Identification of tyrosine residues involved in ligand recognition by the phosphatidylinositol 3-kinase Src homology 3 domain: circular dichroism and UV resonance Raman studies.
Src homology 3 (SH3) domains are small noncatalytic protein modules capable of mediating protein-protein interactions. We previously demonstrated that the association of a ligand peptide RLP1 (RKLPPRPSK) causes environmental and structural changes of Trp55 and some of seven Tyr residues in the phosphatidylinositol 3-kinase (PI3K) SH3 domain by circular dichroism (CD) and 235-nm excited UV resonance Raman (UVRR) spectroscopies [Okishio, N., et al. (2000) Biopolymers 57, 208-217]. In this work, the affected Tyr residues were identified as Tyr12, Tyr14, and Tyr73 by the CD analysis of a series of mutants, in which every single Tyr residue was replaced by a Phe residue. Among these three residues, Tyr14 was found to be a main contributor to the UVRR spectral change upon the RLP1 binding. Interestingly, CD and UVRR analyses revealed that RLP1 associates with the Y14F and Y14H mutants in different ways. These results suggest that Tyr14 plays a crucial role in the ligand recognition, and the amino acid substitution at Tyr14 affects the mode of PI3K SH3-ligand interaction. Our findings give an insight into how SH3 domains can produce diversity and specificity to transduce signaling within cells.
Okishio
N
N
Department of Biochemistry, School of Medicine, Kanazawa University Faculty of Medicine, Kanazawa, Ishikawa 920-8640, Japan.
Tanaka
T
T
Nagai
M
M
Fukuda
R
R
Nagatomo
S
S
Kitagawa
T
T
D019943:Amino Acid Substitution / Q000235:genetics; D002942:Circular Dichroism; D006639:Histidine / Q000235:genetics; D006801:Humans; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018909:src Homology Domains / Q000235:genetics
D008024:Ligands; D014443:Tyrosine; D010649:Phenylalanine; D006639:Histidine; D019869:Phosphatidylinositol 3-Kinases