title: Tetrameric assembly of hGBP1 is crucial for both stimulated GMP formation and antiviral activity.
abstract: Interferon-γ inducible human guanylate binding protein-1 (hGBP1) shows a unique characteristic that hydrolyses 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 chimaeras that are defective in both tetramerization and enhanced GMP formation. Unlike wild-type protein, chimaeras did not show allosteric interactions, indicating that tetramerization and enhanced GMP formation are allosterically coupled. Hence, we propose that after the cleavage of the first phosphoanhydride 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 do not inhibit proliferation of hepatitis C virus, indicating critical role of a tetramer in the antiviral activity. Thus, the present 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.
authors.affiliation: National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
authors.forename: Esha
authors.initials: E
authors.lastname: Pandita
authors.affiliation: National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
authors.forename: Sudeepa
authors.initials: S
authors.lastname: Rajan
authors.affiliation: National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
authors.forename: Safikur
authors.initials: S
authors.lastname: Rahman
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
authors.forename: Ranajoy
authors.initials: R
authors.lastname: Mullick
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
authors.forename: Saumitra
authors.initials: S
authors.lastname: Das
authors.affiliation: National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India apurbaksau@gmail.com apurba@nii.res.in.
authors.forename: Apurba Kumar
authors.initials: AK
authors.lastname: Sau
mesh_terms: D000998:Antiviral Agents / Q000378:metabolism / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D006153:Guanosine Diphosphate / Q000378:metabolism; D006157:Guanosine Monophosphate / Q000378:metabolism*; D016174:Hepacivirus / Q000187:drug effects; D006801:Humans; D011485:Protein Binding; D055503:Protein Multimerization / Q000502:physiology; D017433:Protein Structure, Secondary
chemical_list: D000998:Antiviral Agents; C490287:GBP1 protein, human; D006153:Guanosine Diphosphate; D006157:Guanosine Monophosphate; D019204:GTP-Binding Proteins
keywords: conformational changes; fluorescence; human guanylate binding protein; regulation of GTP hydrolysis