A single amino acid in the SH3 domain of Hck determines its high affinity and specificity in binding to HIV-1 Nef protein (original) (raw)

Abstract

We have examined the differential binding of Hck and Fyn to HIV-1 Nef to elucidate the structural basis of SH3 binding affinity and specificity. Full-length Nef bound to Hck SH3 with the highest affinity reported for an SH3-mediated interaction (KD 250 nM). In contrast to Hck, affinity of the highly homologous Fyn SH3 for Nef was too weak (KD > 20 microM) to be accurately determined. We show that this distinct specificity lies in a variable loop, the 'RT loop', positioned close to conserved SH3 residues implicated in the binding of proline-rich (PxxP) motifs. A mutant Fyn SH3 with a single amino acid substitution (R96I) in its RT loop had an affinity (KD 380 nM) for Nef comparable with that of Hck SH3. Based on additional mutagenesis studies we propose that the selective recognition of Nef by Hck SH3 is determined by hydrophobic interactions involving an isoleucine residue in its RT loop. Although Nef contains a PxxP motif which is necessary for the interaction with Hck SH3, high affinity binding was only observed for intact Nef protein. The binding of a peptide containing the Nef PxxP motif showed > 300-fold weaker affinity for Hck SH3 than full-length Nef.

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  1. Alexandropoulos K., Cheng G., Baltimore D. Proline-rich sequences that bind to Src homology 3 domains with individual specificities. Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3110–3114. doi: 10.1073/pnas.92.8.3110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cheng G., Ye Z. S., Baltimore D. Binding of Bruton's tyrosine kinase to Fyn, Lyn, or Hck through a Src homology 3 domain-mediated interaction. Proc Natl Acad Sci U S A. 1994 Aug 16;91(17):8152–8155. doi: 10.1073/pnas.91.17.8152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cicchetti P., Mayer B. J., Thiel G., Baltimore D. Identification of a protein that binds to the SH3 region of Abl and is similar to Bcr and GAP-rho. Science. 1992 Aug 7;257(5071):803–806. doi: 10.1126/science.1379745. [DOI] [PubMed] [Google Scholar]
  4. Cohen G. B., Ren R., Baltimore D. Modular binding domains in signal transduction proteins. Cell. 1995 Jan 27;80(2):237–248. doi: 10.1016/0092-8674(95)90406-9. [DOI] [PubMed] [Google Scholar]
  5. Eck M. J., Atwell S. K., Shoelson S. E., Harrison S. C. Structure of the regulatory domains of the Src-family tyrosine kinase Lck. Nature. 1994 Apr 21;368(6473):764–769. doi: 10.1038/368764a0. [DOI] [PubMed] [Google Scholar]
  6. Erpel T., Superti-Furga G., Courtneidge S. A. Mutational analysis of the Src SH3 domain: the same residues of the ligand binding surface are important for intra- and intermolecular interactions. EMBO J. 1995 Mar 1;14(5):963–975. doi: 10.1002/j.1460-2075.1995.tb07077.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Feng S., Chen J. K., Yu H., Simon J. A., Schreiber S. L. Two binding orientations for peptides to the Src SH3 domain: development of a general model for SH3-ligand interactions. Science. 1994 Nov 18;266(5188):1241–1247. doi: 10.1126/science.7526465. [DOI] [PubMed] [Google Scholar]
  8. Huang Y., Zhang L., Ho D. D. Characterization of nef sequences in long-term survivors of human immunodeficiency virus type 1 infection. J Virol. 1995 Jan;69(1):93–100. doi: 10.1128/jvi.69.1.93-100.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Jones T. A., Zou J. Y., Cowan S. W., Kjeldgaard M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A. 1991 Mar 1;47(Pt 2):110–119. doi: 10.1107/s0108767390010224. [DOI] [PubMed] [Google Scholar]
