Solution Nuclear Magnetic Resonance Structure of the GATase Subunit and Structural Basis of the Interaction between GATase and ATPPase Subunits in a two-subunit-type GMPS from Methanocaldococcus jannaschii
2013
Ali, Rustam | Kumar, Sanjeev | Balaram, Hemalatha | Sarma, Siddhartha P.
The solution structure of the monomeric glutamine amidotransferase (GATase) subunit of the Methanocaldococcus janaschii (Mj) guanosine monophosphate synthetase (GMPS) has been determined using high-resolution nuclear magnetic resonance methods. Gel filtration chromatography and ¹⁵N backbone relaxation studies have shown that the Mj GATase subunit is present in solution as a 21 kDa (188-residue) monomer. 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. Furthermore, 99.4% of the backbone dihedral angles are present in the allowed region of the Ramachandran map, indicating the stereochemical quality of the structure. The core of the tertiary structure of the GATase is composed of a seven-stranded mixed β-sheet that is fenced by five α-helices. The Mj GATase is similar in structure to the Pyrococcus horikoshi (Ph) GATase subunit. Nuclear magnetic resonance (NMR) chemical shift perturbations and changes in line width were monitored to identify residues on GATase that were responsible for interaction with magnesium and the ATPPase subunit, respectively. These interaction studies showed that a common surface exists for the metal ion binding as well as for the protein–protein interaction. The dissociation constant for the GATase–Mg²⁺ interaction has been found to be ∼1 mM, which implies that interaction is very weak and falls in the fast chemical exchange regime. The GATase–ATPPase interaction, on the other hand, falls in the intermediate chemical exchange regime on the NMR time scale. The implication of this interaction in terms of the regulation of the GATase activity of holo GMPS is discussed.
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