New Structural Insights into Formation of the Key Actin Regulating WIP

Articles Cite This: ACS Chem. Biol. XXXX, XXX, XXX−XXX

New Structural Insights into Formation of the Key Actin Regulating WIP-WASp Complex Determined by NMR and Molecular Imaging Adi Halle-Bikovski,†,§ Sophia Fried,‡,§ Eva Rozentur-Shkop,† Guy Biber,‡ Hadassa Shaked,† Noah Joseph,‡ Mira Barda-Saad,*,‡,§ and Jordan H. Chill*,†,§ †

Department of Chemistry, and ‡Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat Gan, 52900, Israel S Supporting Information *

ABSTRACT: Wiskott−Aldrich syndrome protein (WASp) is exclusively expressed in hematopoietic cells and responsible for actin-dependent processes, including cellular activation, migration, and invasiveness. The C-terminal domain of WASp-Interacting Protein (WIP) binds to WASp and regulates its activity by shielding it from degradation in a phosphorylation dependent manner as we previously demonstrated. Mutations in the WAS-encoding gene lead to the primary immunodeficiencies Wiskott−Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT). Here, we shed a first structural light upon this function of WIP using nuclear magnetic resonance (NMR) and in vivo molecular imaging. Coexpression of fragments WASp(20−158) and WIP(442− 492) allowed the purification and structural characterization of a natively folded complex, determined to form a characteristic pleckstrin homology domain with a mixed α/β-fold and central two-winged β-sheet. The WIP-derived peptide, unstructured in its free form, wraps around and interacts with WASp through short structural elements. Förster resonance energy transfer (FRET) and biochemical experiments demonstrated that, of these elements, WIP residues 454−456 are the major contributor to WASp affinity, and the previously overlooked residues 449−451 were found to have the largest effect upon WASp ubiquitylation and, presumably, degradation. Results obtained from this complementary combination of technologies link WIP-WASp affinity to protection from degradation. Our findings about the nature of WIP·WASp complex formation are relevant for ongoing efforts to understand hematopoietic cell behavior, paving the way for new therapeutic approaches to WAS and XLT.

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the C-terminal domain of WASp to activate the Arp2/3 complex and trigger actin polymerization.12 The second phosphorylation at position S488 in the EVH1-binding Cterminal region of WIP (WIPC) is mediated by protein kinase C theta (PKCθ), uniquely expressed in T-cells.13,14 This phosphorylation is known to induce a rearrangement of the interaction surface between WASp and WIP, regulating WASp activity.14,15 A recent study showed that S488 phosphorylation triggers a dissociation of the WIP EVH1 binding domain from WASp while leaving intact the actin-mediated second interaction between the actin binding domains at the WIP Nterminus and the WASp C-terminal region.14 The outcome of this partial dissociation is two-fold: (i) activation of WASp and initiation of actin polymerization mediated by the Arp2/3 complex and (ii) cellular ubiquitylation of WASp at two lysine residues, K76 and K81, leading to proteasomal degradation.2,9 Therefore, activation and degradation of WASp are delicately balanced and controlled by WIP as the key switch in the life cycle of T cells. Perturbation of the direct WIP−WASp interaction is involved in the hereditary diseases Wiskott

he Wiskott−Aldrich Syndrome protein (WASp), responsible for cytoskeleton rearrangement upon activation, is a 502-residue polypeptide exclusively expressed in hematopoietic cells. WASp-interacting protein (WIP), the WASp cellular binding partner, is a 503-residue widely expressed member of the mammalian verprolin family consisting of WIP-homologues CR16 and WIRE/WICH. Its C-terminal domain contains a central multiepitope WASp-binding domain which interacts with the N-terminal Wiskott homology-1 domain (WH1, or Enabled/VASP homology-1 [EVH1]) of WASp (Figure 1).1−3 WIP binding to WASp, a central activator of the actinnucleating complex Arp2/3, regulates its cellular distribution and function,2,4−6 as well as its stability by protecting it from ubiquitylation-triggered proteasomal degradation.2,7−9 In resting cells, an estimated 95% of WASp is complexed with WIP.7 Functional levels of WASp were reduced in WIP-deficient mice, whereas WASp mRNA levels were unaffected. Furthermore, WASp activity could be rescued by exogenous addition of WIP.8,10,11 Therefore, this “chaperone” function of WIP is essential for WASp stability. Two phosphorylation events are involved in regulating WASp activity. Phosphorylation at position Y291 of the WASp GBD site releases WASp from an autoinhibitory conformation (in which the WASp C-terminal region is occluded) and allows © XXXX American Chemical Society

Received: June 13, 2017 Accepted: November 13, 2017

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DOI: 10.1021/acschembio.7b00486 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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ACS Chemical Biology

