Boundary sequences of the NADPH oxidase p67(phox) C-terminal SH3 domain play on its specificity.
SH3 domains are found in many signal transduction proteins where they mediate protein-protein binding by recognizing specific peptides rich in proline. Based on the analysis of sequence alignment data, the NADPH oxidase p67(phox) C-terminal SH3 domain possesses a typical compact beta-barrel consisting of five beta-strands arranged in two antiparallel beta-sheets of three and two beta-strands. Multiple amino acid substitutions were made at beta e and its flanking residues to determine the role of the boundary sequences in binding activity and conformational specificity of the domain. Analysis of amino acid P55 indicated that all mutants were completely abolished in their binding activities. The substitution of F58 with Y58 showed no effect of the binding, whereas substitution with stop codon abolished activity. Furthermore, when amino acid V59 was substituted with stop codon, activity was also completely abolished. Substitution of E60 with stop codon showed no effect of binding. Moreover, our data show that V59 particularly could not be replaced by Leu. Taken together, these data suggest that V59 may not only contribute the exact boundary site but also play on the specificity for protein-protein interactions in phagocyte NADPH oxidase.
Hwang
S L
SL
Neurosurgery Department, Kaohsiung Medical University, Kaohsiung, Taiwan, Republic of China
Cheng
T S
TS
Chen
C H
CH
Sun
Y J
YJ
Hsiao
C D
CD
Hong
Y R
YR
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019255:NADPH Oxidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D020798:Two-Hybrid System Techniques; D018909:src Homology Domains
D010750:Phosphoproteins; D011994:Recombinant Proteins; C079789:neutrophil cytosol factor 67K; D019255:NADPH Oxidases