Mona/Gads SH3C Binding to Hematopoietic Progenitor Kinase 1 (HPK1) Combines an Atypical SH3 Binding Motif, R/KXXK, with a Classical PXXP Motif Embedded in a Polyproline Type II (PPII) Helix

Hematopoietic progenitor kinase 1 (HPK1) is implicated in signaling downstream of the T cell receptor. Its non-catalytic, C-terminal half contains several prolinerich motifs, which have been shown to interact with different SH3 domain-containing adaptor proteins in vitro. One of these, Mona/Gads, was also shown to bind HPK1 in mouse T cells in vivo. The region of HPK1 that binds to the Mona/Gads C-terminal SH3 domain has been mapped and shows only very limited similarity to a recently identified high affinity binding motif in SLP-76, another T-cell adaptor. Using isothermal titration calorimetry and x-ray crystallography, the binding of the HPK1 motif to Mona/Gads SH3C has now been characterized in molecular detail. The results indicate that although charge interactions through an RXXK motif are essential for complex formation, a PXXP motif in HPK1 strongly complements binding. This unexpected binding mode therefore differs considerably from the previously described interaction of Mona/Gads SH3C with SLP-76. The crystal structure of the complex highlights the great versatility of SH3 domains, which allows interactions with very different proteins. This currently limits our ability to categorize SH3 binding properties by simple rules. Hematopoietic progenitor kinase 1 (HPK1) is implicated in signaling downstream of the T cell receptor. Its non-catalytic, C-terminal half contains several prolinerich motifs, which have been shown to interact with different SH3 domain-containing adaptor proteins in vitro. One of these, Mona/Gads, was also shown to bind HPK1 in mouse T cells in vivo. The region of HPK1 that binds to the Mona/Gads C-terminal SH3 domain has been mapped and shows only very limited similarity to a recently identified high affinity binding motif in SLP-76, another T-cell adaptor. Using isothermal titration calorimetry and x-ray crystallography, the binding of the HPK1 motif to Mona/Gads SH3C has now been characterized in molecular detail. The results indicate that although charge interactions through an RXXK motif are essential for complex formation, a PXXP motif in HPK1 strongly complements binding. This unexpected binding mode therefore differs considerably from the previously described interaction of Mona/Gads SH3C with SLP-76. The crystal structure of the complex highlights the great versatility of SH3 domains, which allows interactions with very different proteins. This currently limits our ability to categorize SH3 binding properties by simple rules. Hematopoietic progenitor kinase 1 (HPK1 1The abbreviations used are: HPK1, hematopoietic progenitor kinase 1; MAP, mitogen-activated protein; MAP4K, MAP kinase kinase kinase kinase; GCK, germinal center kinase; JNK, c-Jun N-terminal kinase; ITC, isothermal titration calorimetry; PPII, polyproline type II. ; Human Genome Organization gene symbol MAP4K1) is a member of the germinal center kinase (GCK) family within the large superfamily of STE20 and p21-activated kinases (Refs. 1Dan I. Watanabe N.M. Kusumi A. Trends Cell Biol. 2001; 11: 220-230Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar and 2Sells M.A. Chernoff J. Trends in Cell Biology. 