Affinity and specificity requirements for the first Src homology 3 domain of the Crk proteins

B. S. Knudsen, J. Zheng, S. M. Feller, J. P. Mayer, S. K. Burrell, David Cowburn, H. Hanafusa

Research output: Contribution to journalArticle

89 Citations (Scopus)

Abstract

The specificity of SH3 domain complex formation plays an important role in determining signal transduction events. We have previously identified a highly specific interaction between the first CrkSH3 domain [CrkSH3(1)] and proline-rich sequences in the guanine nucleotide exchange factor C3G. A 10 amino acid peptide derived from the first proline-rich sequence (P3P4P5A6L7P8P9K10K11R12) bound with a K(d) of 1.89 ± 0.06 μM and fully retained the high affinity and unique selectivity for the CrkSH3(1) domain. Mutational analysis showed that P5, P8, L7 and K10 are critical for high affinity binding. A conservative mutation, K10R, significantly decreased the affinity for the CrkSH3(1) domain while increasing the affinity for Grb2. Comparative binding studies with the K10R and K10A mutant peptides to c-Crk and v-Crk further suggested that K10 binds via a charge-dependent and a charge-independent interaction to the RT loop of the CrkSH3(1) domain. Besides determining important structural features necessary for high affinity and specificity binding to the CrkSH3(1) domain, our results also demonstrate that a conservative mutation in a single amino acid can significantly alter the specificity of an SH3 binding peptide.

Original languageEnglish (US)
Pages (from-to)2191-2198
Number of pages8
JournalEMBO Journal
Volume14
Issue number10
StatePublished - 1995
Externally publishedYes

Fingerprint

src Homology Domains
Proline
Peptides
Guanine Nucleotide-Releasing Factor 2
Amino Acids
Signal transduction
Mutation
Proteins
Signal Transduction

Keywords

  • C3G
  • Crk
  • Proline-rich sequence
  • SH3 domain

ASJC Scopus subject areas

  • Cell Biology
  • Genetics

Cite this

Knudsen, B. S., Zheng, J., Feller, S. M., Mayer, J. P., Burrell, S. K., Cowburn, D., & Hanafusa, H. (1995). Affinity and specificity requirements for the first Src homology 3 domain of the Crk proteins. EMBO Journal, 14(10), 2191-2198.

Affinity and specificity requirements for the first Src homology 3 domain of the Crk proteins. / Knudsen, B. S.; Zheng, J.; Feller, S. M.; Mayer, J. P.; Burrell, S. K.; Cowburn, David; Hanafusa, H.

In: EMBO Journal, Vol. 14, No. 10, 1995, p. 2191-2198.

Research output: Contribution to journalArticle

Knudsen, BS, Zheng, J, Feller, SM, Mayer, JP, Burrell, SK, Cowburn, D & Hanafusa, H 1995, 'Affinity and specificity requirements for the first Src homology 3 domain of the Crk proteins', EMBO Journal, vol. 14, no. 10, pp. 2191-2198.
Knudsen BS, Zheng J, Feller SM, Mayer JP, Burrell SK, Cowburn D et al. Affinity and specificity requirements for the first Src homology 3 domain of the Crk proteins. EMBO Journal. 1995;14(10):2191-2198.
Knudsen, B. S. ; Zheng, J. ; Feller, S. M. ; Mayer, J. P. ; Burrell, S. K. ; Cowburn, David ; Hanafusa, H. / Affinity and specificity requirements for the first Src homology 3 domain of the Crk proteins. In: EMBO Journal. 1995 ; Vol. 14, No. 10. pp. 2191-2198.
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