Insights into open/closed conformations of the catalytically active human guanylate kinase as investigated by small-angle X-ray scattering.

Rohit Jain,Andreas Menzel,Ivan Rajkovic,Nazimuddin Khan,Manfred Konrad,Simone Techert

doi:10.1007/s00249-015-1079-9

Abstract

Bio-catalysis is the outcome of a subtle interplay between internal motions in enzymes and chemical kinetics. Small-angle X-ray scattering (SAXS) investigation of an enzyme’s internal motions during catalysis offers an integral view of the protein’s structural plasticity, dynamics, and function, which is useful for understanding allosteric effects and developing novel medicines. Guanylate kinase (GMPK) is an essential enzyme involved in the guanine nucleotide metabolism of unicellular and multicellular organisms. It is also required for the intracellular activation of numerous antiviral and anticancer purine nucleoside analog prodrugs. Catalytically active recombinant human GMPK (hGMPK) was purified for the first time and changes in the size and shape of open/closed hGMPK were tracked by SAXS. The binding of substrates (GMP + AMPPNP or Ap5G or GMP + ADP) resulted in the compaction of size and shape of hGMPK. The structural changes between open and completely closed hGMPK conformation were confirmed by observing differences in the hGMPK secondary structures with circular dichroism spectroscopy.Graphical abstractElectronic supplementary materialThe online version of this article (doi:10.1007/s00249-015-1079-9) contains supplementary material, which is available to authorized users.

Highlights

Guanylate kinase (GMPK, ATP:GMP phosphotransferase, EC 2.7.4.8) is an essential enzyme involved in the guanine nucleotide metabolism of unicellular and multicellular organisms
We evaluated the reconstructed Small-angle X-ray scattering (SAXS) models of different human GMPK (hGMPK) conformations with the crystal structure of its murine homologue, the fully closed form of mGMPK (Sekulic et al 2002)
The activity of recombinant hGMPK presented in this work is in contrast to previous reports where hGMPK was found inactive (Ardiani et al 2009; Brady et al 1996)

Summary

Introduction

Guanylate kinase (GMPK, ATP:GMP phosphotransferase, EC 2.7.4.8) is an essential enzyme involved in the guanine nucleotide metabolism of unicellular and multicellular organisms. We evaluated the reconstructed SAXS models (ab initio) of different hGMPK conformations with the crystal structure of its murine homologue, the fully closed form of mGMPK (Sekulic et al 2002). Different hGMPK conformations in the presence and absence of substrates and analogs were measured at the third-generation cSAXS beamline: No ligand (open form), GMP + AMPPNP (completely closed form 1), Ap5G (completely closed form 2), and GMP + ADP (completely closed form 3).

Results

Conclusion