Structural Insights Into the Specific Binding of Huntingtin Proline-Rich

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provided by Elsevier - Publisher Connector Structure 14, 1755–1765, December 2006 ª2006 Elsevier Ltd All rights reserved DOI 10.1016/j.str.2006.09.014 Structural Insights into the Speciﬁc Binding of Huntingtin Proline-Rich Region with the SH3 and WW Domains

Yong-Guang Gao,1,3 Xian-Zhong Yan,2,* Ai-Xin Song,1 Human Htt is a large multidomain protein of 3144 Yong-Gang Chang,1,3 Xue-Chao Gao,1,3 Nan Jiang,2 amino acid residues with a polyQ domain at the N termi- Qi Zhang,2 and Hong-Yu Hu1,* nus (MacDonald et al., 1993). The polyQ domain ranges 1 State Key Laboratory of Molecular Biology from 11 to 34 glutamine residues in unaffected individ- Institute of Biochemistry and Cell Biology uals, whereas that of HD patients extends to 37 or Shanghai Institutes for Biological Sciences more glutamine residues (Bates et al., 2002). A proline- Chinese Academy of Sciences rich region (PRR) containing 40 residues (normally resi- Shanghai 200031 dues 41–80) directly follows the polyQ domain in se- China quence (Figure 1A). 2 NMR Laboratory Recently, many investigations revealed that mutant National Center of Biomedical Analysis Htt with expanded polyQ impairs the normal interactions 27 Taiping Road of several proteins involved in gene transcription, traf- Beijing 100850 ficking, endocytosis, and cell signaling (Goehler et al., China 2004; Harjes and Wanker, 2003; Li and Li, 2004). Thus, 3 Graduate School of the Chinese Academy of Sciences it is speculative that HD neuropathology is related to 19A Yu Quan Road the interference of the normal function of cellular pro- Beijing 100039 teins by the aberrant Htt protein (Landles and Bates, China 2004; Li et al., 2003; Li and Li, 2004). There are many Htt-interacting partners identified; some contain Src homology 3 (SH3) or tryptophan Summary (WW) domains, such as SH3GL3/endophilin3 (Sittler et al., 1998), protein kinase C and casein kinase sub- The interactions of huntingtin (Htt) with the SH3 do- strate in neurons 1 (PACSIN1/syndapin) (Modregger main- or WW domain-containing proteins have been et al., 2002), HYPA/FBP11 (Faber et al., 1998), PSD-95 implicated in the pathogenesis of Huntington’s dis- (Sun et al., 2001), RasGAP (Liu et al., 1997), and CA150 ease (HD). We report the specific interactions of Htt (Holbert et al., 2001). Among them, SH3GL3 is reported proline-rich region (PRR) with the SH3GL3-SH3 do- to be preferentially expressed in human brain and testis, main and HYPA-WW1-2 domain pair by NMR. The re- and its C-terminal SH3 domain is essential for the inter- sults show that Htt PRR binds with the SH3 domain action with Htt PRR (Sittler et al., 1998). The characteris- through nearly its entire chain, and that the binding re- tics of the interaction between SH3GL3 and Htt and the gion on the domain includes the canonical PxxP-bind- colocalization of these two proteins suggest that ing site and the specificity pocket. The C terminus of SH3GL3 could be involved in the selective neuronal PRR orients to the specificity pocket, whereas the N cell death in HD (Sittler et al., 1998). SH3GL3 was also terminus orients to the PxxP-binding site. Htt PRR found to bind to the shell of the Htt body, suggesting can also specifically bind to WW1-2; the N-terminal that the SH3GL3-associated HD pathology may be portion preferentially binds to WW1, while the C-termi- caused by sequestering the Htt inclusion bodies (Qin nal portion binds to WW2. This study provides struc- et al., 2004). PACSIN1 has been implicated in clathrin- tural insights into the specific interactions between mediated endocytosis (DiProspero et al., 2004), and its Htt PRR and its binding partners as well as the alter- abnormal binding behavior and altered intracellular dis- ation of these interactions that involve PRR, which tribution in pathological tissues suggest that it plays may have implications for the understanding of HD. a role during the early stages of the selective neuropathology of HD (DiProspero et al., 2004; Modregger et al., 2002). Human HYPA interacts with the N-terminal region of Htt protein through its tandem WW domains, Introduction as identified by yeast two-hybrid assay (Faber et al., 1998; Passani et al., 2000). FBP11, the murine ortholog Huntington’s disease (HD) is a dominant neurodegener- of human HYPA, is one of the several proteins that ative disorder characterized by movement abnormali- bind with formins involved in murine limb and kidney ties, cognitive impairment, and psychiatric disturbances development (Bedford, et al., 1997). FBP11 also partici- due to neuronal cell loss, especially in the basal ganglia pates in pre-mRNA splicing (Lin et al., 2004) and regula- and the cerebral cortex (Martin and Gusella, 1986; Von- tion of N-WASP localization (Mizutani et al., 2004). sattel et al., 1985). Accumulating evidence supports the The involvement of PRR in the pathological process of finding that a polyglutamine (polyQ) expansion tract in HD can also be observed from the fact that MW7 scFv, huntingtin (Htt), a ubiquitously expressed protein of yet a monoclonal antibody recognizing Htt PRR, signifi- unknown function, is the cause of this disease (MacDon- cantly inhibits aggregation as well as the cell death ald et al., 1993; DiFiglia et al., 1995). induced by mutant Htt protein (Khoshnan et al., 2002). Another observation indicates that removal of a series of prolines adjacent to the polyQ tract in Htt blocks forma- *Correspondence: [email protected] (H.-Y.H.), [email protected] tion of the shell of the Htt body and redistributes seques- (X.-Z.Y.) tration of many vesicle-associated proteins, a process Structure 1756

Figure 2. 3D Solution Structure of the SH3 Domain from SH3GL3 (A) Backbone atom superposition of the ﬁnal ten structures. The structures are superimposed adopting residues 3–58. (B) Ribbon diagram representation of the SH3 domain of SH3GL3. The canonical PxxP-binding pocket is indicated in a circle, while the speciﬁcity pocket between the RT loop and the n-Src loop is also highlighted.

