP·EVH1 (BMRB accession number 5554) and confirmed using three-dimensional HNCO, HNCA, and CCONH spectra. Side-chain assignments were completed using three-dimensional HCCH-TOCSY (total correlation spectroscopy) and 13C (aromatic)-edited NOESY-HSQC spectra.

Structure Determination—Distance constraints were obtained from three-dimensional 15N-edited NOESY-HSQC, 13C-edited NOESY-HSQC, and 13C (aromatic)-edited NOESY-HSQC spectra ({tau}mix = 80 ms). Backbone {varphi} and {psi} dihedral angle constraints were generated from 1H{alpha}, 13C{alpha}, 13Cbeta, 13C', and 15N shifts using TALOS (25). The structure was solved using CYANA (26) followed by final refinement by X-PLOR in explicit water solvent (27). Coordinates, chemical shifts, and restraints were deposited in the Protein Data Bank (code 2IFS).

Binding Assays—Pulldown experiments using purified N-WASP EVH1 domain and GST fusion proteins with peptides from the WIP WBD were performed as previously described (22). Expression of GFP-WIP WBD proteins and detection of actin tail formation in vaccinia-infected cells was performed as described previously (22).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
NMR Screening of WIP·EVH1 Complexes—Independent analyses of the interaction between N-WASP and WIP identified distinct EVH1 binding sequences, corresponding to WIP residues 451-461 (22) and residues 461-485 (19). The WIP-(461-485)·EVH1 structure revealed an extensive interaction surface unique in the EVH1 domain family (19). Phosphorylation of WIP at Ser488 was also shown to disrupt the WIP-WASP interaction, perhaps controlling WASP stability in the cell (6). This residue was not implicated in the WIP-WASP interaction but is immediately carboxyl-terminal to the WIP461-485 sequence that binds the N-WASP EVH1 domain. To examine the structural role of residues adjacent to the WIP-(461-485) WASP binding domain, we purified a series of 15N-labeled fusion proteins containing WIP fragments corresponding to residues 461-485, 451-485, or 451-493 linked to the amino terminus of the N-WASP EVH1 domain. The two-dimensional 1H-15N HSQC spectrum of the WIP-(451-493)-EVH1 construct contained extremely broad lines and poor chemical shift dispersion and was deemed unsuitable for further analysis. In contrast, HSQC spectra of the complexes with WIP-(461-485) (WIP·EVH1) and WIP-(451-485) (exWIP·EVH1) were of high quality (Fig. 1A), and we compared them to assess whether residues 451-460 participate in the WIP·EVH1 interaction.


Figure 2
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  		FIGURE 2.
NMR structure and dynamics of the exWIP·EVH1 complex. A, ensemble of 20 NMR structures. B, comparison of WIP conformation in the original WIP·EVH1 complex (black ribbon) and the exWIP·EVH1 complex (gray ribbon). Side chains for the 463PPP465 polyproline type II helix are shown. C, heteronuclear 15N-1H NOE values for the WIP·EVH1 complex (upper panel) and exWIP·EVH1 complex (lower panel) plotted as a function of N-WASP and WIP sequences.

 
WIP Residues 451-460 Extend the EVH1 Binding Site—Significant differences in the two-dimensional HSQC spectra for WIP·EVH1 and exWIP·EVH1 (Fig. 1A) are restricted to a small number of residues in three regions of the EVH1 sequence (Fig. 1B). No chemical shift perturbations are observed in residues of the EVH1 domain that contact WIP-(461-485) (Fig. 1C). Instead, shifts induced by WIP residues 451-460 are clustered in a region of the EVH1 domain near WIP residue Asp461 (Fig. 1D). These data are consistent with formation of a complex in which WIP residues 451-460 add to the previous binding interface by continuing to wrap around the EVH1 surface.

