Principal Investigator- Dr. Apurba Kumar Sau https://dspace.nii.res.in//https://dspace.nii.res.in/handle/123456789/26 2026-04-27T15:41:52Z 2026-04-27T15:41:52Z Rashmi, Divya Gupta, Sowmiya Kausar, Tasneem Sau, Apurba Kumar https://dspace.nii.res.in//https://dspace.nii.res.in/handle/123456789/1561 2025-12-05T12:47:52Z 2024-01-01T00:00:00Z

Title: Helical domain of hGBP3 cannot stimulate the second phosphate cleavage of GTP Authors: Rashmi, Divya; Gupta, Sowmiya; Kausar, Tasneem; Sau, Apurba Kumar Abstract: Interferon-gamma-inducible large GTPases, hGBPs, possess antipathogenic and antitumor activities in human cells. Like hGBP1, its closest homolog, hGBP3 has two domains; an N-terminal catalytic domain and a C-terminal helical domain, connected by an intermediate region. The biochemical function of this protein and the role of its domains in substrate hydrolysis have not yet been investigated. Here, we report that while hGBP3 can produce both GDP and GMP, GMP is the minor product, 30% (unlike 85% in hGBP1), indicating that hGBP3 is unable to produce enhanced GMP. To understand which domain(s) are responsible for this deficiency, we created hGBP3 truncated variants. Surprisingly, GMP production was similar upon deletion of the helical domain, suggesting that in contrast to hGBP1, the helical domain of hGBP3 cannot stimulate the second phosphate cleavage of GTP. We conducted computational and solution studies to understand the underlying basis. We found that the regulatory residue W79, present in the catalytic domain, forms an H-bond with the backbone carbonyl of K76 (located in the catalytic loop) of the substrate-bound hGBP3. However, after gamma-phosphate cleavage of GTP, the W79-containing region does not undergo a conformational change, failing to redirect the catalytic loop toward the beta-phosphate. This is necessary for efficient GMP formation because hGBP homologs utilize the same catalytic residue for both phosphate cleavages. We suggest that the lack of specific interdomain contacts mediated by the helical domain prevents the catalytic loop movement, resulting in reduced GMP formation. These findings may provide insight into how hGBP3 contributes to immunity.

2024-01-01T00:00:00Z Sau, Apurba Kumar Mittal, Monika Kausar, Tasneem Rajan, Sudeepa Rashmi, Divya https://dspace.nii.res.in//https://dspace.nii.res.in/handle/123456789/1483 2025-12-05T12:48:59Z 2023-01-01T00:00:00Z

Title: Difference in Catalytic Loop Repositioning Leads to GMP Variation between Two Human GBP Homologues Authors: Sau, Apurba Kumar; Mittal, Monika; Kausar, Tasneem; Rajan, Sudeepa; Rashmi, Divya Abstract: Interferon-gamma-inducible human large GTPases, hGBP1 and hGBP2, have a distinctive feature of hydrolyzing GTP to GDP and GMP through successive phosphate cleavages. In hGBP1, GMP is the major product, which is essential for its anti-pathogenic activities. However, its close homologue hGBP2 produces significantly less GMP, despite having a similar active site architecture. The molecular basis for less GMP formation and catalytic residue(s) in hGBP2 are not fully explored. To address these issues, we performed systematic biochemical, biophysical, and microsecond simulation studies. Our data suggest that the less GMP formation in hGBP2 is due to the lack of H-bond formation between the W79 side-chain (located near the active site) and main-chain carbonyl of K76 (present in the catalytic loop) in the substrate-bound hGBP2. The absence of this H-bond could not redirect the catalytic loop toward the beta phosphate after the cleavage of gamma-phosphate, a step essential for enhanced GMP formation. Furthermore, based on the mutational and structural analyses, this study for the first time indicates that the same residue, T75, mediates both phosphate cleavages in hGBP2 and hGBP1. This suggests the conservation of the catalytic residue in hGBP homologues. These findings emphasize the indispensable role of correct catalytic loop repositioning for efficient beta phosphate cleavage. This led us to propose a new substrate hydrolysis mechanism by hGBP1 and hGBP2, which may also be helpful to understand the GTP hydrolysis in other hGBP homologues. Overall, the study could provide insight into how these two close homologues play crucial roles in host-mediated immunity through different mechanisms.

2023-01-01T00:00:00Z Sau, Apurba Kumar https://dspace.nii.res.in//https://dspace.nii.res.in/handle/123456789/1336 2025-12-05T12:45:20Z 2016-01-01T00:00:00Z

Title: Tetrameric assembly of hGBP1 is crucial for both stimulated GMP formation and antiviral activity Authors: Sau, Apurba Kumar 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.

2016-01-01T00:00:00Z Sau, Apurba Kumar https://dspace.nii.res.in//https://dspace.nii.res.in/handle/123456789/1335 2025-12-05T12:46:58Z 2017-01-01T00:00:00Z

Title: Arginase of Helicobacter Gastric Pathogens Uses a Unique Set of Non-catalytic Residues for Catalysis Authors: Sau, Apurba Kumar Abstract: Helicobacter pylori arginase, a bimetallic enzyme, is crucial for pathogenesis of the bacterium in human stomach. Despite conservation of the signature motifs in all arginases, the H. pylori homolog has a non-conserved motif (153ESEEKAWQKLCSL165), whose role was recently shown to be critical for its stability and function. The sequence analysis also reveals the presence of this motif with critical residues in the homolog of other Helicobacter gastric pathogens. However, the underlying mechanism for its significance in catalytic function is not clearly understood. Using H. pylori arginase, our studies reveal that the interactions of His122 and Tyr125 with Trp159 are indispensable for tertiary structural intactness through optimal positioning of the motif and thus for the catalytic function. The single and double mutants of His122 and Tyr125 not only enhanced the solvent accessibility and conformational flexibility of Trp159 in the holo protein, but also showed complete loss of catalytic activity. An intact bimetallic center and unaltered substrate binding indicate that proper positioning of the motif by aromatic-aromatic contact is vital for the generation of a catalytically active conformation. Additionally, the metal ions provide higher stability to the holo protein. We also identified the presence of these two residues exclusively in arginase of other Helicobacter gastric pathogens, which may have similar function. Therefore, to the best of our knowledge, these findings highlight for the first time that arginase of all Helicobacter gastric pathogens utilizes a unique non-catalytic triad for catalysis, which could be exploited for therapeutics.

2017-01-01T00:00:00Z