Results

The Three Portions of Htt PRR Have Different Preferences for the SH3 and WW Domains To identify which portion of the Htt PRR region (residues 41–80) is responsible for the interaction with the SH3 or WW domain, we subdivided the region into three pep- Figure 1. The N-Terminal, Central, and C-Terminal Portions of Htt tide parts, corresponding to the N-terminal (Pept-1), PRR Have Different Binding Preferences for the SH3 and WW central (Pept-2), and C-terminal (Pept-3) portions of Domains Htt PRR, respectively (Figure 1A). The three segmental (A) The domain architecture of the N-terminal Htt protein and the sequences were cloned and expressed as GST fusion amino acid sequence of the PRR region. Six peptides corresponding proteins for a pull-down assay. Figure 1B suggests to different portions of Htt PRR were generated by peptide synthe- that SH3GL3-SH3 and PACSIN1-SH3 only bind to sis. Pept-1, Pept-2, and Pept-3 were also generated and purified Pept-2, not to Pept-1 or Pept-3, while HYPA-WW1-2 as recombinant GST fusion proteins for pull-down assay. (B) GST pull-down assay showing that the tandem WW domains of (the tandem domains of HYPA) binds to Pept-1 and HYPA-WW1-2 interact with GST-Pept-1 and GST-Pept-3, while Pept-3, but not to Pept-2. This is consistent with the SH3GL3-SH3 and PACSIN1-SH3 only interact with GST-Pept-2 previous study that an expanded Pept-2 specifically in vitro. The ‘‘Input’’ lane represents the band from 10% of the binds with PACSIN1-SH3 (Modregger et al., 2002). The amount of protein in each sample. results imply that the three portions of Htt PRR have different binding affinities for the SH3 domain of SH3GL3 or PACSIN1 and the tandem WW domains that may be related to neuronal dysfunction (Qin et al., of HYPA. 2004). The typical PRR segment recognized by SH3 or WW Solution Structure of the SH3GL3-SH3 Domain domains contains less than 10 amino acid residues To study the interaction between SH3GL3-SH3 and Htt (Kay et al., 2000), and the molecular mechanism under- PRR in detail, we solved the structure of the SH3 domain lying the specific recognition has been elucidated and in solution by heteronuclear multidimensional NMR reviewed (Ilsley et al., 2002; Musacchio, 2002; Sudol, techniques. A summary of the NMR experimental re- 1996; Zarrinpar et al., 2003). However, many proteins, straints for structure calculation and statistics is pre- such as Htt, formin (Bedford et al., 1997), and N-WASP sented in Table S1 (see the Supplemental Data available (Bompard and Caron, 2004), contain PRR far longer with this article online). All ten of the lowest-energy final than 10 residues. How these long PRRs recognize vari- structures converge with an NOE or dihedral angle viola- ous SH3 and WW domains remains largely unknown. tion no greater than 0.3 A˚ or 5, respectively. The aver- Obtaining the structural knowledge of the interaction age root-mean-square deviations (rmsds) for the ten between Htt PRR and the SH3 or WW domain will be structures for backbone and all heavy atoms are 0.64 the first step toward understanding how the abnormally and 1.76 A˚ , respectively. Figure 2A depicts a superimpo- expanded polyQ tract interferes with the normal function sition of the ten lowest-energy structures and a ribbon of cellular proteins that contain the SH3 or WW domain. representation of one of the ten NMR structures. The so- We have determined the solution structure of the SH3 lution structure of SH3GL3-SH3 maintains a typical SH3 domain of SH3GL3 and have assigned the backbone fold containing five b strands organized in an antiparallel resonances of the WW domain pair of HYPA by hetero- b barrel structure with a short 310 helix between the last nuclear NMR. Based on the structure, the binding spec- two b strands (Figure 2B). Similar to most SH3 fold do- ificities between Htt PRR and these domains have been mains (Dalgarno et al., 1997), this structure also contains elucidated in detail. This study reveals that Htt PRR rec- two characteristic loops, namely, the RT loop between ognizes the SH3 domain and the WW domain pair with b1 and b2 and the n-Src loop between b2 and b3. There high specificities, but with different mechanisms. is also a broad pocket called the specificity pocket Binding Specificity of Huntingtin PRR 1757

Figure 3. Summary of the Chemical Shift Per- turbations and Surface Mapping for Interac- tions between SH3GL3-SH3 and Various Pro- line-Rich Peptides (A–F) The chemical shift perturbations of the SH3 domain upon addition of (A) Pept-1, (B) Pept-2, (C) Pept-3, (D) Pept-1-2, (E) Pept-2- 3, and (F) Pept-1-2-3 to a peptide:protein molar ratio of 8:1. (G–J) The surface mapping of the signiﬁcantly perturbed residues (red) in the presence of (G) Pept-1, Pept-2, or Pept-3; (H) Pept-1-2; (I) Pept-2-3; and (J) Pept-1-2-3. The residues with chemical shift changes larger than the average are depicted on the surfaces of the structure. (K) The titration curves for Pept-1-2-3 binding to the SH3 domain. By ﬁtting the data to a bimolecular binding algorithm, the dissociation

constant (KD) of Pept-1-2-3 binding to SH3GL3-SH3 is 680 6 80 mM.