Structure and Backbone Dynamics of the exWIP·EVH1 Complex—We determined the exWIP·EVH1 structure by NMR spectroscopy (Fig. 2A and Table 1). Overall, the exWIP·EVH1 complex closely resembles the previously determined WIP·EVH1 structure, with the only obvious difference arising from the presence of additional WIP residues (Fig. 2B). For residues of the EVH1 domain, the backbone r.m.s.d. between the two structures is ~1.5 Å, and the conformation of WIP residues common to both structures (461-485) is similar (backbone r.m.s.d. ~ 1.5 Å). A cis configuration was observed for the His457-Pro458 peptide bond in the amino-terminal extension of the WIP sequence. The cis linkage, initially detected using the difference between the proline 13Cbeta and 13C{gamma} chemical shifts (>9 ppm) (28), was verified by structure calculations in which numerous short- and medium-range NOE distance constraints were satisfied only when the cis configuration was enforced.


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  		TABLE 1
Statistics for 20 conformers of N-WASP EVH1 domain bound to the WASP binding domain of WIP

 
Heteronuclear 15N-1H NOE values exceeding 0.75 for most residues of the exWIP·EVH1 and WIP·EVH1 complexes show that the EVH1 domain is highly ordered (Fig. 2C). Prolines at WIP residues 463-465 and 467 create gaps in the NOE profile for both complexes, but the pattern for residues 466-480 includes a flexible region that reaches a minimum NOE value of ~0.3 at Thr471. Maximal NOE values for WIP residues above 0.7 indicate that those portions of the WIP sequence adopt the same rotational correlation time as the EVH1 domain. WIP segments corresponding to residues 455-468 and 474-479 are ordered as a result of binding to the EVH1 domain and are connected by a short flexible linker. These observations are consistent with the calculated ensemble of structures.

Distinct WIP Epitopes Bind an Elongated EVH1 Surface—The extended WIP sequence wraps more than halfway around the N-WASP EVH1 domain, using three distinct epitopes to form specific contacts (Fig. 3A) totaling 1033 Å2 in buried surface area. Epitope 1 corresponds largely to residues identified by Zettl and Way (22) as an alternative N-WASP binding domain. WIP residues 451-453 display no long-range NOEs and are unconstrained in the family of structures, but the 454FYFHPIS460 segment is well defined and closely associated with the EVH1 domain (Fig. 3B). Aromatic side chains of WIP residues Phe454 and Phe456 bind a hydrophobic surface that includes Val42 and Ala119 of N-WASP. This interaction is reflected in the large 1H and 13C chemical shift changes relative to the original WIP·EVH1 construct for the Ala119 methyl group as well as in new NOE cross-peaks to the Phe454 and Phe456 aromatic protons (Fig. 3C). Hydrophobic WIP residues Phe454, Phe456, and Ile459 contribute 197 Å2, ~20% of the total EVH1 contact surface. In contrast, two nearby aromatic WIP residues, Tyr455 and His457, are solvent-exposed and make no contacts with the EVH1 domain. This amino-terminal WIP epitope is linked to the polyproline sequence (epitope 2) by a well defined structural element consisting of the His457-Pro458 cis-peptide bond followed by a tight helical turn (residues 459-461).

Epitope 2 comprises WIP residues 461-468 and contributes just over 40% of the total buried surface of the exWIP·EVH1 complex (414 Å2). WIP residues 462LPPP465 surround the conserved Trp54 side chain of the EVH1 binding surface, similar to other EVH1·peptide complexes but with the polypeptide chain running in the opposite direction (19). NOE contacts between Pro463 and the N-WASP Trp54 indole NH are absent in the exWIP·EVH1 spectrum (Fig. 3D), suggesting that docking of the LPPP motif onto the conserved tryptophan side chain has been altered by the WIP-(451-460) extension. In an overlay with the original WIP·EVH1 complex, the polyproline helix of the new exWIP·EVH1 structure is shifted and unwound slightly. As illustrated in Fig. 3D, it appears as though the LPPP motif has been stretched toward the amino-terminally attached epitope 1 binding site.

