Balaji Prakash

Key residues in Mycobacterium tuberculosis protein kinase G play a role in regulating kinase activity and survival in the host.

Protein kinase G (PknG) in Mycobacterium tuberculosis has been shown to modulate phagosome-lysosome fusion. The protein has three distinct domains, an N-terminal Trx domain, a kinase domain, and a C-terminal TPR domain. The present study extensively analyzes the roles of these domains in regulating PknG kinase activity and function. We find that the kinase domain of PknG by itself is inactive, signifying the importance of the flanking domains. Although the deletion of the Trx domain severely impacts the activity of the protein, the C-terminal region also contributes significantly in regulating the activity of the kinase. Apart from this, PknG kinase activity is dependent on the presence of threonine 309 in the p + 1 loop of the activation segment. Mutating the conserved cysteine residues in the Trx motifs makes PknG refractory to changes in the redox environment. In vitro experiments identify threonine 63 as the major phosphorylation site of the protein. Importantly, we find that this is the only site in the protein that is phosphorylated in vivo. Macrophage infection studies reveal that the first 73 residues, the Trx motifs, and the threonine 63 residue are independently essential for modulating PknG-mediated survival of mycobacteria in its host. We have extended these studies to investigate the role of PknG and PknG mutants in the pathogenesis of mycobacteria in mice. Our results reinforce the findings from the macrophage infection experiments, and for the first time demonstrate that the expression of PknG in non-pathogenic mycobacteria allows the continued existence of these bacteria in host tissues.

Structural stabilization of GTP-binding domains in circularly permuted GTPases: Implications for RNA binding

GTP hydrolysis by GTPases requires crucial residues embedded in a conserved G-domain as sequence motifs G1–G5. However, in some of the recently identified GTPases, the motif order is circularly permuted. All possible circular permutations were identified after artificially permuting the classical GTPases and subjecting them to profile Hidden Markov Model searches. This revealed G4–G5–G1–G2–G3 as the only possible circular permutation that can exist in nature. It was also possible to recognize a structural rationale for the absence of other permutations, which either destabilize the invariant GTPase fold or disrupt regions that provide critical residues for GTP binding and hydrolysis, such as Switch-I and Switch-II. The circular permutation relocates Switch-II to the C-terminus and leaves it unfastened, thus affecting GTP binding and hydrolysis. Stabilizing this region would require the presence of an additional domain following Switch-II. Circularly permuted GTPases (cpGTPases) conform to such a requirement and always possess an ‘anchoring’ C-terminal domain. There are four sub-families of cpGTPases, of which three possess an additional domain N-terminal to the G-domain. The biochemical function of these domains, based on available experimental reports and domain recognition analysis carried out here, are suggestive of RNA binding. The features that dictate RNA binding are unique to each subfamily. It is possible that RNA-binding modulates GTP binding or vice versa. In addition, phylogenetic analysis indicates a closer evolutionary relationship between cpGTPases and a set of universally conserved bacterial GTPases that bind the ribosome. It appears that cpGTPases are RNA-binding proteins possessing a means to relate GTP binding to RNA binding.

Analysis of GTPases carrying hydrophobic amino acid substitutions in lieu of the catalytic glutamine: Implications for GTP hydrolysis

Ras superfamily GTP‐binding proteins regulate important signaling events in the cell. Ras, which often serves as a prototype, efficiently hydrolyzes GTP in conjunction with its regulator GAP. A conserved glutamine plays a vital role in GTP hydrolysis in most GTP‐binding proteins. Mutating this glutamine in Ras has oncogenic effects, since it disrupts GTP hydrolysis. The analysis presented here is of GTP‐binding proteins that are a paradox to oncogenic Ras, since they have the catalytic glutamine (Glncat) substituted by a hydrophobic amino acid, yet can hydrolyze GTP efficiently. We term these proteins HAS‐GTPases. Analysis of the amino acid sequences of HAS‐GTPases reveals prominent presence of insertions around the GTP‐binding pocket. Homology modeling studies suggest an interesting means to achieve catalysis despite the drastic hydrophobic substitution replacing the key Glncat of Ras‐like GTPases. The substituted hydrophobic residue adopts a “retracted conformation,” where it is positioned away from the GTP, as its role in catalysis would be unproductive. This conformation is further stabilized by interactions with hydrophobic residues in its vicinity. These interacting residues are strongly conserved and hydrophobic in all HAS‐GTPases, and correspond to residues Asp92 and Tyr96 of Ras. An experimental support for the “retracted conformation” of Switch II arises from the crystal structures of Ylqf and hGBP1. This conformation allows us to hypothesize that, unlike in classical GTPases, catalytic residues could be supplied by regions other than the Switch II (i.e., either the insertions or a neighboring domain). Proteins 2005.