between these two loops. The canonical PxxP-binding the SH3 domain with similar binding behavior suggests pocket is located on the hydrophobic patch that con- that their binding abilities may originate from prolines, tains a cluster of conserved aromatic residues (Tyr10, but not from the nonproline residues. A similar result is Phe12, Trp38, and Tyr54) that are arranged adjacent also obtained from the binding of two double-portion each other in a nearly parallel manner. peptides (Pept-1-2 and Pept-2-3). It is likely that Pept- 1-2 and Pept-2-3 bind to the SH3 domain with similar Binding Specificity of SH3GL3-SH3 with Htt PRR binding sites, but with different orientations. In addition, To elucidate the binding specificity of SH3GL3-SH3 with binding of these short peptides, whether single portion Htt PRR, several truncated peptides of Htt PRR (Fig- or double portions, to the SH3 domain is relatively ure 1A) were tested for interaction with the SH3 domain weak, whereas binding of Pept-2 to the SH3 domain is by using the chemical shift perturbation method. Fig- a little stronger than that of Pept-1 and Pept-3 (Table ure 3 shows the chemical shift mapping on the SH3 do- S2). This may explain why only the binding of Pept-2 main upon binding with various peptides. The effects on with the SH3 domain has been identified by the less sen- some residues, such as Glu15 and Phe49, which have sitive experiment of pull-down assay (Figure 1B). These amide resonances largely affected by the addition of results are consistent with the studies that recognition Pept-1 (Figure 3A), Pept-2 (Figure 3B), or Pept-3 (Fig- of the SH3 domain by most short proline-rich peptides ure 3C), are very similar. The significant residues for is moderately weak and poorly selective, and that the these three titrations are mapped on the domain struc- specificity pocket of the SH3 domain is favorable to pro- ture (Figure 3G), in which the significantly perturbed res- line-rich peptide recognition (Santamaria et al., 2003). idues are located in the RT loop of the SH3 domain. The For this reason, we speculate that the binding of these residues mostly affected by the addition of Pept-1-2 short peptides to the SH3 domain is less specific, and (Figure 3D) cover the canonical PxxP-binding site and that it is the proline residue that contributes to the the specificity pocket between the RT loop and the binding. n-Src loop of SH3GL3-SH3 (Figure 3H). Moreover, As for the full-length PRR (Pept-1-2-3), its binding Pept-2-3 also exhibits similar binding properties with causes perturbation on some additional residues, such Pept-1-2 (Figures 3E and 3I), though it possesses a dif- as Lys23, Glu24, and Asp26 (Figure 3F), which reside on ferent peptide sequence. The result that the three nona- the opposite surface of the PxxP-binding pocket on the peptides, though having different sequences, bind to SH3GL3-SH3 domain. Thus, the binding sites on the Structure 1758

Figure 4. Effects of the Spin-Labeled Peptides on the Backbone Amide Resonances of the SH3 Domain (A) Sequences of Pept-22-N and Pept-22-C. The Cys residue was attached to the N or C terminus of the peptide, and the MTSSL spin was specifically labeled to the thiol group of the Cys residue. N, Cys in the N terminus; C, Cys in the C terminus. (B) Overlay of the HSQC spectra of the SH3 domain complexed with Pept-22-N-MTSSL (blue) and Pept-22-N (red). (C) Overlay of the HSQC spectra of the SH3 domain complexed with Pept-22-C-MTSSL (blue) and Pept-22-C (red). The missing peaks due to the paramagnetic effect are denoted by arrows. (D) Mapping of the residues with peaks missing on the SH3GL3-SH3 domain structure. The residues with peaks missing induced by Pept-22-N- MTSSL are colored in red, and the residues affected by Pept-22-C-MTSSL are colored in blue or red. domain structure cover the entire canonical PxxP- the Asp11 and Glu13 residues. As for Pept-22-C-MTSSL binding site and the specificity pocket and perhaps its binding, many resonances, even including those of opposite surface (Figure 3J). The saturation curves for Asp11 and Glu13, are significantly affected by the spin the chemical shift changes of three representative resi- label, but most of the residues are located in the proxim- dues in SH3GL3-SH3 upon titration with Pept-1-2-3 ity of the specificity pocket of the SH3 domain (Fig- are well fitted with a 1:1 stoichiometry (Figure 3K) and ure 4C). This observation demonstrates that the C termi- give a dissociation constant (KD)ofw0.68 mM. Mutation nus of the peptide contacts the SH3 domain near the of Gln17 to Ala, which is located in the crucial RT loop, specificity pocket (Figure 4D). Taken together, these causes the binding ability to be reduced by w3-fold findings indicate that Htt PRR exhibits a preferred bind- (KD = 2.2 mM). Taken together, the chemical shift pertur- ing mode on the SH3 domain (also see Figure 8A), and bation experiments indicate that the entire Pept-1-2-3 is that the orientation of PRR is consistent with that of necessary for its specific binding to the SH3 domain. the Class II SH3 ligand (Mayer, 2001). When it is shortened, it would loss its binding affinity (Table S2) and specificity (Figure 3). This binding speci- Evidence for the Specific Binding ficity of Htt PRR for the SH3 domain might be required from Intermolecular NOEs for regulating SH3-mediated interactions (Li, 2005). On the basis of filtered-NOESY analysis on the SH3GL3- SH3/Pept-1-2-3 complex (molar ratio of 1:1) (Figure 5A), Orientation of Htt PRR Binding on the SH3 Domain several NOE peaks from 7 residues in the SH3 domain A spin-labeling NMR method (Mahoney et al., 2000) was interacting with Pept-1-2-3 were unambiguously identi- applied to identify the orientation of Htt PRR binding on fied and assigned on the SH3 domain. Among the resi- the surface of the SH3 domain. For ensuring that the dues interacting with Pept-1-2-3 (Figure 5B), Gly8 and binding sites on the SH3 domain are affected by the Asn53 are located in the typical PxxP-binding site, while spin-labeled compound, we produced a pair of 22 resi- Gln17 and Trp38 flank the specificity pocket. Surpris- due peptides, which contain not only the entire central ingly, the residues Cys6, Ile28, and Val59 have also portion of Htt PRR, but also its several flanking amino been found to interact with Pept-1-2-3. These residues acids (Figure 4A), that were spin labeled with MTSSL are located on a concave surface corresponding to the in either the N or C terminus. When the N-terminally binding surface of Pex5p on the Pex13p-SH3 domain labeled peptide Pept-22-N-MTSSL binds to the SH3 (Douangamath et al., 2002; Kami et al., 2002). There is domain, two crosspeaks in the HSQC spectrum corre- a possibility that the extended residues in Pept-1-2-3 sponding to Asp11 and Glu13 are nearly completely also contact the opposite surface of the SH3 domain eliminated (Figure 4B), indicating that the N terminus when it specifically binds to the PxxP-binding site and of the peptide sterically contacts the SH3 domain near the specificity pocket. Binding Specificity of Huntingtin PRR 1759

