Affinity and specificity requirements for the first Src homology 3 domain of the Crk proteins.
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 Kd of 1.89 +/- 0.06 microM 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.
Knudsen
B S
BS
Laboratory of Molecular Oncology, Rockefeller University, New York, NY 10021, USA.
Zheng
J
J
Feller
S M
SM
Mayer
J P
JP
Burrell
S K
SK
Cowburn
D
D
Hanafusa
H
H
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D004252:DNA Mutational Analysis; D051380:GRB2 Adaptor Protein; D016391:Genes, src; D020662:Guanine Nucleotide Exchange Factors; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D050736:Oncogene Protein v-crk; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D050737:Proto-Oncogene Proteins c-crk; D015682:Retroviridae Proteins, Oncogenic / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; D020662:Guanine Nucleotide Exchange Factors; D009842:Oligopeptides; D050736:Oncogene Protein v-crk; D011506:Proteins; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D015682:Retroviridae Proteins, Oncogenic; D011505:Protein-Tyrosine Kinases