Figure 1. Molecular model of the WIP·WASp complex and activation-induced conformational changes. Above, WASp associates with WIP via two separate molecular interfaces (see text). Phosphorylations occur at residue Y291, induced by TCR activation, and at residue S488, which stimulates the partial dissociation of WIP and WASp. Below, amino acid sequence of the WASp-binding domain of WIP, highlighting known binding epitopes and compared to sequences of previous studies. Dotted lines indicate segments in previous studies that could not be observed by NMR.

down cells exhibits no reduction of N-WASp levels, suggesting distinct modes of interaction between WIP and the two WASpfamily members.11 Thus, additional information is needed to enhance our structural understanding of the complex formed between WASp and WIP in hematopoietic cells. Here, in a detailed study of the complex formed between the WASp EVH1 domain and an extended WIP-derived peptide, we address these structural and biological questions. Coexpression of these two polypeptides resulted in a high-affinity and biologically relevant complex amenable to NMR studies. The NMR-based global fold of this complex identified previously unrecognized segments of both proteins contributing to their biological function and mapped the binding interface corresponding to the longer WIP peptide. Binding to WASp induces a conformational change in WIP442−492 from a disordered polypeptide to a sequence of accurately placed structural elements that wrap around WASp and maximize their binding affinity. In addition, the effects of WIP mutants upon complex affinity demonstrated a strong correspondence with both cellular colocalization of the two proteins and ubiquitylation-driven degradation. The implications of our findings upon the biology of the WIP·WASp interaction and the molecular basis of WAS pathophysiology are discussed.

Aldrich Syndrome (WAS) and X-linked thrombocytopenia (XLT),16,17 characterized by an impaired immune response leading to recurrent infections, autoimmune diseases, and hematopoietic malignancies.18,19 WAS and XLT are mainly caused by EVH1 mutations that reduce its affinity to WIP,20,21 leading to an untenable increase in WASp degradation. Furthermore, homologues of WASp are overexpressed in various malignancies, such as melanoma22 and lung23 cancers. These examples highlight the need for a better understanding of the WIP−WASp interaction under homeostasis and in disease. EVH1 domains are members of the Pleckstrin homology (PH) superfamily, a wide-ranging class of mixed-α/β fold proteins involved in binding inositol lipids, phosphotyrosines, and polyproline sequences.24 Within this superfamily, EVH1 domains have evolved as polyproline-sequence binders typically located at the N-terminus of proteins. Polyproline recognition is mediated by a groove formed by β-strands 1, 2, 6, and 7 and an interaction with a highly conserved tryptophan residue.25−27 The structures of several EVH1 domains have been solved, and all exhibit close similarities.26,28−32 Specifically, an investigation of a complex between murine N-WASp, a WASp homologue, and WIP-derived peptides described some of the salient structural features of the complex.32,33 WIP residues 451−485 were shown to wrap around N-WASp and form an extensive binding interface through three distinct interaction regions.33 The WIP peptide orientation was directionally reversed (from N-to-C) in comparison to other EVH1 polyproline ligands.27,32 However, the WIP peptide failed to include functional segments such as the PKCθ phosphorylation motif and an Nterminal segment (residues 446−451) of biological importance.34 Also, the two proteins could be expressed only as a single polypeptide tethered by a flexible linker, suggesting the constrained positioning of the peptide in relation to N-WASp may influence the structure. Finally, despite the obvious similarities between hematopoietic WASp and N-WASp, they may interact differently with WIP. A report of WIP knock-

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RESULTS AND DISCUSSION NMR-Determined Global Fold of the WASp EVH1 Domain. As mentioned previously, the N-WASp EVH1 domain is poorly expressed as an independent polypeptide, since properly positioned covalently tethered WIP peptides were needed to avoid its aggregation and subsequent precipitation.27,32 To avoid detrimental effects of this covalent tethering on the complex structure, we coexpressed an Nterminal fragment of WASp containing the EVH1 domain (residues 20−158) with a peptide spanning residues 442−492 of WIP, resulting in a soluble WIP442−492·WASp20−158 complex with satisfactory yields (Supporting Information, Figure S1). SDS-PAGE analysis (data not shown) established that B


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Figure 2. NMR characterization and assignment of the WIP442−492·WASp20−158 complex. (A) 1H,15N-HSQC spectrum of 0.5 mM 13C,15N-labeled complex with annotations indicating the residue assignments of cross-peaks. Peaks lacking annotations are Arg/Asn/Gln/Trp side chain 1H15N groups or a minority (6%) of unassigned backbone amide protons. (B) Circular dichroism curve for a 10 μM sample of the complex under similar conditions exhibiting a mixed α/β fold. (C). TALOS+-derived secondary structure predictions along the WASp20−158 and WIP442−492 sequences delineate the seven β-strands and two helices in the EVH1 domain and secondary structure in bound WIP. Solvent-exchange protected amide peaks are designated by dots.