Figure 5. Intermolecular NOEs of the SH3GL3-SH3/Pept-1-2-3 Complex (A) Selected portion of an F1fF2e NOESY spectrum of 13C/15N-SH3/unlabeled Pept-1- 2-3 complex (molar ratio of 1:1). Indicated are the observed intermolecular NOEs between residues in the SH3 domain and Pept-1-2-3 (mostly proline side chains). These NOEs are the contributions of both side chain atoms (Gln17, Trp38, and Asn53) and backbone atoms (Cys6, Gly8, Ile28, Trp38, and Val59) in the SH3 domain. (B) Mapping of the contact surface on the SH3GL3-SH3 domain structure with the residues (red) that are detected to have intermolecular NOEs with Pept-1-2-3 in the complex.

Backbone Dynamics of the HYPA WW Domain Pair chemical shift perturbation method. The plots of the Since Htt PRR binds to the WW domain pair of HYPA, it chemical shift changes versus residue number of the will be interesting to study how Htt PRR interacts with WW domain pair upon titration of various peptides at a this domain pair. The structure of the FBP11-WW1 do- peptide:protein molar ratio of 8:1 are displayed in Fig- main has been elucidated (Pires, et al., 2005), but that ure 6. The addition of increasing amounts of the peptide of WW2 and the domain orientation in the domain pair ligand PPTPPPLPP results in chemical shift changes of remain unsolved. To explore whether these small WW a number of residues dispersed in both the WW1 and domains are orientationally flexible with respect to WW2 domains (Figure 6A). During titration, the amide each other in the WW domain pair of HYPA, we deter- resonances of some residues exhibit a little broadening 1 15 mined the relaxation rates (R1 and R2) and { H}- N in the line widths. This line broadening may be due to NOEs for the WW1-2 domain pair (Figure S1). Generally, the chemical exchange at an intermediate time regime large NOE values (>0.7) reflect slow internal motion, between the ligand-free and -bound states. Notably, whereas small values indicate substantial internal the chemical shift changes for the residues in WW1 are motion and negative values are indicative of fully dis- in some aspects similar to those for the corresponding ordered regions (Bruschweiler, 2003). The average residues in WW2 at every titration point. This indicates NOE value of WW1 (residues 13–42) is w0.52, suggest- that both WW domains have similar binding capabilities ing that WW1 in the free state is marginally stable, which for peptide PPTPPPLPP. In other words, the two WW has been proved by the following titration experiments. domains have no selectivity for the peptide from formin, WW2 (residues 54–83) is more poorly folded than WW1, which is consistent with the previous alanine-scanning as indicated by its smaller NOE values (an average of mutagenesis analysis (Bedford et al., 1997). 0.45). Moreover, the single WW1 domain is structured, As for the peptides from Htt PRR, titration of Pept-1 as the 1H-15N HSQC spectrum shows wide dispersion; causes chemical shift changes of a number of residues however, the single WW2 is fully disordered, as indi- in both domains, but more prominently in the WW1 do- cated by severe line broadening and peak overlapping main (Figure 6B). During titration, the amide resonances (data not shown). The linker region (residues 43–53) ex- in WW1 exhibits large chemical shift changes with slight hibits even faster internal motions than both domains, line broadening, in contrast to the large broadenings ob- and the average NOE value is only 0.33. A similar result served for the corresponding resonances in WW2 (Fig- can also be obtained from the relaxation data of the ure 7A). In particular, some amide resonances in WW2, R2:R1 ratio (Figure S1). The different dynamics of WW1 such as Ser61, Asp62, Tyr69, and Arg77, rapidly dis- and WW2 in the domain pair infers that they have differ- appear from the spectrum at the first titration point ent properties for binding with PRR-containing partners. and reappear at a peptide:protein ratio higher than 4:1. Titration of Pept-2 results in only small or no chemical Binding Specificity of the WW Domain Pair shift changes of the residues in both domains, even at with Htt PRR a Pept-2:WW1-2 ratio of 8:1 (Figure 6C). There is no sub- To understand the binding properties of the HYPA WW stantial line broadening in any of the resonances either domain pair with Htt PRR in detail, we studied binding of (Figure 7B). This indicates that Pept-2 has no or very the three nonapeptides and Pept-1-2-3 from Htt PRR as weak interaction with the WW1-2 domain pair, which is well as peptide PPTPPPLPP from the PRR of formin consistent with the result from the pull-down experiment (Bedford et al., 1997; Pires et al., 2005) by using the (Figure 1B). Titration of Pept-3 also causes chemical Structure 1760