1997; 7: 162-167Abstract Full Text PDF PubMed Scopus (265) Google Scholar). As its name indicates, HPK1 expression is prominent in hematopoietic cells. Initial cloning reports implicated HPK1 in the activation of stress kinases (SAPKs/JNKs) (3Hu M.C. Qiu W.R. Wang X. Meyer C.F. Tan T.H. Genes Dev. 1996; 10: 2251-2264Crossref PubMed Scopus (195) Google Scholar, 4Kiefer F. Tibbles L.A. Anafi M. Janssen A. Zanke B.W. Lassam N. Pawson T. Woodgett J.R. Iscove N.N. EMBO J. 1996; 15: 7013-7025Crossref PubMed Scopus (200) Google Scholar). Since then, HPK1 activity has been associated with lymphocyte antigen receptor signaling (BCR, TCR; Refs. 5Liu S.K. Smith C.A. Arnold R. Kiefer F. McGlade C.J. J. Immunol. 2000; 165: 1417-1426Crossref PubMed Scopus (57) Google Scholar, 6Ma W. Xia C. Ling P. Qiu M. Luo Y. Tan T.H. Liu M. Oncogene. 2001; 20: 1703-1714Crossref PubMed Scopus (44) Google Scholar, 7Tsuji S. Okamoto M. Yamada K. Okamoto N. Goitsuka R. Arnold R. Kiefer F. Kitamura D. J. Exp. Med. 2001; 194: 529-539Crossref PubMed Scopus (55) Google Scholar, 8Kiefer F. Vogel W.F. Arnold R. Transpl. Immunol. 2002; 9: 69-82Crossref PubMed Scopus (18) Google Scholar, 9Han J. Kori R. Shui J.W. Chen Y.R. Yao Z. Tan T.H. J. Biol. Chem. 2003; 278: 52195-52202Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) and in signaling induced by transforming growth factor-β (10Wang W. Zhou G. Hu M.C. Yao Z. Tan T.H. J. Biol. Chem. 1997; 272: 22771-22775Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 11Zhou G. Lee S.C. Yao Z. Tan T.H. J. Biol. Chem. 1999; 274: 13133-13138Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). It has also been shown that physiological concentrations of prostaglandin E2 raise HPK1 kinase activity in T cells and myeloid cells by a yet undefined mechanism, possibly leading to the suppression of fos gene transcription (12Sawasdikosol S. Russo K.M. Burakoff S.J. Blood. 2003; 101: 3687-3689Crossref PubMed Scopus (24) Google Scholar). In addition, a single report suggests a role for HPK1 in erythropoietin-induced signaling of two cell lines (13Nagata Y. Kiefer F. Watanabe T. Todokoro K. Blood. 1999; 93: 3347-3354Crossref PubMed Google Scholar), but this finding remains unconfirmed for primary erythroid progenitor cells. HPK1 is presumed to act as a MAP4K (MAP kinase kinase kinase kinase). Its potential substrates include the mixed lineage kinase MLK3, the mitogenic kinase MEKK1, the transforming growth factor-β activated kinase TAK1 (8Kiefer F. Vogel W.F. Arnold R. Transpl. Immunol. 2002; 9: 69-82Crossref PubMed Scopus (18) Google Scholar), and also c-Jun. 2E. K. Schmidt and S. M. Feller, unpublished data. Furthermore, HPK1 is activated by caspase cleavage (14Chen Y.R. Meyer C.F. Ahmed B. Yao Z. Tan T.H. Oncogene. 1999; 18: 7370-7377Crossref PubMed Scopus (67) Google Scholar, 15Arnold R. Liou J. Drexler H.C. Weiss A. Kiefer F. J. Biol. Chem. 2001; 276: 14675-14684Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) and functions as a regulator of NFκB signaling (15Arnold R. Liou J. Drexler H.C. Weiss A. Kiefer F. J. Biol. Chem. 2001; 276: 14675-14684Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 16Hu M.C. Wang Y. Qiu W.R. Mikhail A. Meyer C.F. Tan T.H. Oncogene. 1999; 18: 5514-5524Crossref PubMed Scopus (48) Google Scholar). Despite these reports, the in vivo functions and signaling pathways of HPK1 remain only marginally defined. HPK1(-/-) mice have been generated by targeted gene disruption but exhibit no obvious phenotype. 3F. Kiefer, personal communication. This may not be entirely surprising, given that HPK1 is just one member of a family of similar kinases with hematopoietic expression (1Dan I. Watanabe N.M. Kusumi A. Trends Cell Biol. 2001; 11: 220-230Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar, 2Sells M.A. Chernoff J. Trends in Cell Biology. 