WASp20−158 coeluted with His6-tagged-WIP442−492 on a Ni2+affinity column and did not dissociate even in 2−4 M urea solution. The 1H,15N-HSQC spectrum of this complex (177 residues excluding purification tags and proline residues) exhibited ∼160 cross-peaks with some overlap, including several well-resolved peaks in the 9−10 1H ppm range consistent with a β-sheet structure and an overlapping central region consistent with α-helical structures (Figure 2A). A circular dichroism (CD) curve of EVH1/WIP was consistent with a polypeptide containing both α-helical segments and βsheet structure. Based on the 190−240 nm region of the curve, the complex was predicted to contain 18 and 37% of residues in α-helical and β-sheet conformations, respectively (Figure 2B). All these suggested that a native high-affinity complex had been formed.

Applying standard multidimensional triple resonance NMR experiments35,36 to uniformly 13C,15N- and 2H,13C,15N-labeled WIP442−492·WASp20−158 samples successfully determined its backbone resonance frequencies. A demonstration of the “sequential walk” using connectivities of adjacent 13Cα and 13 β C resonances to the backbone amides enabling the assignment of each cross-peak to its appropriate residue appears in the Supporting Information (Figure S2). Assignment completeness (submitted to the BioMagResBank database, accession code 27268) was 95% for nonproline 15N nuclei, 93% for 13C nuclei (94% when including the β-positions), and 87% for 1H nuclei (77% when including the β-positions). NMRderived backbone chemical shifts are an excellent predictor of protein secondary structure,37−40 and the assignment data were analyzed using the TALOS+ platform41 to determine helical segments and β-sheets along the WASp EVH1 backbone C


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ACS Chemical Biology (Figure 2C). The EVH1 domain is comprised of seven βstrands spanning residues 43−56, 63−66, 71−76, 82−88, 95− 101, 113−118, and 124−128 (designated β1−β7), which are flanked by long α-helices spanning residues 30−38 (α1) and 131−149 (α2). Chemical shifts along the longest β-strand were consistent with a backbone kink at residue V50 that splits β1 in two, β1N (43−49) and β1C (51−56). The global fold was deduced from the exposure of amide protons to a solvent exchange experiment, indicating inner and outer strands (Figures 2C and 3) and 4−7 proton−proton connectivities

important in the complex between DOCK180 and the ELMO PH domain.45 Binding to WASp Triggers a Conformational Change in WIP442−492. We had previously shown residues 407−503 of free WIP to be intrinsically disordered with some structural propensity in the core WASp-binding segment.34 Focusing on the conformation of bound WIP442−492, we compared its secondary chemical shifts in its free state and in complex with WASp20−158 under identical sample conditions. To facilitate this comparison, we prepared a complex in which WIP442−492 was selectively labeled with NMR-isotopes 15N and 13C while WASp20−158 remained unlabeled. This was achieved by producing an unlabeled weaker mutant complex (mutation at WIP residues 475−477, vide inf ra) followed by competitive titration with wildtype doubly labeled 13C,15N-WIP442−492 and purification of the stronger complex by size-exclusion chromatography. This revealed conformational changes resulting from the interaction of WIP442−492 with the EVH1 domain. The fingerprint HSQC spectrum of free WIP442−492 exhibits the poor spectral dispersion typical of an unstructured polypeptide but in complex with WASp20−158 behaves as a well-folded protein (Figure 4A). WASp-induced resonance perturbations

Figure 3. Determination of the WIP442−492·WASp20−158 global fold. Left, overlay of 1H,15N-HSQC spectra of the WASp20−158·WIP442−492 complex before (gray) and 24 h after (red) exposure to 2H2O. As an example, residues of the fully protected β3 strand are annotated. Right, schematic topology of the global fold of WASp20−158 (the EVH1 domain). β-strands (light gray) and helices (dark gray) are labeled according to the text. Amide protons protected from exchange as seen in the HSQC spectrum after exposure to 2H2O are shown as short arrows in the direction of the amide bond.

Figure 4. WASp-induced changes in WIP conformation. (A) Comparison of 1H−15N-HSQC spectra for free (red) and complexed (black) WIP442−492, demonstrating the change to a structured conformation. (B) Top, chemical shifts changes along the WIP442−492 sequence induced by binding to WASp20−158, expressed as ΔHN = (ΔH2 + (ΔN/5)2)1/2, where ΔH and ΔN are the individual changes in 1H and 15N shifts, respectively. TALOS-based predictions for helical (middle) and β-strand (bottom) content are shown for bound and free WIP442−492 in dark gray and red bars, respectively.