intermediate chemical exchange on the NMR timescale, while the corresponding resonances undergo fast exchange upon binding with Pept-3 (Figures 7A and 7C). As a rough approximation, tight binding might correlate with slow ligand exchange, and weak binding might correlate with fast exchange (Fielding, 2003). More- over, the chemical shift changes of WW1 caused by Pept-1 binding are large, in contrast to the small chemical shift changes caused by Pept-3 binding in each titration step (Figure 7E). The titration curves of WW1 caused by Pept-1 binding show that the binding tends to reach saturation at a lower Pept-1 concentration than the concentration of Pept-3 in the curves of WW1 caused by Pept-3 binding. Collectively, these results demonstrate that Pept-1 binds to WW1 more tightly than Pept-3. Since WW2 is not as stable in the ligand-free state as WW1, which is indicated by the dynamics analysis (Fig- ure S1), it is possible that WW2 undergoes confor- mational exchange on the NMR timescale even in the ligand-free state. Upon peptide binding, the chemical exchange between the ligand-bound and -free states makes it complicated to compare how WW2 binds to Pept-1 and Pept-3. Upon addition of Pept-1, a number of resonances in WW2 undergo chemical shift change and line broadening (Figure 7A). Some of them (e.g., Ser61 and Asp62) disappear and finally reappear at the later titrations; some (e.g., Tyr69 and Glu83) do not reappear even at the last titration point (peptide:protein ratio of 8:1). However, upon addition of Pept-3, the corresponding peaks in WW2 do not disappear, but their chemical shifts do change (Figure 7C). Interestingly, the intensities of these peaks increase considerably upon binding. This indicates that, in contrast to Pept-1, a small amount of Pept-3 could transform WW2 from a relatively unstable state to the rather stable bound state. From this aspect, Pept-3 is a preferential ligand for WW2 binding. The titration curves for chemical shift changes of WW2 upon addition of Pept-2 or Pept-3 clearly indicate that WW2 preferentially binds to Pept-3, not Pept-2 Figure 6. Chemical Shift Perturbation of the HYPA WW Domain Pair (Figure 7F). Taken together, these findings show that upon Titration of Various Proline-Rich Peptides WW2 in the ligand-free state is less stable than WW1 (A–E) The chemical shift changes of WW1-2 versus its residue num- in the form of tandem domains. Pept-1 is preferential ber are shown upon addition of peptide (A) PPTPPPLPP, (B) Pept-1, to WW1 binding, while Pept-3 is preferential to WW2 (C) Pept-2, (D) Pept-3, and (E) Pept-1-2-3 to a peptide:protein ratio of 8:1. The negative data for WW2 in (B) represent the disappearance of binding; however, Pept-2 has no or very weak binding peaks due to line broadening. capability for both WW1 and WW2. Thus, Pept-1-2-3 should exhibit a preferred binding mode on the HYPA shift changes of many residues in both domains (Fig- WW domain pair (also see Figure 8B). ure 6D). During titration, some resonances in WW2, such as Ser61 and Tyr69, exhibit increased peak intensi- Discussion ties (Figure 7C), whereas the corresponding resonances (Ser20 and Tyr28) in WW1 show no intensity change. In- Multiple Binding Sites of the SH3GL3-SH3 Domain terestingly, titration of Pept-1-2-3 causes significant Currently, three different ligand-binding sites on diverse chemical shift changes of many resonances in both do- SH3 domains have been identified. The first is the mains (Figure 6E). The resonances of these residues in canonical PxxP-binding site that is constructed by the both domains rapidly disappear from the spectrum at conserved aromatic residues. The second is the speci- a peptide:protein ratio below 1:1 and reappear at a ratio ficity pocket between the RT loop and the n-Src loop, higher than 4:1 (Figure 7D). The intensities of these reap- such as the binding site of p67phox SH3(C) for the peared peaks at the last titration are significantly en- a-helical portion of the p47phox tail peptide (Kami et al., hanced compared to the peaks in the ligand-free form. 2002). The third site, analogous to the binding site of This indicates that binding of Pept-1-2-3 leads WW1-2, Pex5p on the Pex13p-SH3 domain, is located on the especially the WW2 part, to achieve a well-folded struc- opposite concave surface of the specificity pocket ture in the complex. consisting of the b1 and b2 strands and the C terminus Upon binding with Pept-1, a number of resonances (Barnett et al., 2000; Douangamath et al., 2002; Kami (e.g., Thr16, Ser20, and Tyr28) in WW1 experience an et al., 2002). Our data provide convincing evidence to Binding Specificity of Huntingtin PRR 1761