E. coli HflX interacts with 50S ribosomal subunits in presence of nucleotides

HflX is a GTP binding protein of unknown function. Based on the presence of the hflX gene in hflA operon, HflX was believed to be involved in the lytic-lysogenic decision during phage infection in Escherichia coli. We find that E. coli HflX binds 16S and 23S rRNA – the RNA components of 30S and 50S ribosomal subunits. Here, using purified ribosomal subunits, we show that HflX specifically interacts with the 50S. This finding is in line with the homology of HflX to GTPases involved in ribosome biogenesis. However, HflX-50S interaction is not limited to a specific nucleotide-bound state of the protein, and the presence of any of the nucleotides GTP/GDP/ATP/ADP is sufficient. In this respect, HflX is different from other GTPases. While E. coli HflX binds and hydrolyses both ATP and GTP, only the GTP hydrolysis activity is stimulated by 50S binding. This work uncovers interesting attributes of HflX in ribosome binding.

Distinct GDP/GTP bound states of the tandem G-domains of EngA regulate ribosome binding

EngA, a unique GTPase containing a KH-domain preceded by two consecutive G-domains, displays distinct nucleotide binding and hydrolysis activities. So far, Escherichia coli EngA is reported to bind the 50S ribosomal subunit in the guanosine-5′-trihosphate (GTP) bound state. Here, for the first time, using mutations that allow isolating the activities of the two G-domains, GD1 and GD2, we show that apart from 50S, EngA also binds the 30S and 70S subunits. We identify that the key requirement for any EngA–ribosome association is GTP binding to GD2. In this state, EngA displays a weak 50S association, which is further stabilized when GD1 too binds GTP. Exchanging bound GTP with guanosine-5′-diphosphate (GDP), at GD1, results in interactions with 50S, 30S and 70S. Therefore, it appears that GD1 employs GTP hydrolysis as a means to regulate the differential specificity of EngA to either 50S alone or to 50S, 30S and 70S subunits. Furthermore, using constructs lacking either GD1 or both GD1 and GD2, we infer that GD1, when bound to GTP and GDP, adopts distinct conformations to mask or unmask the 30S binding site on EngA. Our results suggest a model where distinct nucleotide-bound states of the two G-domains regulate formation of specific EngA–ribosome complexes.

Deciphering the Catalytic Machinery in 30S Ribosome Assembly GTPase YqeH

Background YqeH, a circularly permuted GTPase (cpGTPase), which is conserved across bacteria and eukaryotes including humans is important for the maturation of small (30S) ribosomal subunit in Bacillus subtilis. Recently, we have shown that it binds 30S in a GTP/GDP dependent fashion. However, the catalytic machinery employed to hydrolyze GTP is not recognized for any of the cpGTPases, including YqeH. This is because they possess a hydrophobic substitution in place of a catalytic glutamine (present in Ras-like GTPases). Such GTPases were categorized as HAS-GTPases and were proposed to follow a catalytic mechanism, different from the Ras-like proteins. Methodology/Principal Findings MnmE, another HAS-GTPase, but not circularly permuted, utilizes a potassium ion and water mediated interactions to drive GTP hydrolysis. Though the G-domain of MnmE and YqeH share only ∼25% sequence identity, the conservation of characteristic sequence motifs between them prompted us to probe GTP hydrolysis machinery in YqeH, by employing homology modeling in conjunction with biochemical experiments. Here, we show that YqeH too, uses a potassium ion to drive GTP hydrolysis and stabilize the transition state. However, unlike MnmE, it does not dimerize in the transition state, suggesting alternative ways to stabilize switches I and II. Furthermore, we identify a potential catalytic residue in Asp-57, whose recognition, in the absence of structural information, was non-trivial due to the circular permutation in YqeH. Interestingly, when compared with MnmE, helix α2 that presents Asp-57 is relocated towards the N-terminus in YqeH. An analysis of the YqeH homology model, suggests that despite such relocation, Asp-57 may facilitate water mediated catalysis, similarly as the catalytic Glu-282 of MnmE. Indeed, an abolished catalysis by D57I mutant supports this inference. Conclusions/Significance An uncommon means to achieve GTP hydrolysis utilizing a K+ ion has so far been demonstrated only for MnmE. Here, we show that YqeH also utilizes a similar mechanism. While the catalytic machinery is similar in both, mechanistic differences may arise based on the way they are deployed. It appears that K+ driven mechanism emerges as an alternative theme to stabilize the transition state and hydrolyze GTP in a subset of GTPases, such as the HAS-GTPases.

A charge reversal differentiates (p)ppGpp synthesis by monofunctional and bifunctional Rel proteins.