1997; 7: 162-167Abstract Full Text PDF PubMed Scopus (265) Google Scholar). Close relatives with overlapping expression patterns include GCK, kinase homologous to SPS1/STE20 (syn. GCKR), HPK1/GCK-like kinase (named NIK in mice) and germinal center-like kinase. The targeted disruption of multiple GCK family kinase genes in mice may eventually shed more light on HPK1 functions. Physical interactions of HPK1 with other intracellular proteins in vitro and in vivo have been reported in numerous studies. The T-cell adaptor SLP-76 and its B-cell relative BLNK, also known as SLP-65 or BASH, are functionally important interaction partners of HPK1 and crucial in the signal transmission of TCR and BCR, respectively (5Liu S.K. Smith C.A. Arnold R. Kiefer F. McGlade C.J. J. Immunol. 2000; 165: 1417-1426Crossref PubMed Scopus (57) Google Scholar, 7Tsuji S. Okamoto M. Yamada K. Okamoto N. Goitsuka R. Arnold R. Kiefer F. Kitamura D. J. Exp. Med. 2001; 194: 529-539Crossref PubMed Scopus (55) Google Scholar, 17Liou J. Kiefer F. Dang A. Hashimoto A. Cobb M.H. Kurosaki T. Weiss A. Immunity. 2000; 12: 399-408Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 18Sauer K. Liou J. Singh S.B. Yablonski D. Weiss A. Perlmutter R.M. J. Biol. Chem. 2001; 276: 45207-45216Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). After receptor activation, SLP-76 and BLNK bind via their SH2 domains to the phosphorylated tyrosine 397 of human HPK1 (18Sauer K. Liou J. Singh S.B. Yablonski D. Weiss A. Perlmutter R.M. J. Biol. Chem. 2001; 276: 45207-45216Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Other protein complexes are formed via four proline-rich motifs in the non-catalytic region of HPK1. SH3 domain-containing adaptors, including Crk, CRKL, Grb2, Nck, Mona/Gads (syn. Grap-2, GrpL, Grf40, GRID), and HIP-55 have been shown to bind HPK1 (5Liu S.K. Smith C.A. Arnold R. Kiefer F. McGlade C.J. J. Immunol. 2000; 165: 1417-1426Crossref PubMed Scopus (57) Google Scholar, 6Ma W. Xia C. Ling P. Qiu M. Luo Y. Tan T.H. Liu M. Oncogene. 2001; 20: 1703-1714Crossref PubMed Scopus (44) Google Scholar, 9Han J. Kori R. Shui J.W. Chen Y.R. Yao Z. Tan T.H. J. Biol. Chem. 2003; 278: 52195-52202Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 19Anafi M. Kiefer F. Gish G.D. Mbamalu G. Iscove N.N. Pawson T. J. Biol. Chem. 1997; 272: 27804-27811Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 20Ensenat D. Yao Z. Wang X.S. Kori R. Zhou G. Lee S.C. Tan T.H. J. Biol. Chem. 1999; 274: 33945-33950Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 21Oehrl W. Kardinal C. Ruf S. Adermann K. Groffen J. Feng G.S. Blenis J. Tan T.H. Feller S.M. Oncogene. 1998; 17: 1893-1901Crossref PubMed Scopus (52) Google Scholar, 22Ling P. Yao Z. Meyer C.F. Wang X.S. Oehrl W. Feller S.M. Tan T.H. Mol. Cell. Biol. 1999; 19: 1359-1368Crossref PubMed Scopus (78) Google Scholar). Of these, Mona/Gads binding to HPK1 was clearly demonstrated in mouse T cells by co-immunoprecipitation of the endogenous proteins (5Liu S.K. Smith C.A. Arnold R. Kiefer F. McGlade C.J. J. Immunol. 2000; 165: 1417-1426Crossref PubMed Scopus (57) Google Scholar). The same study implicated the fourth proline-rich motif in the non-catalytic part of HPK1 as the binding region for Mona/Gads but did not provide details regarding the HPK1 residues crucial for high affinity binding. We and others have recently shown that the SH3C domain of Mona/Gads can interact with nanomolar affinity with a SLP-76 motif, which lacks a typical PXXP motif (23Liu Q. Berry D. Nash P. Pawson T. McGlade C.J. Li S.S. Mol. Cell. 2003; 11: 471-481Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 24Harkiolaki M. Lewitzky M. Gilbert R.J. Jones E.Y. Bourette R.P. Mouchiroud G. Sondermann H. Moarefi I. Feller S.M. EMBO J. 2003; 22: 2571-2582Crossref PubMed Scopus (95) Google Scholar). In this study, the interaction of HPK1 with Mona/Gads was initially analyzed by isothermal