Figure 7. Summary of the Spectral Perturbations for Interactions between the HYPA WW Domain Pair and Various Peptides (A) Overlay of a representative region of the 1H-15N HSQC spectra of HYPA-WW1-2 upon titration of Pept-1. The traces show the peak move- ments from free peptide (black) to a peptide:protein ratio of 8:1 (purple). The changes of some typical peaks in the WW1 and WW2 domains are indicated by labeling the residues. Most peaks in WW1 (such as T16, S20, Y27, and Y28) undergo chemical shift changes, while many peaks in WW2 (S61, D62, Y69, R77, and E83) tend to disappear. (B) Titration of Pept-2 to HYPA-WW1-2. Pept-2 has no or very little effect on the peaks in both WW1 and WW2 domains. (C) Titration of Pept-3 to HYPA-WW1-2. Some peaks in WW1 (T16 and S20) exhibit chemical shift changes, while some peaks in WW2 (S61, D62, Y69, and R77) exhibit line broadening. (D) Titration of Pept-1-2-3 to HYPA-WW1-2. Many peaks in both domains are significantly affected by the addition of Pept-1-2-3. Most of the peaks first disappear and then reappear due to the intermediate exchange. The intensities of some of the reappeared peaks at the last titration (purple) are significantly enhanced compared to the peaks in the ligand-free form (black). (E) Plot of weighted average chemical shift changes for three typical residues in WW1 (T16, S20, and Y28) versus the concentration of Pept-1 (red), Pept-2 (green), and Pept-3 (blue). (F) Plot of average chemical shift changes for residues in WW2 (Y59, Y69, and R77) versus the concentration of Pept-2 (green) and Pept-3 (blue). Due to the large line broadening, the titration curve for chemical shift changes of the residues in WW2 upon the addition of Pept-1 to WW1-2 is not included. support that Htt PRR specifically binds to the SH3 do- Characteristics of the Interaction of the HYPA main, and that the binding sites include the canonical WW Domain Pair PxxP-binding site and the specificity pocket (Figure 8A). Many proteins possess arrays of multiple WW domains; Since the PRR chain is much longer than the canonical it is likely that the tandem WW domains cooperate with SH3 ligands, it is likely that it can contact the SH3 do- each other to achieve further specific recognition. To our main on the third site, as indicated by the results of the knowledge, Htt PRR is the first example reported as a li- intermolecular NOE experiment. The C terminus of gand for binding of the WW domain pair. The N-terminal PRR orients to the specificity pocket, whereas the N ter- portion of Htt PRR preferentially binds to WW1, while the minus orients to the PxxP-binding site, which is consis- C-terminal portion binds to WW2; however, the central tent with what is observed in the Class II SH3 ligand portion remains unoccupied (Figure 8B). Up to date, (Mayer, 2001). This kind of binding mode may allow only two structures of the tandem WW domain pair, PRR motifs (generally in the PPII helix) to bind with var- Prp40 (Wiesner et al., 2002) and Su(dx) (Fedoroff et al., ious SH3 domains that are more diverse and selective. 2004), have been solved. In the case of HYPA, both Structure 1762