  10. Kato J. Y., Takeya T., Grandori C., Iba H., Levy J. B., Hanafusa H. Amino acid substitutions sufficient to convert the nontransforming p60c-src protein to a transforming protein. Mol Cell Biol. 1986 Dec;6(12):4155–4160. doi: 10.1128/mcb.6.12.4155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kawakami T., Pennington C. Y., Robbins K. C. Isolation and oncogenic potential of a novel human src-like gene. Mol Cell Biol. 1986 Dec;6(12):4195–4201. doi: 10.1128/mcb.6.12.4195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Knudsen B. S., Feller S. M., Hanafusa H. Four proline-rich sequences of the guanine-nucleotide exchange factor C3G bind with unique specificity to the first Src homology 3 domain of Crk. J Biol Chem. 1994 Dec 30;269(52):32781–32787. [PubMed] [Google Scholar]
  13. Knudsen B. S., Zheng J., Feller S. M., Mayer J. P., Burrell S. K., Cowburn D., Hanafusa H. Affinity and specificity requirements for the first Src homology 3 domain of the Crk proteins. EMBO J. 1995 May 15;14(10):2191–2198. doi: 10.1002/j.1460-2075.1995.tb07213.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ladbury J. E., Lemmon M. A., Zhou M., Green J., Botfield M. C., Schlessinger J. Measurement of the binding of tyrosyl phosphopeptides to SH2 domains: a reappraisal. Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3199–3203. doi: 10.1073/pnas.92.8.3199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lemmon M. A., Ladbury J. E. Thermodynamic studies of tyrosyl-phosphopeptide binding to the SH2 domain of p56lck. Biochemistry. 1994 May 3;33(17):5070–5076. doi: 10.1021/bi00183a010. [DOI] [PubMed] [Google Scholar]
  16. Lim W. A., Richards F. M., Fox R. O. Structural determinants of peptide-binding orientation and of sequence specificity in SH3 domains. Nature. 1994 Nov 24;372(6504):375–379. doi: 10.1038/372375a0. [DOI] [PubMed] [Google Scholar]
  17. Mayer B. J., Baltimore D. Signalling through SH2 and SH3 domains. Trends Cell Biol. 1993 Jan;3(1):8–13. doi: 10.1016/0962-8924(93)90194-6. [DOI] [PubMed] [Google Scholar]
  18. Murphy S. M., Bergman M., Morgan D. O. Suppression of c-Src activity by C-terminal Src kinase involves the c-Src SH2 and SH3 domains: analysis with Saccharomyces cerevisiae. Mol Cell Biol. 1993 Sep;13(9):5290–5300. doi: 10.1128/mcb.13.9.5290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Musacchio A., Saraste M., Wilmanns M. High-resolution crystal structures of tyrosine kinase SH3 domains complexed with proline-rich peptides. Nat Struct Biol. 1994 Aug;1(8):546–551. doi: 10.1038/nsb0894-546. [DOI] [PubMed] [Google Scholar]
  20. Musacchio A., Wilmanns M., Saraste M. Structure and function of the SH3 domain. Prog Biophys Mol Biol. 1994;61(3):283–297. doi: 10.1016/0079-6107(94)90003-5. [DOI] [PubMed] [Google Scholar]
  21. Noble M. E., Musacchio A., Saraste M., Courtneidge S. A., Wierenga R. K. Crystal structure of the SH3 domain in human Fyn; comparison of the three-dimensional structures of SH3 domains in tyrosine kinases and spectrin. EMBO J. 1993 Jul;12(7):2617–2624. doi: 10.2210/pdb1shf/pdb. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. O'Shannessy D. J., Brigham-Burke M., Soneson K. K., Hensley P., Brooks I. Determination of rate and equilibrium binding constants for macromolecular interactions using surface plasmon resonance: use of nonlinear least squares analysis methods. Anal Biochem. 1993 Aug 1;212(2):457–468. doi: 10.1006/abio.1993.1355. [DOI] [PubMed] [Google Scholar]
  23. Okada M., Howell B. W., Broome M. A., Cooper J. A. Deletion of the SH3 domain of Src interferes with regulation by the phosphorylated carboxyl-terminal tyrosine. J Biol Chem. 1993 Aug 25;268(24):18070–18075. [PubMed] [Google Scholar]
  24. Pawson T., Schlessingert J. SH2 and SH3 domains. Curr Biol. 1993 Jul 1;3(7):434–442. doi: 10.1016/0960-9822(93)90350-w. [DOI] [PubMed] [Google Scholar]
  25. Potts W. M., Reynolds A. B., Lansing T. J., Parsons J. T. Activation of pp60c-src transforming potential by mutations altering the structure of an amino terminal domain containing residues 90-95. Oncogene Res. 1988;3(4):343–355. [PubMed] [Google Scholar]