titration calorimetry (ITC). Subsequently, a suitable fragment of HPK1 in complex with Mona/Gads SH3C was crystallized and studied by x-ray crystallography. The structure of the Mona/Gads SH3C-HPK1 peptide complex was solved to 1.5-Å resolution. The data show that, different from the interaction of Mona/Gads SH3C with SLP-76, a PXXP motif, which is part of a polyproline type II helix, is essential for complex formation of murine Mona/Gads SH3C and HPK1. Alanine scanning and crystal contacts define the residues PXVPXRXXK as key sites for complex formation. Peptide synthesis as well as expression and purification of murine Mona/Gads SH3C were done as described previously (24Harkiolaki M. Lewitzky M. Gilbert R.J. Jones E.Y. Bourette R.P. Mouchiroud G. Sondermann H. Moarefi I. Feller S.M. EMBO J. 2003; 22: 2571-2582Crossref PubMed Scopus (95) Google Scholar). The pGEX-2T expression vector for GST-Grb2 SH3N (human amino acids 3–57) is a gift of O. Janssen. Expression and glutathione-Sepharose purification of this fusion protein was done under the same conditions as for Mona/Gads SH3C. The functionality of the construct had been previously confirmed in precipitation experiments using Sos-containing cell lysate. ITC was done essentially as described previously (24Harkiolaki M. Lewitzky M. Gilbert R.J. Jones E.Y. Bourette R.P. Mouchiroud G. Sondermann H. Moarefi I. Feller S.M. EMBO J. 2003; 22: 2571-2582Crossref PubMed Scopus (95) Google Scholar). Briefly, ITC measurements were performed on a VP-ITC MicroCalorimeter (MicroCal, Northhampton, MA). 1.43 ml of 0.05 mm affinity-purified GST-SH3 domain in ITC buffer (25 mm HEPES-KOH, pH 7.5, 100 mm potassium acetate, 5 mm magnesium acetate) was clarified for 10 min at 20,800 × g and degassed for 10 min with Thermovac (MicroCal) before being transferred into the sample chamber. Synthetic peptides were diluted to 0.5 mm in ITC buffer, clarified, and degassed, and ∼300 μl was loaded into the syringe. During the measurements, 5- and 10-μl aliquots of peptide solution were titrated once and 28 times, respectively, into the sample chamber at an equilibrium temperature of 25 °C. The heat of dilution was negligible. Total heat generated and Kd values were calculated in ORIGIN (V5.0). For crystallization, the Mona/Gads SH3C construct at 15 mg/ml was mixed with the 16-residue HPK1 peptide P5 at a 1:3 molar ratio. Crystals were obtained with the sitting drop vapor diffusion method at 20 °C overnight under equilibration conditions of 0.1 m HEPES, pH 7.5, 2 m (NH4)2SO4 (Index Screen, Hampton Research). These were cryoprotected through transfer into mother liquor containing 20% glycerol and vitrified. Single wavelength data were collected on BM14 at the European Synchrotron Radiation Facility, Grenoble, France (see also Table II). The structure was solved by molecular replacement using AMoRe (25Navaza J. Saludjian P. Methods Enzymol. 1997; 276: 581-594Crossref PubMed Scopus (368) Google Scholar) with 1OEB.pdb as the search model. The atomic model was built using the program O (26Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard Acta Crystallogr. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar) and refined with Refmac (27Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. D. 1997; 53: 240-255Crossref PubMed Scopus (13910) Google Scholar); see Table II for details. Generation of structure and density representations, as well as calculation of buried surface areas and surface representations, were done as described previously (24Harkiolaki M. Lewitzky M. Gilbert R.J. Jones E.Y. Bourette R.P. Mouchiroud G. Sondermann H. Moarefi I. Feller S.M. EMBO J. 2003; 22: 2571-2582Crossref PubMed Scopus (95) Google