between each two adjacent β-strands derived from a 15N-edited NOESY-HSQC experiment (Figure 3). All available data point to a model in which the seven β-strands are arranged in two βsheets, one involving β1N−β3−β4−β5 and the other involving β2−β1C−β7−β6. The kinked β1 (supported also by the NOESY data) anchors the two β-sheets in position, forming the characteristic β-sandwich fold. In addition, extreme solvent exchange protection (observable in 2H2O after 24 h) for the amide protons of α2 residues A134, F137, and V141 indicated a packing of the hydrophobic surface of this helix against the βsandwich structure. This “capping” of the winged fold by the Cterminal helix is a hallmark of EVH1 domains and their parent superfold pleckstrin homology (PH) domain and reminiscent of other available structures,24,26,28−31 including the aforementioned N-WASp.32,33 In contrast, the N-terminal helix (residues E31NQRLFE37) has not been reported to date in the polyproline-binding EVH1 family. It is, however, a known feature of PH domains, as seen (among others) in residues D 1 8 LQALLK 2 4 of phospholipase C 4 2 and residues A71DEEAVR77 in the related Drosophila phosphotyrosine binding domain Numb.43 Sequence similarity between these three homologous N-terminal segments hints to the presence of an amphiphilic helix at the final four residues. Interestingly, N-WASp residues E23NESLFT29 preceding the reported structure conform to the same pattern, suggesting that it too possesses such an N-terminal helix that was not included in the earlier structural work. Finally, bacterial homologues of PH domains exhibit a homologous N-terminal helix,44 and a similar motif was reported and determined to be functionally

span WIP residues 449−488, suggesting this is the segment involved in the complex binding interface (Figure 4A). Significantly, this extended binding segment now includes a fourth previously overlooked binding epitope (residues 449− 452) as well as the PKCθ phosphorylation site, consistent with earlier findings14 that this WIP segment is proximal to the EVH1 domain in the WIP-bound inhibited state of WASp. Whereas unbound WIP442−492 shows only marginal secondary structure, WASp induces a folding of WIP into a helical conformation at residues 449−452, 458−461, and 475−480, and an extended β-like conformation at residues 454−456 and 468−474 (Figure 4B). The latter change is particularly striking in comparison to the disorder observed for these residues in free WIP442−492 and consistent with a rigidification of the 462− 465 and 474−478 binding segments upon interaction with the EVH1 domain. Overall, the TALOS+ predictions for secondary structure in the complex, 23 and 31% for α-helical and β-sheet structure, respectively, are in relatively good agreement with the CD results. Probing the Contribution of WIP Binding Epitopes to Its Biological Function. Within the aforementioned WASpinteracting WIP segment, four binding epitopes can be D


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ACS Chemical Biology identified:33 (i) the extended segment F454YFHPI459, (ii) the polyproline motif L462PPP465, (iii) residues K473SYPSK478, and (iv) a novel binding epitope involving WIP residues 449−452. To better understand how these epitopes cooperate to perform the chaperone function of WIP, we mutated key residues from each WIP epitope (Table 1) and transfected WASp-expressing Table 1. WIP Mutants Used to Investigate Its WASp-Binding Chaperone Function mutant WIP449−451 WIP454−456 WIP463−465 WIP475−477

binding epitope 448

DEWES452 454 FYFHPIS460 462 LPPPEP467 473 KSYPSK478

mutated residues

mutation

449−451 454−456 463−465 475−477

EWE → GSG FYF → GSA PPP → APA YPS → GGA

cell lines with mutant WIP-encoding plasmids. This enabled us to assess the effects of various WIP mutations upon cellular behavior using both biochemical and molecular imaging analyses of the WIP/WASp interaction in cellular settings. The utility of this approach was demonstrated by immunoprecipitation comparison of binding to WASp by wildtype (WIPwt) and mutant WIP variants in lysates of cells expressing YFPtagged WIP wt or the indicated mutant WIP variants. WIPwt showed a high level of precipitated WASp as expected,14 and a similar level of precipitation was observed for WIP475−477. In contrast, a substantial reduction in precipitated WASp was observed for the other three WIP mutants (Figure 5A). These results suggest that these mutated residues are critical for the interaction of WIP with WASp and that our cellular approach can be used to address the importance of WIP epitopes to inhibitory colocalization with WASp prior to phosphorylationtriggered activation. FRET Analysis of Epitope Contribution to the WIP442−492·WASp20−158 Complex. Since the biochemical approach described above is limiting on the temporal level and in sensitivity, we employed a Förster resonance energy transfer (FRET)-based molecular imaging approach that enables us to follow the dynamics of the WIP−WASp interaction within their cellular context. Using the previously developed FRET-based assay,14 cells stably expressing CFPWASp were transfected with plasmids encoding YFP-tagged wildtype or mutant WIP (in both cases, the fluorescent tag is tethered to the protein N-terminal end), and cells were plated over stimulatory coverslips (coated with an anti-CD3 antibody) and fixed after 2 min of activation (Figure 6). Signaling clusters were recruited to the stimulatory plane for all five forms of WIP (Figure 6A). A high average FRET efficiency was observed after 2 min of activation between CFP-WASp and either YFP-WIPwt (30.4 ± 2.6%) or YFP-WIP475−477 (25.9 ± 2.5% P = 0.23), indicative (based upon previous results14) of a native WIP− WASp interaction. In contrast, the FRET efficiency was significantly reduced for both YFP-WIP449−451 and YFPWIP463−465 (16.9 ± 3.3% and 14.8 ± 3.6%, P ≤ 0.008 and P ≤ 0.001, respectively) and dramatically so for YFP-WIP454−456 (3.7 ± 2.1%, P ≤ 0.001; Figure 6A,B). Thus, WIP449−451, WIP454−456, and WIP463−465 have important roles in the formation and stabilization of the WIP·WASp complex. Effects of WIP Mutations upon WASp Ubiquitylation. To address the contribution of WIP epitopes to the shielding of WASp from degradation, we performed a modified immunoprecipitation experiment. Cells were treated with WIP 3′ UTRspecific siRNA, thus silencing endogenous WIP expression

