Development of a universal phosphorylated peptide-binding protein for simultaneous assay of kinases.
This study describes the development of a universal phosphorylated peptide-binding protein designed to simultaneously detect serine, threonine and tyrosine kinases. The Escherichia coli alkaline phosphatase (EAP) is a well-defined nonspecific phosphated monoesterase and Ser-, Thr- or Tyr-phosphorylated peptides served as substrates for EAP in preliminary experiments. Based on the known catalytic mechanism of EAP, the recombinant site-directed mutant EAP-S102L was generated, whose catalytic activity was blocked, but its binding ability was preserved. For EAP-S102L the catalytic rate constant, k(cat), was reduced by a factor of 1000, while the Michaelis-Menten constant, K(m), remained almost unchanged. Crystallographic analysis of the EAP-S102L/phophorylated peptide complex revealed that EAP-S102L could bind the phosphate group of the phosphorylated peptide but lacked nucleophilic attack potential which was essential for the catalytic ability of EAP. Finally, by combining the fluorescence-labeled EAP-S102L with non-phophorylated peptide chips, kinases could be detected from tumor cell samples. The recombinant EAP-S102L construct is perhaps the first functional binding protein derived from a native enzyme, illustrating how one single mutation tremendously alters protein function.
Li
Wei
W
State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
Bi
Lijun
L
Wang
Wenhua
W
Li
Yongjin
Y
Zhou
Yafeng
Y
Wei
Hongping
H
Jiang
Tao
T
Bai
Lin
L
Chen
Yuanyuan
Y
Zhang
Zhiping
Z
Yuan
Xinghua
X
Xiao
Jianping
J
Zhang
Xian-En
XE
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006801:Humans; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000032:analysis* / Q000378:metabolism; D017354:Point Mutation; D040081:Protein Array Analysis; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000379:methods*
D001426:Bacterial Proteins; D010455:Peptides; D011994:Recombinant Proteins; D010770:Phosphotransferases; D000469:Alkaline Phosphatase