Epitope 3 is separated from the other binding elements by a flexible linker and contributes slightly less than 40% of the contact surface area (374 Å2), mainly from Lys473, Tyr475, Pro476, and Ser477 (Fig. 3A). Although Lys478 does not make extensive contact with the EVH1 surface, it is positioned by the WIP-(475-478) helical turn to form a salt bridge with Glu90 of N-WASP (Fig. 3E).

Binding of Individual WIP Epitopes to N-WASP—The NMR structure and 15N-1H NOE values (Fig. 2) show that WIP WBD epitopes 1 and 2 form a well defined structural unit connected by a cis-peptide bond and single helical turn, whereas epitope 3 is connected by a flexible linker that does not contribute to the binding interface. Our previous studies showed that WIP peptides containing either epitope 1 (22) or both epitopes 2 and 3 (19) could bind the N-WASP EVH1 domain. Because epitope 3 contributes more than one-third of the total WIP·EVH1 binding surface, it seemed possible that it could bind as an isolated unit as observed for epitope 1. We used GST pulldown experiments to compare the binding of four WIP fragments from the WBD to the N-WASP EVH1 domain (Fig. 4, A and B). As demonstrated previously, WIP fragments A (residues 451-461) and B (461-485) bind strongly, but fragments C (459-472) and D (471-485), corresponding to epitopes 2 and 3 in isolation, do not. Thus, despite contributing a smaller portion of the total interaction surface, epitope 1 binds the N-WASP EVH1 domain independently, whereas epitopes 2 and 3 generate a stable N-WASP·WIP complex only when linked.

Multiple WIP WBD Epitopes Are Required for N-WASP Binding in Vivo—WIP-(451-485) corresponds to a region of high sequence conservation in the verprolin family (Fig. 4C), and residues throughout the WIP WBD form specific N-WASP contacts. However, our in vitro binding results suggest that some of those contacts may be functionally redundant. To determine whether the entire WIP WBD is necessary for N-WASP binding in the cell, we mutated key conserved residues from each WIP epitope and measured the functional impact of WIP WBD overexpression on vaccinia actin tail formation. Recruitment of N-WASP to vaccinia is mediated by WIP binding to the EVH1 domain, and expression of the intact WIP WBD efficiently disrupts N-WASP recruitment and blocks actin tail formation (29). As shown previously (22), simultaneous alanine substitution for Phe454 and Phe456 eliminates almost completely the ability of WIP WBD to block actin tail formation (Fig. 4D). Likewise, substitution of Pro465, which contributes more than any other individual WIP residue to the binding interface, has the greatest impact in the actin tail formation assay. In contrast, substitution of Asp461 or Leu462, which connect epitopes 1 and 2, has only a modest effect. This demonstrates that the binding interactions of WIP WBD epitope 1 are not sufficient for the formation of a functional N-WASP·WIP complex. Similarly, the K478A substitution in epitope 3 diminishes the inhibition of actin tail formation by 50% relative to the wild-type WIP WBD. Although this effect is less dramatic than observed for P465A, it suggests that functional N-WASP·WIP complexes require contributions from all three WIP WBD epitopes (Fig. 4E).