Experimental Procedures

Protein Expression and Purification The encoding sequence for the SH3GL3-SH3 domain, corresponding to residues 285–344 of SH3GL3, was amplified via PCR and was cloned into the pET-22b expression plasmid by using the NdeI/XhoI cloning sites. The SH3 sequence was renumbered from 285–344 to 1–60, which is hereinafter used for describing the SH3 domain. The plasmid was transformed into E. coli BL21 (DE3). Cells harboring the plasmid were grown at 37 C. When OD600 reached w0.6, SH3GL3-SH3 expression was induced by adding IPTG to a final concentration of 0.1 mM. The culture was then shaken for 10 hr at 25C. Figure 8. Schematic Representation of the Specific Interactions of After cell lysis, the protein was purified through a nickel-NTA column Htt PRR with the SH3 Domain and the WW Domain Pair (QIAGEN), followed by a 16/40 Superose 12 column (Amersham 15 15 13 (A and B) In the SH3 domain, almost all of the residues of the PRR Pharmacia). N- and N/ C-labeled SH3GL3-SH3 proteins were 15 chain contribute to SH3 binding, and the binding sites on the SH3 prepared by using the M9 minimal medium containing NH4Cl 13 domain include the typical PxxP-binding site and the specificity and/or C6-D-glucose as the sole nitrogen and/or carbon resource, pocket. The interaction is oriented such that the C terminus of PRR respectively. points toward the specificity pocket, while the N-terminus is proximal The encoding sequence of the WW1-2 domain, corresponding to to the PxxP-binding site. In the case of the WW domain pair, two WW residues 136–224 of HYPA/FBP11, was amplified via PCR and was domains cooperatively bind to Htt PRR; WW1 specifically binds to cloned into the pET-3a plasmid by using the NdeI/BamHI sites. the N-terminal portion, and WW2 binds to the C-terminal portion. The WW1-2 sequence was renumbered from 136–224 to 1–89, which is hereinafter used for describing the HYPA-WW1-2 domain. The WW domains are marginally folded, as indicated by the plasmid was transformed into E. coli BL21 (DE3). Cells containing w 1 15 the plasmid were grown at 37 C until an OD600 of 0.8 was reached. wide dispersion of amide resonances in the H- N Expression was induced by adding IPTG to a final concentration of HSQC spectrum. These domains, especially WW2, are 0.1 mM. The culture was then shaken for 10 hr at 25C. The protein not as stable as those in the ligand-bound states. The was purified sequentially by heat denaturation, ion-exchange chro- current study also provides insights into the dynamics matography on a Q-Sepharose Fast Flow column and gel filtration and ligand recognition of the WW domain pair. on a 16/60 Superdex 75 column (Amersham Pharmacia). The encoding sequences of the WW1 and WW2 domains, corresponding to residues 10–42 and 51–83 of WW1-2, respectively, were amplified Alteration of the Interactions Involving PRR via PCR and were cloned into the pGBTNH plasmid by using the Has Implications for the HD Pathology BamHI/XhoI cloning sites (Bao et al., 2006). The respective plasmid Since the aberrant interactions involved in Htt PRR and was transformed into E. coli BL21 (DE3). Cells harboring each plas- w its SH3 domain- or WW domain-containing partners mid were grown at 37 C. When OD600 reached 0.8, WW1 or WW2 might be associated with the pathology of HD, it is inter- expression was induced by adding IPTG. After cell lysis, the super- esting to know how the polyQ tract alters the normal in- natant was loaded onto a nickel-NTA column (QIAGEN). After washing, followed by on-column thrombin cleavage for removing the GB1 teraction between Htt PRR and the SH3 or WW domain. tag, the protein was eluted and subjected to chromatography on Our current result showing that the N terminus of Htt a 16/40 Superose 12 column (Amersham Pharmacia). PRR, occurring after the polyQ domain, is critical for binding to the SH3 domain and the WW domain pair Synthesis of Proline-Rich Peptides implies that the specific interactions are susceptible The peptides (PPTPPPLPP, Pept-1, Pept-2, Pept-3, Pept-1-2, Pept- to the length of the polyQ tract. In the Htt-interacting 2-3, and Pept-1-2-3) were obtained from automatic solid-phase partners, all of the SH3 domain- or WW domain-contain- peptide synthesis on 2-Cl-Trt resin by using the FMOC strategy. The N-terminal amino group and the C-terminal carboxyl group of ing partners, except PSD-95, bind more tightly to Htt the peptides were retained unmodified. After release of the peptides with elongated polyQ stretch (Li and Li, 2004). It is pos- from the resin by using AcOH/TFE/DCM, the peptides were purified sible that the elongated polyQ stretch makes the entire by reverse-phase HPLC by following standard protocols involving PRR region more accessible to these SH3 domain- or a water/acetonitrile solvent system containing 0.1% trifluoroacetic WW domain-containing partners and thus enhances acid. Pooled fractions of the pure peptides were lyophilized and their binding. analyzed by electrospray mass spectrometry to verify identity and homogeneity. Since Htt PRR is proximal to the polyQ tract, it is likely that alteration of the interactions involving PRR is Spin Labeling associated with the HD pathology. The expanded polyQ For producing the two peptides Pept-22-N and Pept-22-C, the tract makes the PRR region more available to the WW- genes encoding these peptides were chemically synthesized and in- containing proteins or to MW7, a monoclonal antibody serted into pGBTNH vectors by using the BamHI/XhoI cloning sites recognizing Htt PRR, and significantly inhibits aggrega- (Bao et al., 2006). Then, the cysteine-containing peptides were tion as well as the cell death induced by the mutant Htt obtained according to the method for production of the WW1 and WW2 domains described above. To label the peptides with MTSSL protein (Khoshnan et al., 2002). In addition, removal of ((1-oxyl-2,2,5,5-tetramethyl pyrroline-3-methyl)methanethiosulfo- a series of prolines adjacent to the polyQ region in Htt nate, Toronto Research Chemicals, Canada), a 10-fold molar excess blocks formation of the shell of the Htt body and redis- of MTSSL was added to each peptide in a 20 mM sodium phosphate tributes the sequestration of many vesicle-associated buffer containing 50 mM NaCl (pH 8.0). The mixture was incubated proteins, a process that may be related to neuronal dys- for longer than 12 hr at 4C, and the free compound was removed function (Qin et al., 2004). Considering that interactions by size-exclusion chromatography. The resulting peptides were ver- ified by mass spectrometry. involving Htt PRR are susceptible to polyQ length (Feany and La Spada, 2003; Li et al., 2003; Sugars and Rubinsz- GST Pull-Down Assay tein, 2003), we speculate that the alteration of these The generation of GST fusion proteins containing short peptides specific interactions is one of the substantial causes corresponding to the N-terminal, central, and C-terminal portions of for HD pathology. Htt PRR was carried out as described previously (Modregger et al., Binding Specificity of Huntingtin PRR 1763

2002). For pull-down assay, the GST fusion proteins were added to WW1-2 was dissolved to a concentration of 0.25 mM in the above- the glutathione Sepharose 4B beads (Amersham Biosciences) in mentioned NMR buffer, and the proline-rich peptides were added a PBS buffer (10 mM sodium phosphate, 100 mM NaCl, 5 mM di- stepwise to give the peptide:protein molar ratios ranging from 0:1 thiothreitol), and the suspension was agitated at 4C for 30 min. to 8:1; each step was monitored by 2D 1H-15N HSQC spectrum. The GST fusion protein-bound beads were washed three times in The combined average chemical shift changes were calculated as 2 2 1/2 the same buffer to remove any unbound protein. An equimolar Ddave = ([0.17DdN] +[DdH] ) , where DdH and DdN are the chemical amount of SH3 or WW1-2 was added, and the suspension was agi- shift changes in the 1H and 15N dimensions, respectively. The titra- tated at 4C for an additional 30 min. The resin was recovered by tion curves were ﬁtted assuming a bimolecular binding event as de- centrifugation, and, after excessive washing, the sample was resus- scribed (Liu et al., 1999). Dissociation constants (KD) were obtained pended in the sample buffer and subjected to SDS-PAGE in a 15% by analyzing the chemical shift changes of three typical residues for gel, followed by Coomassie staining. SH3GL3-SH3 or its Q17A mutant upon addition of each peptide.

