Deciphering Structure-Function Relationships in a Two-Subunit-Type GMP Synthetase by Solution NMR Spectroscopy

Abstract

The guanosine monophosphate synthetase (GMPS) is a class I glutamine amidotransferase, involved in the de-novo purine nucleotide biosynthesis. The enzyme catalyzes the biochemical transformation of xantosine (XMP) into guanosine monophosphate (GMP) in presence of ATP, Mg2+ and glutamine. All GMPSs consist of two catalytic sites 1) for GATase activity 2) for the ATPPase activity. The two catalytic sites may be housed in the same polypeptide (two-domain-type) or in separate polypeptides (two-subunit-type). Most of the studies have been performed on two-domain-type GMPSs, while only one study has been reported from two-subunit-type GMPS (Maruoka et al. 2009). The two-subunit-type GMPS presents an example where the component reactions of a single enzymatic reaction are carried out by two distinct subunits. In order to get better understanding of structural aspects and mechanistic principle that governs the GMPS activity in two-subunit-type GMPSs, we initiated the study by taking GMPS of Methanocaldococcus jannaschii as a model system. The GMPS of M. jannaschii (Mj) is a two-subunit-type protein. The GATase subunit catalyzes the hydrolysis of glutamine to produce glutamate and ammonia. The ATPPase subunit catalyses the amination of XMP to produce GMP using the ammonia generated in GATase subunit. Since the two component reactions are catalysed by two separate subunits and are coupled in the way that product of one reaction (ammonia) acts as a nucleophile in the second reaction. The cross-talk between these two subunits in order to maximise the efficiency of overall GMPS warrants investigation. The GATase activity is tightly regulated by the interaction with ATPPase domain/subunit, in all GMPS except in the case of P. falciparum. This interaction is facilitated by substrate binding to the ATPPase domain/subunit. Though, the conditions for the interaction between two subunits is known in a two-subunit-type GMP synthetase from P. horikoshii, the structural basis of substrate dependent interaction is not known. As a first step to understand the structural basis of interaction between the Mj GATase and Mj ATPPase subunits, we have determined the structure of Mj GATase (21 kDa) subunit using high resolution, multinuclear, multidimensional NMR spectroscopy. Sequence specific resonance assignments were obtained through analysis of various 2D and 3D hetero-nuclear multidimensional NMR experiments. NMR based distance restraints were obtained from assignment of correlations observed in NOE based experiments. Data were acquired on isotopically enriched samples of Mj GATase. The structure of Mj GATase (2lxn) was solved by using cyana-3.0 using NMR based restraints as input for the structure calculation. The ensemble of 20 lowest-energy structures showed root-mean-square deviations of 0.35±0.06 Å for backbone atoms and 0.8±0.06 Å for all heavy atoms. Attempts were also made to obtain assignments for the 69.6 kDa dimeric ATPPase subunit. Partial assignments have been obtained for this subunit. The GATase subunit is catalytically inactive. So far, there has been only one published report on a two-subunit-type GMPS from P. horikashii. The study has shown that the catalytic activity of GATase is regulated by the GATase-ATPPase interaction which is facilitated by the substrate binding to the ATPPase subunit. For the first time, we have provided the structural basis of interaction between GATase-ATPPase (112 kDa) in a two-subunit-type GMPS. Observed line width changes were used to identify residues in GATase residues that are involved in the Mj GATase-ATPPase interaction. Our data provides a possible explanation for conformational changes observed in the Mj GATase subunit upon GATase-ATPPase interaction that lead to GATase activation. Ammonia is generated in GATase subunit and is very reactive and labile. Thus, the faithful transportation of ammonia from GATase to ATPPase subunit is very crucial for optimal GMPS activity. Till date, a PDB query for GMPS retrieves only one structure which belongs to two-subunit-type GMPS, where authors have determined the structures of GATase and ATPPase subunits separately. However, the structure of holo-GMPS is not determined yet. Using interface information from experimental data and HADDOCK, we have constructed a model for the holo-GMPS from M. jannaschii. A possible ammonia channel has been deduced using the programs MOLE 2.0 and CAVER 2.0. This ammonia channel has a length of 46 Å, which is well within the range of the lengths calculated for similar channels in other glutamine amidotransferase. It had been suggested earlier that in addition to the magnesium required for charge stabilization of ATP, additional binding sites were present on GMPS. The effect of excess Mg2+ requirement on the GMPS activity has been studied in two-domain-type GMPS. However, the interaction between GATase and Mg2+ has been not investigated in any GMPS. This prompted us to investigate the effect of MgCl2 on Mj GATase subunit. For the first time, using chemical shift perturbation, we have established interaction between Mj GATase and Mg2+. The dissociation constant (Kd) of the Mj GATase-Mg2+ interaction was determined. The Kd value was found to be 1 mM, which indicates a very weak interaction. The substrate of the GATase subunit is glutamine. The condition of the hydrolysis of the glutamine is known in GMPS. However, the binding of the glutamine and associated conformational changes in GATase have been not studied in GMPS. Furthermore, till date there is no structure available for the glutamine bound GMPS/GATase. Using isotope edited one dimensional and two-dimensional NMR spectroscopy; we have shown that the Mj GATase catalytic residues are not in a compatible conformation to bind with glutamine. Thus, a conformational change in Mj GATase subunit is a pre-requisite condition for the binding of glutamine. These conformational changes are brought by the Mj GATase-ATPPase interaction.