Figure 5. WASP binding and WASP stability affected by WIP mutations. (A) Top, Jurkat E6.1 cells expressing YFP-tagged wildtype or mutant WIP (see Table 1) were stimulated with anti-CD3 and anti CD28 antibodies before being subjected to immunoprecipitation (IP) with an anti-GFP antibody (exhibiting reactivity with YFP). Samples were analyzed by Western immunoblotting (IB) with the indicated antibodies. Blots are from one experiment representing three independent experiments. Bottom, whole-cell lysates before immunoprecipitation were analyzed by Western blotting with anti GFP and anti-GAPDH (as loading control) antibodies. (B) Top, Jurkat E6.1 cells expressing YFP-tagged wildtype or mutant WIP were treated with WIP 3′ UTR-specific siRNA to gene silence specifically the endogenous WIP (without affecting the exogenous WIP). Cells were stimulated with anti-CD3 and anti-CD28 antibodies and immunoprecipitated with anti-WASP antibodies. Samples were analyzed by Western blotting with anti-ubiquitin (Ub) and anti-WASP antibodies. Bottom, whole-cell lysates before immunoprecipitation were analyzed by Western blotting with anti-WIP and anti-GAPDH antibodies.

without affecting the expression levels of YFP-WIPwt or the mutant variants. Immunoprecipitation of WASp was performed in cells expressing YFP-WIPwt after activation and followed by Western immunoblotting (IB) analysis with anti-WASp or antiubiquitin antibodies. WASp degradation (known to be ubiquitylation-dependent as previously described9) was enhanced in cells expressing all mutated WIP forms. As the typical striated pattern representing massive ubiquitylation demonstrated, WIP449−451 exhibited a dramatic increase in ubiquitylation, and a smaller yet significant increase was seen for WIP454−456 (Figure 5B, top). These results clearly indicate that disruption of the WIP−WASp interaction by mutations in WIP is correlated with and crucially impacts WASp ubiquitylation. Since WASp ubiquitylation leads to its degradation via the proteasome pathway,9 we can conclude that WIP residues that are critical for its binding to WASp directly affect its stability and degradation. Mapping of the WIP442−492 Binding Interface upon WASp20−158 by NMR. To place these biological implications of WIP mutants in their structural context, we followed mutationinduced structural variations in the WIP442−492·WASp20−158 complex using NMR chemical shift perturbations in the fingerprint 1H,15N-HSQC spectra. Strikingly, although all four E


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insufficient to protect WASp from aggregation or degradation. In the remaining three coexpressed mutant complexes, we compared chemical shifts in the fingerprint spectrum between the wildtype WIP442−492·WASp20−158 complex and the analogous complexes formed with mutant WIP polypeptides. The majority of observed changes in WIP resonances (excluding the mutated residues themselves) were minor in comparison to those seen for free WIP, suggesting that complex stability was maintained (Figure 7). When considering significant changes in WASp resonances, WIP449−451 caused shifts in the α1 helix and the β4/β5 loop, and WIP463−465 caused shifts centered on residue W64 in the β2 strand and the β6/β7 linker and β7 strand. WIP475−477 was responsible for the broadest distribution of spectral changes, inducing significant changes in the β1/β2 linker, the β4 and β5 strands, the β5/β6 loop, and β6/β7 turn (Figure 7). On a qualitative level, these changes identify interaction surfaces in the WIP·WASp complex, suggesting that residues 449−451 contact the α1 helix and the first β-sheet (β1N−β3−β4−β5), residues 463−465 contact the second βsheet (β2−β1C−β6−β7), and residues 475−477 and the preceding linker residues wrap around the β-sandwich cleft. Combined with the earlier secondary and tertiary structural characterization, overall the data suggest a rudimentary model for the WIP·WASp complex, in which the WIP polypeptide wraps around the double-winged structure of the EVH1 domain, interacting significantly with both halves of WASp (Figure 8). This is consistent with the binding mode of other polyproline motifs with EVH1 homologues. A key β2 Trp residue is present in the polyproline binding sites of Mena (W23),28 Homer (W24),30 and N-WASp (W54),32 which together with homologous residues on the β1C strand (M14/ Y16, F14/I16, Y46/A48, respectively) and β6 strand (F77/ Q79, F74/Q76, F104/T106, respectively) form the groove in which the polyproline helix resides. In addition, the direction of the polyproline helix within this groove echoes that reported earlier for N-WASp.32 A Combined Structural and Cellular View of WIPMediated Chaperoning of WASp. A significant strong point of our approach is the synergistic combination of structural NMR and cellular molecular imaging methods aimed at elucidating the nature of the WIP·WASp complex. Since effects of PKCθ phosphorylation upon this complex have been previously described,14 here we focused on the contribution of four WIP epitopes upon complex formation. Both immunoprecipitation and FRET identified residues 454−456 as most important for stabilization of WASp and residues 449− 451 and 463−465 as major contributors to the interaction. Thus, the WIP−WASp interaction in lysates and in live cells exhibited an identical pattern, ranking the epitopes in terms of binding contribution as epitope 454−456 > epitope 449−451 ∼ epitope 463−465 > epitope 475−477. In agreement with this ranking, mutation of residues 454−456 entirely abolished bacterial expression of the complex. On the structural level, residues 463−465 exhibited interactions with residues from the β2 and β6 strands, consistent with the location of the polyproline-segment binding groove in other EVH1 domains,24,26,28−31 and mutation of residues 449−451 resulted in a similar intensity of resonance perturbations. Mutating residues 475−477 effected changes at WASp residues 98−107, consistent with the reported WIP(K478)−N−WASp(E100) salt bridge.32 The widespread WIP475−477-induced HSQC changescontrary to other mutants, also quite significant in WIP itselfappear to contrast with the relative insensitivity of

