Mapping the putative binding site for uPA protein in Esophageal Cancer-Related Gene 2 by heteronuclear NMR method.
Esophageal Cancer-Related Gene 2 (ECRG2) is a novel member of the KAZAL-type serine proteinase inhibitor family and plays an important role in the inhibition of human esophageal cancer cell proliferation. The previous studies have shown that ECRG2 can bind the urokinase-type plasminogen activator (uPA)/plasmin system and inhibit its activity. In this study, the strategy of cloning, overexpression, and purification of ECRG2 for obtaining a properly folded ECRG2 with accurately formed disulfide bonds was established. The heteronuclear NMR experiments were performed with isotope labeled ECRG2 to investigate the binding interface of the protein with uPA. The sequence regions of ECRG2 for uPA binding were determined. Analysis indicates that the uPA-binding loops of ECRG2 are in correspondence with the reactive site loops for binding of serine proteinase in turkey ovomucoid third domain (OMTKY3). The structural similarity of ECRG2 to OMTKY3 was identified and a model for ECRG2 was proposed.
Geng
Yong
Y
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China.
Feng
Yingang
Y
Xie
Tao
T
Dai
Yuanyuan
Y
Wang
Jinfeng
J
Lu
Shih-Hsin
SH
D001665:Binding Sites / Q000502:physiology; D003001:Cloning, Molecular; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D005341:Fibrinolysin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010449:Peptide Mapping / Q000379:methods; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000502:physiology; D053491:Proteinase Inhibitory Proteins, Secretory; D000074162:Serine Peptidase Inhibitors, Kazal Type; D040681:Structural Homology, Protein; D025521:Tumor Suppressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D004220:Disulfides; D053491:Proteinase Inhibitory Proteins, Secretory; C473799:SPINK7 protein, human; D000074162:Serine Peptidase Inhibitors, Kazal Type; D025521:Tumor Suppressor Proteins; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator