Insight into temperature dependence of GTPase activity in human guanylate binding protein-1.

Anjana Rani,Esha Pandita,Safikur Rahman,Apurba Kumar Sau,Shashank Deep,Karl-Wilhelm Koch

doi:10.1371/journal.pone.0040487

Anjana Rani, Esha Pandita + Show 4 more

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https://doi.org/10.1371/journal.pone.0040487

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Abstract

Interferon-γ induced human guanylate binding protein-1(hGBP1) belongs to a family of dynamin related large GTPases. Unlike all other GTPases, hGBP1 hydrolyzes GTP to a mixture of GDP and GMP with GMP being the major product at 37°C but GDP became significant when the hydrolysis reaction was carried out at 15°C. The hydrolysis reaction in hGBP1 is believed to involve with a number of catalytic steps. To investigate the effect of temperature in the product formation and on the different catalytic complexes of hGBP1, we carried out temperature dependent GTPase assays, mutational analysis, chemical and thermal denaturation studies. The Arrhenius plot for both GDP and GMP interestingly showed nonlinear behaviour, suggesting that the product formation from the GTP-bound enzyme complex is associated with at least more than one step. The negative activation energy for GDP formation and GTPase assay with external GDP together indicate that GDP formation occurs through the reversible dissociation of GDP-bound enzyme dimer to monomer, which further reversibly dissociates to give the product. Denaturation studies of different catalytic complexes show that unlike other complexes the free energy of GDP-bound hGBP1 decreases significantly at lower temperature. GDP formation is found to be dependent on the free energy of the GDP-bound enzyme complex. The decrease in the free energy of this complex at low temperature compared to at high is the reason for higher GDP formation at low temperature. Thermal denaturation studies also suggest that the difference in the free energy of the GTP-bound enzyme dimer compared to its monomer plays a crucial role in the product formation; higher stability favours GMP but lower favours GDP. Thus, this study provides the first thermodynamic insight into the effect of temperature in the product formation of hGBP1.

Highlights

Human guanylate binding protein-1 belongs to a family of large farnesylated GTPases that is induced by interferonc, an immunomodulatory cytokine [1,2,3]
We addressed the following key issues in Human guanylate binding protein-1 (hGBP1); i) how does temperature control the GTPase activity in hGBP1? ii) unlike GMP, why GDP formation is unusually different with temperature (GDP is lower and higher at 37 and 15uC, respectively) and iii) whether the stability of the nucleotide bound dimeric protein has a role in GMP formation? The temperature dependent kinetic assays using radiolabeled (a-32P) GTP on hGBP1 as well as assays with external GDP reveal that GDP formation takes place as a result of the reversible dissociation of GDP-bound enzyme dimer to GDP-bound enzyme monomer that subsequently dissociates into the free enzyme and GDP
The data were fitted to a Hill equation to obtain the kinetic parameters kcat and Km at each temperature and the Arrhenius plots were made for each product

Summary

Introduction

Human guanylate binding protein-1 (hGBP1) belongs to a family of large farnesylated GTPases that is induced by interferonc, an immunomodulatory cytokine [1,2,3]. It is a member of the Dynamin superfamily with molecular mass of about 65–70 kDa [4]. The protein exists as a monomer and dimer in the absence and presence of the analogue GppNHp, respectively [6] This protein has been shown to inhibit VSV (vesicular stomatitis virus) and EMCV (Encephalomyocarditis virus) in HeLa cells [8,9]. It controls the endothelial cell proliferation by inhibiting the matrix metalloproteinase-1 [10,11]

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