Scholar).Table IIData collection and refinementCrystal dataSpace groupC2Unit cell parameters (Å)a = 71.46, b = 27.44, c = 33.04α = γ = 90°, β = 104.21°Monomers/assymetric unit1Solvent content (%)29.0Data collectionX-ray sourceBM14 (ESRF)Image plate systemMAR CCD 133 mmWavelength (Å)0.979All dataaValues in parenthesis correspond to the highest resolution shellResolution range (Å)20–1.50 (1.55–1.50)Unique reflections9855Average multiplicity3.4Completeness (%)97.4 (98.2)I/σ(I)25.2 (14.7)RmergebRmerge = ΣjΣh (|Ij,h – 〈Ih〉|)/ΣjΣh(〈Ih〉), where h is the unique reflection index, Ij,h is the intensity of the symmetry-related reflection, and 〈Ihrang; is the mean intensity (%)3.0 (6.5)RefinementRworkcR = Σh‖Fo|h – |Fc|h|/Σh|Fo|h, where h defines the unique reflections/RfreedCalculated on random 5% of the data (%)20.7/22.1Atoms protein/peptide/water atoms475/126/63r.m.s.d.eRoot-mean-square deviation bonds (Å)0.012 (0.021)fTarget valuesr.m.s.d.eRoot-mean-square deviation angles (°)1.11 (1.96)fTarget valuesAverage B main/side chain/peptide (Å2)14.46/18.72/24.21Model quality (Ramachandran plot)gValues from PROCHECK (35)Most favored (%)94.7Additional allowed (%)5.3a Values in parenthesis correspond to the highest resolution shellb Rmerge = ΣjΣh (|Ij,h – 〈Ih〉|)/ΣjΣh(〈Ih〉), where h is the unique reflection index, Ij,h is the intensity of the symmetry-related reflection, and 〈Ihrang; is the mean intensityc R = Σh‖Fo|h – |Fc|h|/Σh|Fo|h, where h defines the unique reflectionsd Calculated on random 5% of the datae Root-mean-square deviationf Target valuesg Values from PROCHECK (35Laskowski R.A. Macarthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) Open table in a new tab Characterization of the Sequence Motif in HPK1 Required for Mona/Gads SH3C Binding—Until now, the SH3C domain of Mona/Gads is the only domain of this adaptor known to mediate protein complex formation independent of tyrosine phosphorylation. Previously, we have shown that the Mona/Gads SH3C domain binds with very high affinity to a PX2DRX2KP motif from SLP-76 and that all of the specified residues contribute considerably to complex formation (24Harkiolaki M. Lewitzky M. Gilbert R.J. Jones E.Y. Bourette R.P. Mouchiroud G. Sondermann H. Moarefi I. Feller S.M. EMBO J. 2003; 22: 2571-2582Crossref PubMed Scopus (95) Google Scholar). However, HPK1, which also binds to Mona/Gads independently of tyrosine phosphorylation, lacks such a PX2DRX2KP motif (Fig. 1A). Instead, the implicated region in HPK1 contains only an RXXK motif in the mouse protein and a KXXK motif in the human protein. Additionally, HPK1 proteins have a cluster of proline residues N-terminal to the R/KXXK motif, which is not present in SLP-76, and could possibly contribute to Mona/Gads SH3C binding. To test this hypothesis, peptides corresponding to 26 amino acids of the relevant region in mouse and human HPK1 (Table I, P1 and P2) were analyzed for binding by ITC. Both peptides bound to Mona/Gads SH3C with affinities of 4 and 21 μm, respectively. These are typical affinities for known SH3 domain-peptide interactions.Table IBinding affinities of mouse HPK1 peptides for SH3 domains Peptide binding to SH3 domains was analyzed by isothermal titration calorimetry as indicated under "Experimental Procedures." Mouse Mona/Gads SH3C (amino acids 256–322) and Grb2 SH3N (amino acids 3–57; sequence identical between mouse and human) were expressed as glutathione S-transferase (GST) fusion proteins. nbd, no binding detectable; tlq, affinity too low for accurate quantification, marginal binding; S.D. = standard deviation for two measurements; HAGBP, high affinity Grb2 SH3N binding peptide, similar to Sos sequence. The PXXPXR motif (arrows) is