NMR Spectroscopy Supplemental Data To solve the solution structure of the SH3GL3-SH3 domain, a Supplemental Data include one figure and two tables and are available 15 13 N/ C-labled sample containing w1 mM protein in a phosphate at http://www.structure.org/cgi/content/full/14/12/1755/DC1/. buffer (10 mM sodium phosphate, 100 mM NaCl, 5 mM dithiothreitol, and 0.05% w/v sodium azide [pH 6.0] in 92% H2O/8% D2O or 100% Acknowledgments D2O) was used for NMR experiments. All NMR data acquisition was carried out at 27C on a Varian INOVA 600 spectrometer equipped We thank Dr. K.H. Sze for valuable discussions and Mr. J.Y. Zhao, with three RF channels and a triple-resonance pulsed-field gradient 1 15 13 Ms. X.J. Lin, and W.J. Bao for technical help. We also thank Dr. M. probe. The backbone and side chain H, N, and C resonances Plomann for providing the plasmid pGEX-SH3. This work was sup- were assigned based on the spectra of 3D HNCO, HNCACB, 15 ported by grants from the 863 Hi-Tech program (2002BA711A13), CBCA(CO)NH, H(CCO)NH, C(CO)NH and 3D N TOCSY-HSQC, the Chinese Academy of Sciences (KSCX1-SW-17 and KSCX2- HCCH-TOCSY. NOE distance restraints for structure calculations 15 13 SW-209), and the Shanghai Commission of Science and Technology were obtained from 3D N-edited NOESY and C-edited NOESY 13 (03JC14081). (aliphatic C regions). Intermolecular NOEs between SH3GL3-SH3 and Pept-1-2-3 were obtained from 2D-filtered/edited NOESY experiments as described (Peterson et al., 2004). Received: September 12, 2006 For HYPA-WW1-2, 15N- or 15N/13C-labeled sample containing Revised: September 12, 2006 w1 mM protein in a phosphate buffer (10 mM sodium phosphate, Accepted: September 27, 2006 100 mM NaCl, 5 mM dithiothreitol, and 0.05% w/v sodium azide Published: December 12, 2006

[pH 6.0] in 92% H2O/8% D2Oor100%D2O) was used for NMR experiments. The backbone resonances were assigned based on the References spectra of 3D HNCO, HN(CA)CO, HNCACB, CBCA(CO)NH and 3D 15 15 N TOCSY-HSQC, N-edited NOESY. Bao, W.J., Gao, Y.G., Chang, Y.G., Zhang, T.Y., Lin, X.J., Yan, X.Z., and Hu, H.Y. (2006). Highly efficient expression and purification sys- Structure Calculation and Analysis tem of small-size protein domains in Escherichia coli for biochemical The NMRPipe software suite (Delaglio et al., 1995) was applied to characterization. Protein Expr. Purif. 47, 599–606. process the NMR data, and NMRView (Johnson and Blevins, 1994) Barnett, P., Bottger, G., Klein, A.T., Tabak, H.F., and Distel, B. (2000). software packages were used for resonance peak picking and The peroxisomal membrane protein Pex13p shows a novel mode of data analysis. The CNS program (Brunger et al., 1998) with the SH3 interaction. EMBO J. 19, 6382–6391. ARIA module (Nilges et al., 1997) was adopted to assign NOE peaks and to calculate structures. The protein structures were assessed by Bates, G., Harper, P., and Jones, L. (2002). Huntington’s Disease using PROCHECK (Laskowski et al., 1996) and were displayed by (Oxford, UK: Oxford University Press). the MOLMOL program (Koradi et al., 1996). Hydrogen bond re- Bedford, M.T., Chan, D.C., and Leder, P. (1997). FBP WW domains straints (two per hydrogen bond) were generated by a combination and the Abl SH3 domain bind to a specific class of proline-rich of H/D exchange data, medium-range NOEs, and chemical shift ligands. EMBO J. 16, 2376–2383. index. Backbone dihedral angle restraints (f and 4) were derived Bompard, G., and Caron, E. (2004). Regulation of WASP/WAVE from the TALOS program (Cornilescu et al., 1999). For SH3GL3- proteins: making a long story short. J. Cell Biol. 166, 957–962. SH3, the restraints used for structural calculation are summarized Brunger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., in Table S1. The structural calculation in combination with iterative Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., NOE peak assignments was performed for 9 cycles, and a total of Pannu, N.S., et al. (1998). Crystallography & NMR system: a new 200 structures were finally obtained. Ten structures of the lowest software suite for macromolecular structure determination. Acta energies, which exhibit no NOE violation > 0.3 A˚ and no dihedral Crystallogr. D Biol. Crystallogr. 54, 905–921. violation > 5, were selected. Bruschweiler, R. (2003). New approaches to the dynamic interpreta- Determination of Backbone Relaxation Parameters tion and prediction of NMR relaxation data from proteins. Curr. Opin. All 15N relaxation data for WW1-2 were acquired at 25C by using 2D Struct. Biol. 13, 175–183. proton-detected heteronuclear NMR experiments implementing the Cornilescu, G., Delaglio, F., and Bax, A. (1999). Protein backbone standard pulse sequences (Farrow et al., 1994). A recycle delay of angle restraints from searching a database for chemical shift and

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Accession Numbers

The coordinates and structure factors for SH3GL3-SH3 are available in the Protein Data Bank under ID code 2EW3.