Differential ligand recognition by the Src and phosphatidylinositol 3-kinase Src homology 3 domains: circular dichroism and ultraviolet resonance Raman studies.
Src homology 3 (SH3) domains recognize Pro-rich motifs using a hydrophobic cleft which contains several conserved aromatic residues. To investigate how aromatic residues contribute to ligand recognition, circular dichroism (CD) and 235 nm excited ultraviolet resonance Raman spectroscopies have been applied to Src and phosphatidylinositol 3-kinase (PI3K) SH3s. The CD analysis shows that Src SH3 binds to RPLPPLP (R-core) using aromatic residues with a dissociation constant (K(d)) of 10 microM. Moreover, intensity increases of the Trp and Tyr Raman bands suggest that the interaction is mediated by hydrophobic contacts and/or hydrogen bond formation with both Trp and Tyr residues. In the interaction of Src SH3 with VSLARRPLPPLP (VSL12) (K(d) 0.8 microM), Trp118 appears to form a strong hydrogen bond with VSL12, judging from significant intensity increases of the Trp Raman bands and the reported complex structure. In contrast, PI3K SH3 binds to R-core and VSL12 with lower affinities (K(d) 34 and 18 microM, respectively), and the interactions are suggested to be mediated mainly by hydrophobic contacts and/or hydrogen bond formation with Tyr residue(s). In the D21N mutant (Asp21 --> Asn) of PI3K SH3, whose hydrophobic cleft is deformed, Trp55 is shown to be responsible for the interaction with VSL12 by intensity increases of the Trp Raman bands. However, the affinity is severely decreased (K(d) 330 microM). These observations imply that SH3 domains associate with their ligands with distinct use of aromatic residues and that hydrogen bond formation with an SH3-conserved Trp residue in the well-ordered hydrophobic cleft is important for stable complex formation.
Okishio
Nobuyuki
N
Department of Biochemistry, School of Medicine, Kanazawa University Faculty of Medicine, Kanazawa, Ishikawa 920-8640, Japan. okishio@med.kanazawa-u.ac.jp
Tanaka
Toshiyuki
T
Fukuda
Ryuji
R
Nagai
Masako
M
D019943:Amino Acid Substitution / Q000235:genetics; D001216:Asparagine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D002942:Circular Dichroism; D007700:Kinetics; D008024:Ligands; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013059:Spectrum Analysis, Raman; D014364:Tryptophan / Q000235:genetics; D014443:Tyrosine / Q000235:genetics; D018909:src Homology Domains / Q000235:genetics
D008024:Ligands; D010446:Peptide Fragments; D001224:Aspartic Acid; D014443:Tyrosine; D001216:Asparagine; D014364:Tryptophan; D019869:Phosphatidylinositol 3-Kinases; D016392:Proto-Oncogene Proteins pp60(c-src)