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Mapping the putative binding site for uPA protein in Esophageal Cancer-Related Gene 2 by heteronuclear NMR method
Yong Geng1, Yingang Feng, Tao Xie
1National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China.
Abstract:
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.
Insights
Esophageal Cancer-Related Gene 2 (ECRG2) inhibits esophageal cancer cell growth by binding the urokinase-type plasminogen activator (uPA) system. This study identified ECRG2
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Esophageal Cancer-Related Gene 2 (ECRG2) is a KAZAL-type serine proteinase inhibitor.
- ECRG2 is known to inhibit human esophageal cancer cell proliferation.
- Previous research indicates ECRG2 binds and inhibits the urokinase-type plasminogen activator (uPA)/plasmin system.
Purpose of the Study:
- To establish a method for obtaining properly folded ECRG2 with correct disulfide bonds.
- To investigate the binding interface between ECRG2 and uPA using NMR.
- To determine the specific sequence regions of ECRG2 responsible for uPA binding.
Main Methods:
- Cloning, overexpression, and purification of ECRG2.
- Isotope labeling of ECRG2 for NMR experiments.
- Heteronuclear Nuclear Magnetic Resonance (NMR) spectroscopy to analyze protein-protein interactions.
Main Results:
- A strategy for producing functional ECRG2 was successfully established.
- The binding interface between ECRG2 and uPA was investigated.
- Specific sequence regions (loops) of ECRG2 involved in uPA binding were identified.
Conclusions:
- The identified uPA-binding loops of ECRG2 correspond to the reactive site loops of homologous serine proteinase inhibitors.
- Structural similarity between ECRG2 and turkey ovomucoid third domain (OMTKY3) was observed.
- A structural model for ECRG2 was proposed based on these findings.