Figure 3
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  		FIGURE 3.
Distinct epitopes in the N-WASP binding domain of WIP. A, stereoview of the exWIP·EVH1 complex. Epitopes correspond to WIP amino acids 454-459 (1, magenta), 461-468 (2, gold), and 473-478 (3, blue). The percentage of total N-WASP contact surface area is plotted for each WIP residue. B, NOE contacts between the aromatic WIP residues Phe454 and Phe456 (magenta) and Ala119 (cyan) from N-WASP. C, the Phe454 and Phe456 phenyl rings give rise to new NOEs and induce dramatic chemical shift changes in the Ala119 1Hbeta ({Delta}{delta} = 1 ppm) and 13Cbeta ({Delta}{delta} = 0.5 ppm) resonances in the exWIP·EVH1 complex. D, NOEs between the Trp54 indole NH of EVH1 and the WIP 462LPPP465 polyproline helix (blue contours) are significantly weaker in the exWIP·EVH1 complex (gold contours). The epitope 2 polyproline helix is unwound and is translated slightly toward the WIP amino terminus in the exWIP·EVH1 structure (gold) relative to the original WIP·EVH1 complex (light blue). E, WIP epitope 3 (474YPSK477) makes a conserved salt bridge to N-WASP residue Glu90.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
N-WASP promotes the formation of branched actin filaments by activating the Arp2/3 complex. Autoinhibited N-WASP (2) is typically found in a complex with WIP, a member of the verprolin family (14). A conserved proline-rich domain near the WIP carboxyl terminus stabilizes N-WASP by binding its amino-terminal EVH1 domain (6, 15). Activation of N-WASP requires Cdc42 binding to relieve inhibitory intramolecular interactions and may also involve disruption of the WIP·N-WASP complex by Toca-1 binding (15) or PKC{theta}-mediated WIP phosphorylation (6). Although WIP plays an important role in WASP/N-WASP function, many details about this complex remain obscure.


Figure 4
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  		FIGURE 4.
Mutations in each epitope of the WIP WBD disrupt N-WASP binding. A, schematic of human WIP and epitope fragments used in GST pulldown experiments with N-WASP EVH1 domain. B, Coomassie-stained gel showing that the EVH1 domain of N-WASP binds to GST·peptide fusions corresponding to WIP fragments A and B (but not C or D). C, sequence conservation in the WBD of the human verprolin family. Conservative substitutions are indicated by gray shading. D, vaccinia-induced actin tail formation in cells overexpressing the WIP WBD and mutant WBD constructs. E, mutations that impair the ability of WIP WBD to block vaccinia-induced tail formation are highlighted on the NMR structure.

 
We previously showed that the WIP polyproline motif binds N-WASP in the opposite direction relative to other EVH1·peptide complexes (19). Specific interactions between WIP residues that are carboxyl-terminal to the polyproline motif (473KSYPSK478) and the EVH1 domain were thought to specify the reversal of the peptide ligand orientation. Here we find that conserved WIP residues amino-terminal to the proline-rich sequence make specific EVH1 contacts in an elongated version of the binding site observed in the original complex (Fig. 3). A similar example of a scaffolding interaction with bidirectional binding of composite linear motifs was observed recently for complexes that regulate mitogen-activated protein kinase pathways in yeast (30). These structures demonstrate how semi-independent linear recognition motifs (31) can be used to make composite recognition motifs that are still recognized in an extended conformation with extremely high specificity.

These three WIP epitopes comprise an ~30-residue conserved WASP binding domain in the verprolin family (Fig. 4C). Although our in vitro WIP binding studies showed that peptides corresponding to epitope 1 alone (Fig. 4B) or to epitopes 2 and 3 (19) could bind N-WASP, other reports suggest that verprolins require the entire WBD to form functional complexes in vivo. For example, mutations in epitope 1 disrupted WIP·WASP binding (22), and deletion of epitopes 2 and 3 abolished WIP binding to WASP in NK cells (32). Likewise, mutations in WIRE of either the conserved aromatic residues of epitope 1 or the polyproline motif of epitope 2 lost the ability to localize WASP to filamentous actin structures at the cell edge (23). Using our knowledge of the three-dimensional structure, we tested this hypothesis by separately mutating conserved residues in each epitope of the WIP-EVH1 interface. Disruption of any one of the three epitopes reduced the ability of the WBD to compete for N-WASP binding in a vaccinia actin tail formation assay (Fig. 4D).

In lymphocytes, more than 95% of WASP is bound to WIP (6). Most of the mutations associated with Wiskott-Aldrich syndrome map to the EVH1 domain of WASP (16, 33) and probably disrupt its ability to bind WIP (19). Interestingly, mutations that cause the most severe disease in WAS patients also result in the greatest reductions in WASP levels in circulating platelets, even though WASP mRNA levels remain normal in each case (16). Lutskiy et al. (16) therefore conclude that WIP plays a WASP-protective role in leukocytes and that disesase-causing mutations lead to degradation of the WASP protein and profound cytoskeletal defects. A similar interdependence of WIP-WASP protein levels was also recently observed in Caenorhabditis elegans (34).