considered to be crucial for Grb2 SH3N binding.View Large Image Figure ViewerDownload (PPT) Open table in a new tab The essential Mona/Gads SH3C binding region in mouse HPK1 was further mapped by analyzing shorter overlapping HPK1 fragments (Table I, P3–P8). A representative example of an ITC measurement is shown in Fig. 1B. P7, a 14-amino-acid motif, was the shortest sequence with an affinity significantly below 10 μm. Further truncation by 3 amino acids (P8) resulted in a 4–5-fold decreased affinity. An alanine scan through P8 subsequently defined the motif PXVPXRX2K as essential for mouse HPK1 binding to Mona/Gads SH3C (Table I, P9–P19). As discussed above, HPK1 has several proline-rich stretches in the non-catalytic part of the protein. In one study, using human HPK1, a region different from the site implicated in the mouse HPK1 has been suggested as the preferential binding site for Mona/Gads SH3C (6Ma W. Xia C. Ling P. Qiu M. Luo Y. Tan T.H. Liu M. Oncogene. 2001; 20: 1703-1714Crossref PubMed Scopus (44) Google Scholar). A corresponding peptide (P20) was therefore also tested for binding to Mona/Gads SH3C by ITC. However, the affinity of this peptide was considerably lower than that of P1, and no further studies were performed with it. The Mona/Gads SH3C binding motif identified in mouse HPK1 clearly overlaps with the PXXPXR core consensus motif for binding of the N-terminal SH3 domain of the Mona/Gads-relative Grb2 as defined previously in several studies (Ref. 28Posern G. Zheng J. Knudsen B.S. Kardinal C. Muller K.B. Voss J. Shishido T. Cowburn D. Cheng G. Wang B. Kruh G.D. Burrell S.K. Jacobson C.A. Lenz D.M. Zamborelli T.J. Adermann K. Hanafusa H. Feller S.M. Oncogene. 1998; 16: 1903-1912Crossref PubMed Scopus (71) Google Scholar and references therein). Grb2 appears to be ubiquitously expressed and might therefore compete with Mona/Gads for some binding partners. Consequently, the affinity of the Grb2 SH3N domain for two HPK1-derived peptides (Table IB, P5 and P8) was also determined. Surprisingly only marginal binding was detected, although an ITC measurement with another PXX- PXR-containing peptide (Table IB, P21) clearly showed that the expressed Grb2 SH3N domain is functional. The significant differences between the HPK1 sequence that binds to Mona/Gads SH3C and the well characterized SH3C binding motif in SLP-76 prompted us to further analyze the Mona/Gads SH3C-HPK1 peptide interaction by x-ray crystallography. Structure of the Mona/Gads SH3C Domain in Complex with an HPK1-derived Peptide—Diffracting crystals were obtained from a complex of Mona/Gads SH3C with HPK1-derived peptide P5. Data and refinement statistics are summarized in Table II. The structure of the complex was solved to 1.5-Å resolution by molecular replacement. A schematic overview of the SH3 domain in complex with the HPK1 peptide is shown in Fig. 2. The structure of the SH3C domain itself remains unaffected by HPK1 peptide binding, retaining all the structural characteristics described previously (24Harkiolaki M. Lewitzky M. Gilbert R.J. Jones E.Y. Bourette R.P. Mouchiroud G. Sondermann H. Moarefi I. Feller S.M. EMBO J. 2003; 22: 2571-2582Crossref PubMed Scopus (95) Google Scholar). The typical β-barrel SH3 domain presents the same binding pocket to the HPK1 peptide as in the case of the SLP-76 peptide (stereo view shown in Fig. 3A), although the interactions between domain and peptide in this structure vary considerably from those established in the previous complex (24Harkiolaki M. Lewitzky M. Gilbert R.J. Jones E.Y. Bourette R.P. Mouchiroud G. Sondermann H. Moarefi I. Feller S.M. EMBO J. 2003; 22: 2571-2582Crossref PubMed Scopus (95) Google Scholar). Sixteen