  26. Ren R., Mayer B. J., Cicchetti P., Baltimore D. Identification of a ten-amino acid proline-rich SH3 binding site. Science. 1993 Feb 19;259(5098):1157–1161. doi: 10.1126/science.8438166. [DOI] [PubMed] [Google Scholar]
  27. Rickles R. J., Botfield M. C., Weng Z., Taylor J. A., Green O. M., Brugge J. S., Zoller M. J. Identification of Src, Fyn, Lyn, PI3K and Abl SH3 domain ligands using phage display libraries. EMBO J. 1994 Dec 1;13(23):5598–5604. doi: 10.1002/j.1460-2075.1994.tb06897.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Saksela K., Cheng G., Baltimore D. Proline-rich (PxxP) motifs in HIV-1 Nef bind to SH3 domains of a subset of Src kinases and are required for the enhanced growth of Nef+ viruses but not for down-regulation of CD4. EMBO J. 1995 Feb 1;14(3):484–491. doi: 10.1002/j.1460-2075.1995.tb07024.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schlessinger J. SH2/SH3 signaling proteins. Curr Opin Genet Dev. 1994 Feb;4(1):25–30. doi: 10.1016/0959-437x(94)90087-6. [DOI] [PubMed] [Google Scholar]
  30. Shugars D. C., Smith M. S., Glueck D. H., Nantermet P. V., Seillier-Moiseiwitsch F., Swanstrom R. Analysis of human immunodeficiency virus type 1 nef gene sequences present in vivo. J Virol. 1993 Aug;67(8):4639–4650. doi: 10.1128/jvi.67.8.4639-4650.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Superti-Furga G., Fumagalli S., Koegl M., Courtneidge S. A., Draetta G. Csk inhibition of c-Src activity requires both the SH2 and SH3 domains of Src. EMBO J. 1993 Jul;12(7):2625–2634. doi: 10.1002/j.1460-2075.1993.tb05923.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Terasawa H., Kohda D., Hatanaka H., Tsuchiya S., Ogura K., Nagata K., Ishii S., Mandiyan V., Ullrich A., Schlessinger J. Structure of the N-terminal SH3 domain of GRB2 complexed with a peptide from the guanine nucleotide releasing factor Sos. Nat Struct Biol. 1994 Dec;1(12):891–897. doi: 10.1038/nsb1294-891. [DOI] [PubMed] [Google Scholar]
  33. Weng Z., Thomas S. M., Rickles R. J., Taylor J. A., Brauer A. W., Seidel-Dugan C., Michael W. M., Dreyfuss G., Brugge J. S. Identification of Src, Fyn, and Lyn SH3-binding proteins: implications for a function of SH3 domains. Mol Cell Biol. 1994 Jul;14(7):4509–4521. doi: 10.1128/mcb.14.7.4509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wiseman T., Williston S., Brandts J. F., Lin L. N. Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal Biochem. 1989 May 15;179(1):131–137. doi: 10.1016/0003-2697(89)90213-3. [DOI] [PubMed] [Google Scholar]
  35. Wittekind M., Mapelli C., Farmer B. T., 2nd, Suen K. L., Goldfarb V., Tsao J., Lavoie T., Barbacid M., Meyers C. A., Mueller L. Orientation of peptide fragments from Sos proteins bound to the N-terminal SH3 domain of Grb2 determined by NMR spectroscopy. Biochemistry. 1994 Nov 22;33(46):13531–13539. doi: 10.1021/bi00250a004. [DOI] [PubMed] [Google Scholar]
  36. Wolber V., Rensland H., Brandmeier B., Sagemann M., Hoffmann R., Kalbitzer H. R., Wittinghofer A. Expression, purification and biochemical characterisation of the human immunodeficiency virus 1 nef gene product. Eur J Biochem. 1992 May 1;205(3):1115–1121. doi: 10.1111/j.1432-1033.1992.tb16880.x. [DOI] [PubMed] [Google Scholar]
  37. Wu X., Knudsen B., Feller S. M., Zheng J., Sali A., Cowburn D., Hanafusa H., Kuriyan J. Structural basis for the specific interaction of lysine-containing proline-rich peptides with the N-terminal SH3 domain of c-Crk. Structure. 1995 Feb 15;3(2):215–226. doi: 10.1016/s0969-2126(01)00151-4. [DOI] [PubMed] [Google Scholar]
  38. Yu H., Chen J. K., Feng S., Dalgarno D. C., Brauer A. W., Schreiber S. L. Structural basis for the binding of proline-rich peptides to SH3 domains. Cell. 1994 Mar 11;76(5):933–945. doi: 10.1016/0092-8674(94)90367-0. [DOI] [PubMed] [Google Scholar]