Interferon-gamma inducible human guanylate binding protein-1 (hGBP1) shows a unique characteristic that hydrolyzes GTP to a mixture of GDP and GMP through successive cleavages, with GMP being the major product. Like other large GTPases, hGBP1 undergoes oligomerization upon substrate hydrolysis, which is essential for the stimulation of activity. It also exhibits antiviral activity against many viruses including hepatitis C. However, which oligomeric form is responsible for the stimulated activity leading to enhanced GMP formation and its influence on antiviral activity, are not properly understood. Using mutant and truncated proteins, our data indicate that transition state-induced tetramerization is associated with higher rate of GMP formation. This is supported by chimeras, where they are defective in both tetramerization and enhanced GMP formation. Unlike wild type protein, chimeras did not show allosteric interactions, indicating that tetramerization and enhanced GMP formation are allosterically coupled. Hence, we propose that after the cleavage of first phophoanhydride bond GDP.Pi-bound protein dimers transiently associate to form a tetramer that acts as an allosteric switch for higher rate of GMP formation. Biochemical and biophysical studies reveal that sequential conformational changes and interdomain communications regulate tetramer formation via dimer. Our studies also show that overexpression of the mutants, defective in tetramer formation in Rep2a cells don't inhibit proliferation of hepatitis C virus, indicating critical role of a tetramer in the antiviral activity. Thus, this study not only highlights the importance of hGBP1 tetramer in stimulated GMP formation, but also demonstrates its role in the antiviral activity against hepatitis C virus.
- Human guanylate binding protein
- conformational changes
- regulation of GTP hydrolysis
- ©2016 The Author(s)
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