Role of Individual Domains and Identification of Internal Gap in Human Guanylate Binding Protein-1

Abdullah, Nazish; Srinivasan, Bharani; Modiano, Nir; Cresswell, Peter; Sau, Apurba Kumar

doi:10.1016/j.jmb.2008.12.060

Cited by 30 publications

(67 citation statements)

References 35 publications

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“…The high Hill coefficient for a dimeric protein could be due to the formation of further higher order structure during the course of hydrolysis. The higher degree of cooperativity has been reported in many cases, where the Hill coefficient was determined to be 1.8 or 4 for a dimeric or tetrameric protein, respectively (23,24). The data also indicate that the binding of the substrate to one active site highly regulates the activity of the other.…”

Section: Cooperativity and Concentration-dependent Oligomerization Anmentioning

confidence: 55%

Biochemical studies on Helicobacter pylori arginase: Insight into the difference in activity compared to other arginases

Srivastava

2010

IUBMB Life

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SummaryArginase is a binuclear Mn 21 -metalloenzyme of urea cycle that catalyzes the conversion of L-arginine to L-ornithine and urea. Unlike other arginases, the Helicobacter pylori enzyme is selective for Co 21 , and has lower catalytic activity. To understand the differences in the biochemical properties as well as activity compared to other arginases, we carried out a detailed investigation of different metal reconstituted H. pylori arginases that includes steady-state kinetics, fluorescence measurement, pH-dependent and oligomerization assays. Unlike other arginases (except human at physiological pH), the Co 21 -and Mn 21 -reconstituted H. pylori enzymes exhibit cooperative mechanism of arginine hydrolysis, and undergo self-association and activation with increasing concentrations. Analytical gel-filtration assays in conjunction with the kinetic data showed that the protein exists as a mixture of monomer and dimer with monomer being the major form (other arginases exclusively exist as a trimer or hexamer) but the dimer is associated with higher catalytic activity. The proportion of dimer is found to decrease with increasing salt concentrations indicating that salt bridges play important roles in dimerization of the protein. Furthermore, the fluorescence measurement showed that Co 21 ions play an important role in the local tertiary structure of the protein than Mn 21 . This is consistent with the pH-dependent studies where the Co 21 -enzyme showed a single ionization compared to the double in the Mn 21 -enzyme. Thus, this study presents the detailed biochemical and spectroscopic investigations into the differences in the biochemical properties and activity between H. pylori and other arginases.

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Section: Cooperativity and Concentration-dependent Oligomerization Anmentioning

confidence: 55%

Biochemical studies on Helicobacter pylori arginase: Insight into the difference in activity compared to other arginases

Srivastava

2010

IUBMB Life

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“…Enzymatic studies of various hGBP1 deletion mutants by Abdullah et al support the conclusion that the helical domain is more important for the cleavage of the second phosphate group, but they suggest a different mechanism. 23 Nevertheless, hGBP1(looseα12/13) constitutively associated with a dimer or tetramer through the helical part leads to a different localization within the cell. How far the coupling of enzymatic activity, conformation changes, and self-assembly of hGBP1 are important for changes in subcellular localization and for biological function must be further investigated.…”

Section: Resultsmentioning

confidence: 99%

Mechanism of GTPase-Activity-Induced Self-Assembly of Human Guanylate Binding Protein 1

Vöpel¹,

Syguda²,

Britzen-Laurent³

et al. 2010

Journal of Molecular Biology

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“…This protein has also a unique 103 DxEKGD 108 motif near the active site, which is critical for GMP formation [29]. D108A hydrolyses GTP to GDP and GMP with a ratio of GMP to GDP ∼ 1:1 (for wild type it is ∼5.8:1), whereas D103L·D108L ( Figure 1A) completely abrogates GMP formation [29].…”

Section: Scheme 1 Description Of the Full-length Truncated And Chimamentioning

confidence: 99%

“…We have previously described a dimeric model of hGBP1 that proposes the hydrolysis of GTP as follows [28,29]. After the cleavage of the first phosphoanhydride bond in GTP, there is a competition between the further cleavage of the phosphoanhydride bond in the GDP-bound enzyme dimer to form GMP and dissociation of this dimer into a monomer leading to GDP formation.…”

Section: Scheme 1 Description Of the Full-length Truncated And Chimamentioning

confidence: 99%

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Tetrameric assembly of hGBP1 is crucial for both stimulated GMP formation and antiviral activity

Pandita

Rajan

Rahman

et al. 2016

Biochemical Journal

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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.

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Role of Individual Domains and Identification of Internal Gap in Human Guanylate Binding Protein-1

Cited by 30 publications

References 35 publications

Biochemical studies on Helicobacter pylori arginase: Insight into the difference in activity compared to other arginases

Biochemical studies on Helicobacter pylori arginase: Insight into the difference in activity compared to other arginases

Mechanism of GTPase-Activity-Induced Self-Assembly of Human Guanylate Binding Protein 1

Tetrameric assembly of hGBP1 is crucial for both stimulated GMP formation and antiviral activity

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