Helices on interdomain interface couple catalysis in the ATPPase domain with allostery inPlasmodium falciparumGMP synthetase

Abstract

<jats:title>Abstract</jats:title><jats:p>GMP synthetase catalyzes the substitution of the C2 oxo-group of the purine base in XMP with an amino-group generating GMP, the last step in the biosynthesis of GMP. This reaction involves a series of catalytic events that include hydrolysis of Gln generating ammonia in the glutamine amidotransferase (GATase) domain, activation of XMP to adenyl-XMP intermediate in the ATP pyrophosphatase (ATPPase) domain and reaction of ammonia with the intermediate to generate GMP. Inherent to the functioning of GMP synthetases is bidirectional domain crosstalk, which leads to allosteric activation of the GATase domain by substrates binding to the ATPPase domain, synchronization of the two catalytic events and tunnelling of ammonia from the GATase to the ATPPase domain. Herein, we have taken recourse to the analysis of structures of GMP synthetases, site-directed mutagenesis and, steady-state and transient kinetic assays on the <jats:italic>Plasmodium falciparum</jats:italic> enzyme to decipher the molecular basis of catalysis in the ATPPase domain and domain crosstalk. The results map the residues critical for catalysis in the ATPPase domain to the helices α11 and α12 that are located at the interdomain interface, and the lid-loop that follows α11. This apart, perturbing interdomain interactions involving residues on α11 and α12 impairs GATase activation. These results imply that this arrangement of helices at the domain interface with residues that play roles in ATPPase catalysis as well as domain crosstalk enables coupling ATPPase catalysis with GATase activation. Overall, the study enhances our understanding of GMP synthetases, which are drug targets in many infectious pathogens.</jats:p>

Mechanistic insights into the functioning of GMP synthetase: a two-subunit, allosterically regulated, ammonia tunnelling enzyme

“…28 Following substrate binding, the lid-loop closes on the active site and positions the residues of the lid-loop invariant motif 141 IK(T/S)HHN 146 (MjATPPase numbering) in proximity with the substrates. 28,33 Since the lid-loop is conformationally dynamic, it is disordered in a majority of the crystal structures (Figure S2); however, in the MjATPPase/XMP structure, we could model the residues His145 and Asn146 of the invariant motif (Figure 3C, D and E). The corresponding residues in PfGMPS play roles in catalysing the synthesis of AMP-XMP intermediate and GMP, respectively.…”

“…The dimerization domain is also important for ligand binding as residues on a loop at the Cterminal extreme and inter-dimer interactions of a conserved Arg residue are crucial for binding XMP. 28 In MjATPPase/XMP structure, the interactions of the residues on the C-terminal loop (residues 298-310) with XMP as well as the inter-dimer interactions of Arg294 are conserved (Figure 4B).…”

“…Perturbing these inter-domain interactions in PfGMPS by mutagenesis revealed that the α1-α12 inter-domain saltbridge interactions are conduits for information exchange pertaining to allostery through which the catalytic events in the ATPPase domain are communicated to the GATase domain. 28 Owning to the transient nature of the MjGMPS complex, we resorted to XL-MS and integrative modelling to uncover the structural basis of allostery. Among the 24 inter-subunit cross-links, 14 of them involved residues on the helix Gα1 and the strand Gβ2 of the MjGATase subunit.…”

“…An earlier study using site-directed mutants of PfGMPS has shown that residues on the lid-loop and the subsequent helix (α6 of MjATPPase, residues 166-176) are crucial for the various functions of the ATPPase core domain. 28 Following substrate binding, the lid-loop closes on the active site and positions the residues of the lid-loop invariant motif 141 IK(T/S)HHN 146 (MjATPPase numbering) in proximity with the substrates. 28,33 Since the lid-loop is conformationally dynamic, it is disordered in a majority of the crystal structures (Figure S2); however, in the MjATPPase/XMP structure, we could model the residues His145 and Asn146 of the invariant motif (Figure 3C, D and E).…”

“…14,33,35 The role of the dimerization domain was probed in PfGMPS where the interactions of the residues on the C-terminal loop with XMP were deemed crucial for substrate binding. 28 Moreover, the conformation of the 13residue long C-terminal loop was stabilized by H-bonds with the side chain of Arg539 (PfGMPS numbering) located in the neighbouring chain. Mutation of Arg539 rendered the enzyme inactive on account of impaired substrate binding underscoring the importance of inter-dimer interactions for enzyme function.…”