structured residues comprise the HPK1 peptide in its bound conformation (distinguished from the SH3 domain residues by * in the subsequent text). The distinct structural characteristics within the peptide are a 310 helix from residues Arg-9* to Lys-12* and a polyproline type II (PPII) helix of residues Gln-2* to Pro-7* (Figs. 2 and 3B). Although the 310 helix is also present in the high affinity SLP-76 peptide described previously, the PPII helix within the HPK1 peptide is clearly novel for a Mona/Gads SH3C interaction.Fig. 3Divergent stereo electrostatic potential surface representations of the Mona/Gads SH3C with the docked peptide(s) in stick representation. Negative potential is drawn in red, and positive potential is drawn in blue. A, superposition of a HPK1 peptide P5 (green) and an SLP-76 peptide (yellow) on the Mona/Gads SH3C domain. The SLP-76 peptide structure is derived from 1OEB.pdb. B, secondary structure elements in the HPK1 peptide (P5) are highlighted with yellow (310 helix) and red (PPII helix). C, atom-specific coloring of the P5 residues.View Large Image Figure ViewerDownload (PPT) A multitude of interactions defines the HPK1 peptide-Mona/Gads SH3C domain interface. Starting from the N terminus of the HPK1 peptide (P5), the first 3 residues do not contribute to binding, although Pro-3* engages in a very weak hydrophobic interaction with Tyr-8. Pro-4* appears to be the first residue vital for peptide docking (Fig. 3C). The pyrrolidine ring of this residue rests in a hydrophobic groove formed by Tyr-52 and Tyr-8 stacking against the phenyl ring of the former and positioned at a 90° angle to the latter. This interaction is further reinforced by a hydrogen bond from the backbone carboxyl oxygen of Pro-4* to the hydroxyl of the Tyr-52 side chain. Leu-5* exhibits only a single hydrogen bond docking the peptide main chain to Nδ2 of Asn-51. The next hydrophobic groove along the path of the HPK1 peptide on the domain surface is formed by residues Tyr-52, Pro-59, Phe-10, and Trp-36. Tight hydrophobic interactions in this region maintain the side chains of Val-6*, and to a lesser degree Pro-7*, firmly locked in place, making their presence indispensable for tight peptide binding. The pyrrolidine ring of Pro-8* stacks against the aliphatic portion of the Glu-11* side chain. Hydrogen bonds from the backbone oxygen of Pro-8* to the nitrogen of the indole ring of Trp-36 and the backbone amine of Glu-11* dock the peptide main chain onto the SH3 surface and onto itself, stabilizing the 310 helix of P5, which emerges past this point. The charged interactions of amino acids Arg-9* and Lys-12*, which flank the 310 helix, involve a tight network of hydrogen bonds with residues of the Mona/Gads SH3C. Specifically, Nϵ and Nη1 of Arg-9* form hydrogen bonds to Oϵ1 and Oϵ2 of Glu-17, and Lys-12* Nζ forms hydrogen bonds to Glu-14 Oϵ1, Glu-17 Oϵ2, and Asp-16 Oδ1 in a manner identical to that observed in the binding of the SLP-76 peptide to the same interaction surface (24Harkiolaki M. Lewitzky M. Gilbert R.J. Jones E.Y. Bourette R.P. Mouchiroud G. Sondermann H. Moarefi I. Feller S.M. EMBO J. 2003; 22: 2571-2582Crossref PubMed Scopus (95) Google Scholar). In addition, Lys-12* further stacks against the indole ring of Trp-36. Lys-10* does not contribute to SH3 domain binding at all, whereas the aliphatic portion of the Glu-11* side chain stacks between Lys-16 and Pro-8*. All residues of the 310 helix contribute hydrogen bonds toward the formation of the secondary structure element itself. The 4 C-terminal residues of P5 do not participate in protein docking and have no direct interaction with any of the Mona/Gads residues in the vicinity. The