Figure 6. Structural modification in the WIP·WASP complex caused by WIP mutants. (A) Jurkat E6.1 cells expressing CFP-WASP were transfected with plasmids encoding wt or mutant YFP-WIP (see Table 1) and were plated over a stimulatory coverslip coated with anti-CD3 antibodies and were fixed after 2 min of activation (n = 25 cells). Cells were imaged by confocal microscopy, and FRET efficiency was measured by the donor-sensitized acceptor emission technology (see Materials and Methods for details). (B) Graph summarizing the percentage of FRET efficiency in cells overlaid on the indicated coverslips. Data are means ± SEM from at least three independent experiments. P values were calculated by a two-tailed Student’s t test and are indicated therein.

WIP mutants exhibit similar expression levels as independent polypeptides, of these, WIP454−456 abrogated the expression of the complex, leading us to conclude that WIP454−456 affinity is F


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Figure 7. Chemical shift perturbation analysis of WIP mutants. Changes observed in the 1H−15N-HSQC spectra of 15N-labeled complex acquired at 303 K and 16.4 T for mutant WIP peptides. Changes are expressed as ΔHN = (ΔH2 + (ΔN/5)2)1/2, where ΔH and ΔN are the individual changes in 1H and 15N shifts, respectively. Regions of significant change (defined as ΔHN > 0.15 ppm for at least two residues) are highlighted in color according to secondary structure elements. Filled diamonds (right-hand panels) indicate five residues around the mutation site that are excluded from the analysis. Results are shown for the WIP449−451 (top, red), WIP463−465 (middle, green), and WIP475−477 (bottom, blue) peptides.

increase when residues 449−451 were mutated. Since we could not attribute this solely to the affinity of the complex (e.g., residues 454−456 contribute more to the strength of the complex), the explanation appears to lie on the functional level. Thus, the N-terminal segment of WIP442−492 appears to play an important role in shielding WASp from ubiquitylation. Considering this on the structural level, WIP residues 449− 451 must inhibit one of the contributing interactions involved in ubiquitylation, namely binding of the E3 ligase, noncovalent binding of ubiquitin itself,46 or the actual ubiquitylation site. HSQC changes associated with this mutant were localized to the α1 helix and the β4/β5 loop on the backside of the first βsheet. On the basis of the canonical structure of EVH1 domains, this site does not appear to coincide with the residues proven to undergo ubiquitylation, K76 and K81,14 located in the β3/β4 loop, or with the characteristic ubiquitin-interacting surface of PH domains located on the opposite β-sheet. It is therefore possible that the 449−451 epitope interferes with binding of the ligase to WASp. However, WIP residues preceding our 442−492 segment may also contribute to the shielding of the ubiquitylation sites, and therefore further structural studies are required to confirm this hypothesis and illuminate the molecular basis of inhibition of ubiquitin conjugation by WIP. Concluding Remarks. The interaction between WASp and its chaperone WIP is a pivotal junction in T cells determining the relative rates of WASp release from its autoinhibitory conformation and eventual degradation, controlling actin polymerization and cellular activities including transcription activation, proliferation, migration, and invasiveness. The harmful effects of both reduced levels (due to degradation) and increased levels (due to overexpression) of WASp highlight the crucial role of WIP in hematopoietic cell homeostasis. This was the underlying rationale of the current investigation, aimed at understanding the WIP−WASp interaction on the molecular level using a highly complementary combination of structural and in vivo molecular imaging methods. This first structural