A major regulatory mechanism evolved by microorganisms to combat stress is the regulation mediated by (p)ppGpp (the stringent response molecule), synthesized and hydrolyzed by Rel proteins. These are divided into bifunctional and monofunctional proteins based on the presence or absence of the hydrolysis activity. Although these proteins require Mg2+ for (p)ppGpp synthesis, high Mg2+ was shown to inhibit this reaction in bifunctional Rel proteins from Mycobacterium tuberculosis and Streptococcus equisimilis. This is not a characteristic feature in enzymes that use a dual metal ion mechanism, such as DNA polymerases that are known to carry out a similar pyrophosphate transfer reaction. Comparison of polymerase Polβ and RelSeq structures that share a common fold led to the proposal that the latter would follow a single metal ion mechanism. Surprisingly, in contrast to bifunctional Rel, we did not find inhibition of guanosine 5′-triphosphate, 3′-diphosphate (pppGpp) synthesis at higher Mg2+ in the monofunctional RelA from Escherichia coli. We show that a charge reversal in a conserved motif in the synthesis domains explains this contrast; an RXKD motif in the bifunctional proteins is reversed to an EXDD motif. The differential response of these proteins to Mg2+ could also be noticed in fluorescent nucleotide binding and circular dichroism experiments. In mutants where the motifs were reversed, the differential effect could also be reversed. We infer that although a catalytic Mg2+ is common to both bifunctional and monofunctional proteins, the latter would utilize an additional metal binding site formed by EXDD. This work, for the first time, brings out differences in (p)ppGpp synthesis by the two classes of Rel proteins.

The significance of EXDD and RXKD motif conservation in Rel proteins

Monofunctional and bifunctional classes of Rel proteins catalyze pyrophosphoryl transfer from ATP to 3′-OH of GTP/GDP to synthesize (p)ppGpp, which is essential for normal microbial physiology and survival. Bifunctional proteins additionally catalyze the hydrolysis of (p)ppGpp. We have earlier demonstrated that although both catalyze identical the (p)ppGpp synthesis reaction, they exhibit a differential response to Mg2+ due to a unique charge reversal in the synthesis domain; an RXKD motif in the synthesis domain of bifunctional protein is substituted by an EXDD motif in that of the monofunctional proteins. Here, we show that these motifs also determine substrate specificities (GTP/GDP), cooperativity, and regulation of catalytic activities at the N-terminal region through the C-terminal region. Most importantly, a mutant bifunctional Rel carrying an EXDD instigates a novel catalytic reaction, resulting in the synthesis of pGpp by an independent hydrolysis of the 5′Pα-O-Pβ bond of GTP/GDP or (p)ppGpp. Further experiments with RelA from Escherichia coli wherein EXDD is naturally present also revealed the presence of pGpp, albeit at low levels. This work brings out the biological significance of RXKD/EXDD motif conservation in Rel proteins and reveals an additional catalytic activity for the monofunctional proteins, prompting an extensive investigation for the possible existence and role of pGpp in the biological system.

Circularly permuted GTPase YqeH binds 30S ribosomal subunit: Implications for its role in ribosome assembly

YqeH, a circularly permuted GTPase, is conserved among bacteria and eukaryotes including humans. It was shown to be essential for the assembly of small ribosomal (30S) subunit in bacteria. However, whether YqeH interacts with 30S ribosome and how it may participate in 30S assembly are not known. Here, using co-sedimentation experiments, we report that YqeH co-associates with 30S ribosome in the GTP-bound form. In order to probe whether YqeH functions as RNA chaperone in 30S assembly, we assayed for strand dissociation and annealing activity. While YqeH does not exhibit these activities, it binds a non-specific single and double-stranded RNA, which unlike the 30S binding is independent of GTP/GDP binding and does not affect intrinsic GTP hydrolysis rates. Further, S5, a ribosomal protein which participates during the initial stages of 30S assembly, was found to promote GTP hydrolysis and RNA binding activities of YqeH.

Multi-scale carbon micro/nanofibers-based adsorbents for protein immobilization

In the present study, different proteins, namely, bovine serum albumin (BSA), glucose oxidase (GOx) and the laboratory purified YqeH were immobilized in the phenolic resin precursor-based multi-scale web of activated carbon microfibers (ACFs) and carbon nanofibers (CNFs). These biomolecules are characteristically different from each other, having different structure, number of parent amino acid molecules and isoelectric point. CNF was grown on ACF substrate by chemical vapor deposition, using Ni nanoparticles (Nps) as the catalyst. The ultra-sonication of the CNFs was carried out in acidic medium to remove Ni Nps from the tip of the CNFs to provide additional active sites for adsorption. The prepared material was directly used as an adsorbent for proteins, without requiring any additional treatment. Several analytical techniques were used to characterize the prepared materials, including scanning electron microscopy, Fourier transform infrared spectroscopy, BET surface area, pore-size distribution, and UV-vis spectroscopy. The adsorption capacities of prepared ACFs/CNFs in this study were determined to be approximately 191, 39 and 70 mg/g for BSA, GOx and YqeH, respectively, revealing that the carbon micro-nanofibers forming synthesized multi-scale web are efficient materials for the immobilization of protein molecules.