observed intra-peptide interactions therein only provide structure to this end of the peptide. Data obtained from binding experiments with SH3- and HPK1-derived Ala mutant peptides in solution (Table I, ITC data) and the contact points between SH3 and HPK1 peptide identified in the crystal structure (summarized in Fig. 1A, indicated by arrows) agree in clearly delineating the residues PXVPXRXXK in mouse HPK1 as important for Mona/Gads SH3C binding. This motif differs considerably from the previously identified contact motif PXIDRXXKPXL derived from the SLP-76 protein (24Harkiolaki M. Lewitzky M. Gilbert R.J. Jones E.Y. Bourette R.P. Mouchiroud G. Sondermann H. Moarefi I. Feller S.M. EMBO J. 2003; 22: 2571-2582Crossref PubMed Scopus (95) Google Scholar), although the RXXK core motif is present in both mouse proteins. Nevertheless, it is evident from the human HPK1 sequence (Fig. 1A) that the arginine can be substituted with a lysine, leaving only a single lysine residue as a strictly conserved position. It is therefore not possible to effectively predict additional candidate binding partners of Mona/Gads SH3C. Comparison of the Mona SH3C-HPK1 Peptide Complex with Other SH3 Domain Complexes—We have previously reported the atomic structure of a SLP-76-derived peptide in complex with Mona/Gads SH3C (24Harkiolaki M. Lewitzky M. Gilbert R.J. Jones E.Y. Bourette R.P. Mouchiroud G. Sondermann H. Moarefi I. Feller S.M. EMBO J. 2003; 22: 2571-2582Crossref PubMed Scopus (95) Google Scholar). The HPK1- and SLP-76-derived SH3C binding peptides both share the 310 helical secondary structure, and accordingly, the respective RXXK motifs are positioned in similar spatial arrangements. Strikingly, although the SLP-76 peptide lacks a PPII helical segment, its C-terminal region occupies approximately the same space as the PPII helix on the hydrophobic surface region of the SH3C (Fig. 3A). The buried surface areas at the SH3-peptide interfaces are quite similar, 1048 Å2 for the bound HPK1 peptide as compared with 1143 Å2 for the complexed SLP-76 peptide and therefore do not provide a simple explanation for the considerable differences in binding affinity of the SLP-76 versus the HPK1 peptide (0.181 and 2.4 μm, respectively; Table I, P5, and Ref. 24Harkiolaki M. Lewitzky M. Gilbert R.J. Jones E.Y. Bourette R.P. Mouchiroud G. Sondermann H. Moarefi I. Feller S.M. EMBO J. 2003; 22: 2571-2582Crossref PubMed Scopus (95) Google Scholar. Stable conformational characteristics of the HPK1 peptide can be identified by analysis of the comparative mean square atomic displacement values (〈u2〉), which provide an indication of the positional stability of each atom in the crystal structure (Fig. 4A). Temperature factors (B = 〈u2〉8π2/3) of P5 atoms fluctuate between 9.6 and 30.9 Å2. This variability is introduced by the terminal residues of the peptide, which are not stabilized by binding to any docking regions on the face of the Mona/Gads domain. The core of P5, defined by the bulk of the two secondary structure arrangements present, one 310 and one PPII helix, is extremely stable. Residues 4* to 12*, which are directly involved with SH3 domain binding, exhibit average B values for main chain atoms of 12.6 Å2 with the side chain atoms showing only marginally higher values averaging 13.8 Å2. Although a direct comparison with the previous Mona/Gads SH3C complex with an SLP-76 peptide is not possible due to variability in crystallographic data quality and crystal lattice environment, we can normalize the average B values for the atoms of the peptides implicated in SH3C binding to a common mean value. Comparative values generated thus suggest that the binding stability of the core ele