Patients with WAS display a range of symptoms that typically progress to severe immune dysfunction because of impaired leukocyte chemotaxis and defects in T and natural killer cell engagement with target cells (35). WIP also binds CrkL and is thought to link T cell receptor ligation to WASP activation through assembly of a ZAP-70·CrkL·WIP·WASP complex at the immunological synapse (6). Likewise, Tsuboi (9) recently demonstrated that interactions between WASP and WIP (or WIRE) are essential for monocyte chemotaxis. Whether WIP simply preserves intact WASP at sufficient levels or contributes directly to key signaling events, inflammation and other immuoregulatory processes dependent on lymphocyte activation and leukocyte migration are clearly compromised in WAS patients because of disruption of this complex.

Our previous structural study correlated WAS mutations with the WIP binding surface or core residues that might disrupt the tertiary structure of the EVH1 domain (19). WASP residues at the interface with epitope 1 (e.g. Val42 and Ala119) might be expected to correspond to additional disease-causing mutations that were previously unexplained, because they could interfere with the binding of WIP residues 451-460. No missense mutations for residues making direct contact with epitope 1 have been identified, but a cluster of mutations including Phe118, Glu121, and Glu123 surrounds Ala119 near the top of the {alpha}-helix. Further down the helix, WASP mutations A134T and R138P (N-WASP residues Ala124 and Arg128) were shown to disrupt WIP-WASP binding (36). These substitutions may alter the epitope 1 binding surface by repositioning the helix and the packing of Ala119 between WIP residues Phe454 and Phe456 (Fig. 3B).

Recently identified WAS missense mutations at positions Trp64 and Glu100 (Trp54 and Glu90 in N-WASP numbering) correspond to specific WIP·EVH1 interactions in epitopes 2 and 3, respectively (37). The W64R mutation would disrupt packing of the WIP polyproline motif (Fig. 3D), and it was correlated with a severe WAS phenotype. The E100D substitution associated with mild disease would likely weaken the conserved salt bridge formed with between Glu90 of N-WASP and Lys478 of WIP, as illustrated in Fig. 3E.

WIP and N-WASP are key regulators of the actin cytoskeleton. Their interaction is required for proper signal transduction in multiple cellular contexts. Our results define the recognition interface for functional complexes between WIP and WASP/N-WASP. Each of three adjacent sequence epitopes, conserved across the verprolin family, makes key contacts with the amino-terminal EVH1 domain of WASP/N-WASP. Based on our measurements using specific WIP mutants and analysis of disease severity associated with specific WASP mutations, we conclude that disruption of any of the three binding epitopes will likely result in the loss of WASP protein and defects in hematopoietic cell signaling associated with Wiskott-Aldrich syndrome.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 2IFS) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

Coordinates and related data (accession number 15020) have been deposited in the Biological Magnetic Resonance Bank.

* This work was supported by National Institutes of Health Grants GM55940 and GM62583 (to W. A. L.), the Packard Foundation (to W. A. L.), and Cancer Research UK (to M. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226. Tel.: 414-456-8400; Fax: 414-456-6510; E-mail: bvolkman{at}mcw.edu.

2 The abbreviations used are: WAS, Wiskott-Aldrich syndrome; WASP, Wiskott-Aldrich syndrome protein; N-WASP, neuronal WASP homolog; WIP, WASP-interacting protein; EVH1, enabled/VASP homology-1; WBD, WASP binding domain; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect enhancement spectroscopy; HSQC, heteronuclear single quantum coherence; r.m.s.d., root mean square deviation. Back


   ACKNOWLEDGMENTS
 
We thank Paulette Hayes and Kelly Kjer for assistance with protein expression.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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