A Distinct Motif in a Prokaryotic Small Ras-Like GTPase Highlights Unifying Features of Walker B Motifs in P-Loop NTPases.

A hallmark of the catalytically essential Walker B motif of P-loop NTPases is the presence of an acidic residue (aspartate/glutamate) for efficient Mg2+ coordination. Although the Walker B motif has been identified in well-studied examples of P-loop NTPases, its identity is ambiguous in many families, for example, in the prokaryotic small Ras-like GTPase family of MglA. MglA, belonging to TRAFAC class of P-loop NTPases, possesses a threonine at the position equivalent to Walker B aspartate in eukaryotic Ras-like GTPases. To resolve the identity of the Walker B residue in MglA, we carried out a comprehensive analysis of Mg2+ coordination on P-loop NTPase structures. Atoms in the octahedral coordination of Mg2+ and their interactions comprise a network including water molecules, Walker A, Walker B and switch motifs of P-loop NTPases. Based on the conserved geometry of Mg2+ coordination, we confirm that a conserved aspartate functions as the Walker B residue of MglA, and validate it through mutagenesis and biochemical characterization. Location of the newly identified aspartate is spatially equivalent to the Walker B residue of the ASCE division of P-loop NTPases. Furthermore, similar to the allosteric regulation of the Walker B aspartate conformation in MglA, we identify protein families in which large conformational changes involving Walker B motif potentially function as allosteric regulators. The study unravels conserved features of Mg2+ coordination among divergent families of P-loop NTPases, especially between ancient Ras-like GTPases and ASCE family of ATPases. The conserved geometric features provide a foundation for design of nucleotide-hydrolyzing enzymes.