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.

GTPases are important regulatory proteins that hydrolyze GTP to GDP. A novel GTP-hydrolysis mechanism is employed by MnmE, YqeH and FeoB, where a potassium ion plays a role analogous to the Arginine finger of the Ras-RasGAP system, to accelerate otherwise slow GTP hydrolysis rates. In these proteins, two conserved asparagines and a ‘K-loop’ present in switch-I, were suggested as attributes of GTPases employing a K+-mediated mechanism. Based on their conservation, a similar mechanism was suggested for TEES family GTPases. Recently, in Dynamin, Fzo1 and RbgA, which also conserve these attributes, a similar mechanism was shown to be operative. Here, we probe K+-activated GTP hydrolysis in TEES (TrmE-Era-EngA-YihA-Septin) GTPases – Era, EngB and the two contiguous G-domains, GD1 and GD2 of YphC (EngA homologue) – and also in HflX, another GTPase that also conserves the same attributes. While GD1-YphC and Era exhibit a K+-mediated activation of GTP hydrolysis, surprisingly GD2-YphC, EngB and HflX do not. Therefore, the attributes identified thus far, do not necessarily predict a K+-mechanism in GTPases and hence warrant extensive structural investigations.

It has been hypothesized that early enzymes are more promiscuous than their extant orthologues. Whether or not this hypothesis applies to the translation machinery, the oldest molecular machine of life, is not known. Efficient protein synthesis relies on a cascade of specific interactions between the ribosome and the translation factors. Here, using Elongation factor-Tu (EF-Tu) as a model system, we have explored the evolution of ribosome specificity in translation factors. Employing pre-steady state fast kinetics using quench-flow, we have quantitatively characterized the specificity of two sequence-reconstructed 1.3 to 3.3-billion-year-old ancestral EF-Tus towards two unrelated bacterial ribosomes, mesophilic Escherichia coli and thermophilic Thermus thermophilus. While the modern EF-Tus show clear preference for their respective ribosomes, the ancestral EF-Tus show similar specificity for diverse ribosomes. Also, despite increase in the catalytic activity with temperature, the ribosome specificity of the thermophilic EF-Tus remain virtually unchanged. Our kinetic analysis thus suggests that EF-Tu proteins likely evolved from the catalytically promiscuous, ‘generalist’ ancestors. Furthermore, compatibility of diverse ribosomes with the modern and ancestral EF-Tus suggests that the ribosomal core probably evolved before the diversification of the EF-Tus. This study thus provides important insights regarding the evolution of modern translation machinery.

Transcriptional repressor, LexA, regulates the ‘SOS’ response, an indispensable bacterial DNA damage repair machinery. Compared with its Escherichia coli ortholog, LexA from Mycobacterium tuberculosis (Mtb) possesses a unique N-terminal extension of additional 24 amino acids in its DNA-binding domain (DBD) and 18 amino acids insertion at its hinge region that connects the DBD to the C-terminal dimerization/autoproteolysis domain. Despite the importance of LexA in ‘SOS’ regulation, Mtb LexA remains poorly characterized and the functional importance of its additional amino acids remained elusive. In addition, the lack of data on kinetic parameters of Mtb LexA–DNA interaction prompted us to perform kinetic analyses of Mtb LexA and its deletion variants using Bio-layer Interferometry (BLI). Mtb LexA is seen to bind to different ‘SOS’ boxes, DNA sequences present in the operator regions of damage-inducible genes, with comparable nanomolar affinity. Deletion of 18 amino acids from the linker region is found to affect DNA binding unlike the deletion of the N-terminal stretch of extra 24 amino acids. The conserved RKG motif has been found to be critical for DNA binding. Overall, the present study provides insights into the kinetics of the interaction between Mtb LexA and its target ‘SOS’ boxes. The kinetic parameters obtained for DNA binding of Mtb LexA would be instrumental to clearly understand the mechanism of ‘SOS’ regulation and activation in Mtb.

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