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. 1994 Aug;3(8):1261–1266. doi: 10.1002/pro.5560030812

Stability and peptide binding affinity of an SH3 domain from the Caenorhabditis elegans signaling protein Sem-5.

W A Lim 1, R O Fox 1, F M Richards 1
PMCID: PMC2142924  PMID: 7987221

Abstract

We have determined the thermodynamic stability and peptide binding affinity of the carboxy-terminal Src homology 3 (SH3) domain from the Caenorhabditis elegans signal-transduction protein Sem-5. Despite its small size (62 residues) and lack of disulfide bonds, this domain is highly stable to thermal denaturation--at pH 7.3, the protein has a Tm of 73.1 degrees C. Interestingly, the protein is not maximally stable at neutral pH, but reaches a maximum at around pH 4.7 (Tm approximately equal to 80 degrees C). Increasing ionic strength also stabilizes the protein, suggesting that 1 or more carboxylate ions are involved in a destabilizing electrostatic interaction. By guanidine hydrochloride denaturation, the protein is calculated to have a free energy of unfolding of 4.1 kcal/mol at 25 degrees C. We have also characterized binding of the domain to 2 different length proline-rich peptides from the guanine nucleotide exchange factor, Sos, one of Sem-5's likely physiological ligands in cytoplasmic signal transduction. Upon binding, these peptides cause about a 2-fold increase in fluorescence intensity. Both bind with only modest affinities (Kd approximately equal to 30 microM), lower than some previous estimates for SH3 domains. By fluorescence, the domain also appears to associate with the homopolymer poly-L-proline in a similar fashion.

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Selected References

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  1. Alexander P., Fahnestock S., Lee T., Orban J., Bryan P. Thermodynamic analysis of the folding of the streptococcal protein G IgG-binding domains B1 and B2: why small proteins tend to have high denaturation temperatures. Biochemistry. 1992 Apr 14;31(14):3597–3603. doi: 10.1021/bi00129a007. [DOI] [PubMed] [Google Scholar]
  2. Booker G. W., Gout I., Downing A. K., Driscoll P. C., Boyd J., Waterfield M. D., Campbell I. D. Solution structure and ligand-binding site of the SH3 domain of the p85 alpha subunit of phosphatidylinositol 3-kinase. Cell. 1993 May 21;73(4):813–822. doi: 10.1016/0092-8674(93)90259-s. [DOI] [PubMed] [Google Scholar]
  3. Cantley L. C., Auger K. R., Carpenter C., Duckworth B., Graziani A., Kapeller R., Soltoff S. Oncogenes and signal transduction. Cell. 1991 Jan 25;64(2):281–302. doi: 10.1016/0092-8674(91)90639-g. [DOI] [PubMed] [Google Scholar]
  4. Clark S. G., Stern M. J., Horvitz H. R. C. elegans cell-signalling gene sem-5 encodes a protein with SH2 and SH3 domains. Nature. 1992 Mar 26;356(6367):340–344. doi: 10.1038/356340a0. [DOI] [PubMed] [Google Scholar]
  5. Egan S. E., Giddings B. W., Brooks M. W., Buday L., Sizeland A. M., Weinberg R. A. Association of Sos Ras exchange protein with Grb2 is implicated in tyrosine kinase signal transduction and transformation. Nature. 1993 May 6;363(6424):45–51. doi: 10.1038/363045a0. [DOI] [PubMed] [Google Scholar]
  6. Janin J., Chothia C. The structure of protein-protein recognition sites. J Biol Chem. 1990 Sep 25;265(27):16027–16030. [PubMed] [Google Scholar]
  7. Kohda D., Hatanaka H., Odaka M., Mandiyan V., Ullrich A., Schlessinger J., Inagaki F. Solution structure of the SH3 domain of phospholipase C-gamma. Cell. 1993 Mar 26;72(6):953–960. doi: 10.1016/0092-8674(93)90583-c. [DOI] [PubMed] [Google Scholar]
  8. Koyama S., Yu H., Dalgarno D. C., Shin T. B., Zydowsky L. D., Schreiber S. L. Structure of the PI3K SH3 domain and analysis of the SH3 family. Cell. 1993 Mar 26;72(6):945–952. doi: 10.1016/0092-8674(93)90582-b. [DOI] [PubMed] [Google Scholar]
  9. Lim W. A., Richards F. M. Critical residues in an SH3 domain from Sem-5 suggest a mechanism for proline-rich peptide recognition. Nat Struct Biol. 1994 Apr;1(4):221–225. doi: 10.1038/nsb0494-221. [DOI] [PubMed] [Google Scholar]
  10. Musacchio A., Noble M., Pauptit R., Wierenga R., Saraste M. Crystal structure of a Src-homology 3 (SH3) domain. Nature. 1992 Oct 29;359(6398):851–855. doi: 10.1038/359851a0. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Olivier J. P., Raabe T., Henkemeyer M., Dickson B., Mbamalu G., Margolis B., Schlessinger J., Hafen E., Pawson T. A Drosophila SH2-SH3 adaptor protein implicated in coupling the sevenless tyrosine kinase to an activator of Ras guanine nucleotide exchange, Sos. Cell. 1993 Apr 9;73(1):179–191. doi: 10.1016/0092-8674(93)90170-u. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Privalov P. L. Stability of proteins: small globular proteins. Adv Protein Chem. 1979;33:167–241. doi: 10.1016/s0065-3233(08)60460-x. [DOI] [PubMed] [Google Scholar]
  15. Rozakis-Adcock M., Fernley R., Wade J., Pawson T., Bowtell D. The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1. Nature. 1993 May 6;363(6424):83–85. doi: 10.1038/363083a0. [DOI] [PubMed] [Google Scholar]
  16. Simon M. A., Dodson G. S., Rubin G. M. An SH3-SH2-SH3 protein is required for p21Ras1 activation and binds to sevenless and Sos proteins in vitro. Cell. 1993 Apr 9;73(1):169–177. doi: 10.1016/0092-8674(93)90169-q. [DOI] [PubMed] [Google Scholar]
  17. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  18. Viguera A. R., Martínez J. C., Filimonov V. V., Mateo P. L., Serrano L. Thermodynamic and kinetic analysis of the SH3 domain of spectrin shows a two-state folding transition. Biochemistry. 1994 Mar 1;33(8):2142–2150. doi: 10.1021/bi00174a022. [DOI] [PubMed] [Google Scholar]
  19. Yu H., Rosen M. K., Shin T. B., Seidel-Dugan C., Brugge J. S., Schreiber S. L. Solution structure of the SH3 domain of Src and identification of its ligand-binding site. Science. 1992 Dec 4;258(5088):1665–1668. doi: 10.1126/science.1280858. [DOI] [PubMed] [Google Scholar]

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