Structural Basis Unifying Diverse GTP Hydrolysis Mechanisms

Abstract

Central to biological processes is the regulation rendered by GTPases. Until recently, the GTP hydrolysis mechanism, exemplified by Ras-family (and G-α) GTPases, was thought to be universal. This mechanism utilizes a conserved catalytic Gln supplied "in cis" from the GTPase and an arginine finger "in trans" from a GAP (GTPase activating protein) to stabilize the transition state. However, intriguingly different mechanisms are operative in structurally similar GTPases. MnmE and dynamin like cation-dependent GTPases lack the catalytic Gln and instead employ a Glu/Asp/Ser situated elsewhere and in place of the arginine finger use a K(+) or Na(+) ion. In contrast, Rab33 possesses the Gln but does not utilize it for catalysis; instead, the GAP supplies both a catalytic Gln and an arginine finger in trans. Deciphering the underlying principles that unify seemingly unrelated mechanisms is central to understanding how diverse mechanisms evolve. Here, we recognize that steric hindrance between active site residues is a criterion governing the mechanism employed by a given GTPase. The Arf-ArfGAP structure is testimony to this concept of spatial (in)compatibility of active site residues. This understanding allows us to predict an as yet unreported hydrolysis mechanism and clarifies unexplained observations about catalysis by Rab11 and the need for HAS-GTPases to employ a different mechanism. This understanding would be valuable for experiments in which abolishing GTP hydrolysis or generating constitutively active forms of a GTPase is important.

Nmd3 is a structural mimic of eIF 5A, and activates the cp GTP ase Lsg1 during 60S ribosome biogenesis

“…The model accounts for residues 135 through 503 of Lsg1. We also do not observe density corresponding to the expected K + ion that stabilizes Switch I in this family of GTPases (Anand et al, 2013); however, the absence of K + may be due to the use of the GTP analog GMPPNP, which has been reported to destabilize the coordination shell of the monovalent ion (Kuhle & Ficner, 2014). Lsg1 is a circularly permuted GTPase, with the sequence motifs appearing in the order of G4-G5-G1-G2-G3 (Fig 5A).…”

Autoinhibition and Signaling by the Switch II Motif in the G-protein Chaperone of a Radical B12 Enzyme

“…They are among most common and ancient regulatory proteins in nature (14). An increasing body of evidence demonstrates significant variations in the mechanisms of and factors used for GTP hydrolysis and signal transduction that are not readily predicted by sequence and structural homology (52). This complexity is also evident within the G3E metallochaperone family in that MeaB, which possesses an atypical active site, is stabilized in an autoinhibitory conformation via its switch II motif in the absence of the GAP protein MCM.…”

Invited review: Small GTPases and their GAPs

“…In the crystal structure of dimeric MnmE with the transition state mimetic GDP‐ , a conserved glutamate residue from switch II compensates for the absence of a cis‐glutamine through an indirect water‐mediated polar interaction with the nucleophilic water . These and related examples have been reviewed in the context of potentially unifying principles, including steric hindrance arising from P‐loop substitutions at the position equivalent to Ras Gly12, as a selective pressure for evolution of diverse GAP‐GTPase mechanisms …”