3DPC

Structure of E.coli Alkaline Phosphatase Mutant in Complex with a Phosphorylated Peptide

Summary for 3DPC

• Entry DOI: 10.2210/pdb3dpc/pdb
• Descriptor: Alkaline phosphatase, Phosphorylated Peptide, PHOSPHATE ION, ... (5 entities in total)
• Functional Keywords: alkaline phosphatase, complex structure, protein kinase, hydrolase, magnesium, metal-binding, phosphoprotein
• Biological source: Escherichia coli; More
• Cellular location: Periplasm: P00634
• Total number of polymer chains: 3
• Total formula weight: 97291.99

Authors

Wang, W.H.,Jiang, T. (deposition date: 2008-07-07, release date: 2009-06-16, Last modification date: 2023-11-01)

Primary citation

Li, W.,Bi, L.,Wang, W.,Li, Y.,Zhou, Y.,Wei, H.,Jiang, T.,Bai, L.,Chen, Y.,Zhang, Z.,Yuan, X.,Xiao, J.,Zhang, X.-E.
Development of a universal phosphorylated peptide-binding protein for simultaneous assay of kinases
Biosens.Bioelectron., 24:2871-2877, 2009

PubMed Abstract: 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.

PubMed: 19349157
DOI: 10.1016/j.bios.2009.02.020

Experimental method

X-RAY DIFFRACTION (2.3 Å)