Figure 8. Topological model of the WIP442−492·WASp20−158 complex based on the WASp20−158 global fold and the WIP mutagenesis data. The EVH1 domain is shown in dark (helices) and light (β-sheets) gray; the WIP442−492 backbone in striated burgundy; and the three mutated epitopes, residues 449−451, 463−465, and 475−477, in red, green, and blue, respectively.

biological experiments to this mutation. Notably, K478 was not mutated in this study because of our focus on residues contributing to the buried surface area. Thus, a plausible explanation is that while WIP475−477 binding to WASp is significantly perturbed, the aforementioned salt bridge maintains the WIP−WASp interaction at this contact region, accounting for the biological results. Most intriguing, however, were the results of the ubiquitylation assay identifying how the decrease in complex affinity exposes WASp to tagging with ubiquitin en route to degradation. Although mutations generally increased the levels of ubiquitin tagging, we observed a dramatic ubiquitylation G


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upon reaching an OD600 of 0.8 with 1 mM isopropyl-thio-galactose (IPTG) followed by overnight expression at 27 °C. Harvested cells were lysed by homogenization (C5 homogenizer, Avestin, Ottawa, Canada) in lysis buffer (20 mM NaPi, at pH 7.4, 300 mM NaCl, 10 mM imidazole, 1 mM DTT), and the soluble fraction was applied on a HisTrap Chelating HP column (GE Healthcare, Chicago, IL, USA) charged with Ni2+. The bound protein was eluted using lysis buffer containing 500 mM imidazole and further purified by gel filtration column (Superdex 75 16/60, GE Healthcare) equilibrated in 20 mM NaPi, at pH 7.2, 100 mM NaCl, and 1 mM DTT. Purified WIP442−492 peptide was kept in 10 mM KPi, at pH 7.0, 20 mM NaCl, and 10 mM β-mercaptoethanol and concentrated to 0.4−0.5 mM. Purified complex was kept in 20 mM NaPi, at pH 6.8, 50 mM NaCl, and 1 mM DTT and concentrated to 0.5−0.7 mM. Circular Dichroism Measurements. CD experiments were performed on a Chirascan polarimeter (Applied Photophysics, Surrey, United Kingdom) using a 1 mm path-length quartz cuvette for a 25 μM sample of WIP442−492·WASp20−158 in 20 mM NaPi buffer, at pH 6.8. The experiment was repeated three times and subtracted from a measurement of an identical buffer sample. Results were analyzed using the CDSSTR module of the DichroWeb platform for the 190− 240 nm range.52 NMR Spectroscopy. All 2D- and 3D-NMR measurements were performed on a DRX700 Bruker spectrometer using a cryogenic tripleresonance TCI probehead equipped with z-axis pulsed field gradients. Conditions for all samples were 0.4−0.7 mM of protein in 20 mM NaPi buffer, at pH 6.8, 50 mM NaCl, 1 mM DTT, and 7% 2H2O. Measurements were conducted at 303 K and 16.4 T. 1H,15N-HSQC spectra for sample characterization and for following signal loss during exchange of amide protons in 2H2O-based solvent (as a series of experiments over 36 h) were run for 40 min for sufficient spectral quality. Triple-resonance experiments and acquisition parameters for assignment of backbone nuclei are described in detail in the Supporting Information. Heteronuclear chemical shifts were referenced indirectly against 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS). All spectra were processed using the TopSpin 3.0 package (Bruker BioSpin, Karlsruhe, Germany). Secondary chemical shift analyses were performed using the TALOS+ server.41 Immunoprecipitation and Western Blotting. Immunoprecipitations and Western blot analysis were performed as previously described.9 Relative protein abundance or relative extent of coprecipitated protein was compared to the relevant control. Spreading Assay and Molecular Imaging. Spreading assays were performed as previously described.49,53 Dynamic fluorescent and interference reflection microscopy images were collected on a Zeiss LSM510 Meta confocal microscope. All images were collected with a 63× Plan-Apochromat objective (Carl Zeiss). FRET Analysis, Correction, and Calculation. FRET was measured by the donor-sensitized acceptor fluorescence technique as previously described.49 Briefly, three sets of filters were used, one optimized for donor fluorescence (excitation, 468 nm; emission, 475− 505 nm), a second for acceptor fluorescence (excitation, 514 nm; emission, 530−600 nm), and a third for FRET (excitation, 468 nm; emission, 530−600 nm). FRET was corrected and its efficiency determined as described in detail in the Supporting Information.

view of a hematopoietic WIP·WASp complex improves upon previous work by coexpression and in situ complex formation of the complex, avoiding the need for non-native tethering, and by extending both polypeptide sequences to include previously omitted WIP (N-terminal helical epitope 449−451 and PKCθ phosphorylation site 485−490) and WASp (α1-helix 30−38) segments. The obtained molecular view of these key cellular events is an important first step toward a comprehensive understanding of how WIP and WASp modulate cytoskeletal change with potential impact on therapeutic approaches to immunodeficiencies.

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MATERIALS AND METHODS

Media and Reagents. DNA primers were purchased from Integrated DNA Technologies (Leuven, Belgium). Isotopically labeled chemicals for constructing labeling media, including 2H2O, 13C6labeled glucose, and 15NH4Cl, were purchased from Cambridge Isotope Laboratories (Andover, MA, USA). The Isogro-DCN supplement for triply labeled media was obtained from Sigma-Aldrich (St. Louis, MO, USA). M9 minimal growth medium was prepared as previously described.47 Antibodies and Oligonucleotides. Antibodies and reagents were obtained from the indicated suppliers. The following antibodies were used for imaging and activation: mouse anti-CD3ε (UCHT or HIT3a) and mouse anti-CD28. The following primary antibodies were used for immunoprecipitations and Western blotting: mouse anti-GFP (Roche), mouse anti-WASp D1 (Santa Cruz), rabbit anti-WIP (Santa Cruz), and mouse anti-GAPDH (Biodesign). HRP-conjugated secondary antibodies used include goat anti-mouse (Sigma-Aldrich) and goat antirabbit (Santa Cruz). WIP 3′ UTR specific siRNA duplex was obtained from sigma (CAGGCUAUUGCUUGCUUCA). Expression Vectors and Plasmid Construction. The expression vectors pEYFP-C1, pEYFP-N1, pECFP-C1, and pECFP-N1 were obtained from Clontech, and pcDNA3.1+/Hygro was obtained from Invitrogen. Complementary DNA (cDNA) encoding human WASp was kindly provided by Dr. David Nelson (NCI, NIH Bethesda, MD, USA). Plasmid encoding FLAG-tagged WIP was purchased from Addgene. cDNA’s encoding WIP or WASp were cloned into the expression vectors pECFP or pEYFP to obtain CFP- or YFP-tagged proteins. Aequorea GFP derivatives were rendered monomeric by the A206K substitution, as was previously described.48 WIP point mutations (FYF-GSA 454−456, YPS-GGA 475−477, PPP-APA 463−465, EWE-GSG 448−451) were generated with the Quickchange II XL site-directed mutagenesis kit (Stratagene), and the mutated sequences were introduced into the YFP-WIP construct. All constructs were verified by DNA sequencing. Transfections. Cells were transfected with a Lonza’s Nucleofecter and appropriate Lonza’s solutions. Both transiently transfected T cell cultures, and stable clones were used in this study, as indicated in the figure legends. Transiently transfected cells were used 48 h after transfection. Stable clones were derived from transiently transfected cells with a combination of drug selection and cell sorting. Cells were selected in either neomycin or hygromycin and verified by Western blotting and FACS analysis. Cell Culture. Jurkat E6.1 cells were cultured as described previously.49 The cells were activated with anti-CD3ε antibody (OKT3, 10 μg/mL) and anti-CD28 antibody (10 μg/mL) for 2 min in 37 °C. Expression and Purification of the Complex between WASp20−158 and WIP442−492. Human WIP442−492 protein and its mutants were expressed in a pET28a(+) vector containing a His6-tag using a restriction site-free cloning method.50 After insertion of the WASp20−158 coding sequence into a pACYCDuet-1 expression vector using the transfer-PCR cloning method,51 the WIP442−492·WASp20−158 complex was coexpressed in E. coli BL21(DE3) cells. All samples were expressed in H2O-based M9 minimal medium supplemented with 1 g/L 99% 15NH4Cl and 2.5 g/L 99% 13C6-glucose as appropriate. E. coli BL21 cells were cultured at 37 °C and induced

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.7b00486. SDS-PAGE analysis of WASP/WIP coexpression (Figure S1), spectral strips allowing assignment of the WIP· WASP complex backbone resonances (Figure S2), acquisition parameters for all NMR experiments, and a description of the FRET analysis (PDF) H


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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Jordan H. Chill: 0000-0002-9518-824X Author Contributions §

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank H. Gottlieb and K. Keinan-Adamsky for spectrometer assistance and I. Tabakman for technical assistance. We also acknowledge Y. Peleg (Weizmann Institute, Rehovot, Israel) for advice on coexpression of the WIP−WASp complex. Financial support by the Heritage Legacy fund (Israel Science Foundation award 491/10) is gratefully acknowledged. Establishment of the 700 MHz spectrometer system in the NMR lab was supported by Fundacion Adar and a Converging Technologies award. M.B.-S. acknowledges support of the Israel Science Foundation (award 747/13) and the Chief Scientist Office of the Ministry of Health (award 3-10151). J.H.C. acknowledges support of the Christians for Israel Chair for Medical Research.

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New Structural Insights into Formation of the Key Actin Regulating WIP

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