A revised mechanism for (p)ppGpp synthesis by Rel proteins: The critical role of the 2'-OH of GTP.

Bacterial Rel proteins synthesize hyperphosphorylated guanosine nucleotides, denoted as (p)ppGpp, which by inhibiting energy requiring molecular pathways help bacteria to overcome the depletion of nutrients in its surroundings. (p)ppGpp synthesis by Rel involves transferring a pyrophosphate from ATP to the oxygen of 3'-OH of GTP/GDP. Initially, a conserved glutamate at the active site was believed to generate the nucleophile necessary to accomplish the reaction. Later this role was alluded to a Mg2+ ion. However, no study has unequivocally established a catalytic mechanism for (p)ppGpp synthesis. Here we present a revised mechanism, wherein for the first time we explore a role for 2'-OH of GTP and show how it is important in generating the nucleophile. Through a careful comparison of substrate-bound structures of Rel, we illustrate that the active site does not discriminate GTP from dGTP, for a substrate. Using biochemical studies, we demonstrate that both GTP and dGTP bind to Rel, but only GTP (but not dGTP) can form the product. Reactions performed using GTP analogs substituted with different chemical moieties at the 2' position suggest a clear role for 2'-OH in catalysis by providing an indispensable hydrogen bond; preliminary computational analysis further supports this view. This study elucidating a catalytic role for 2'-OH of GTP in (p)ppGpp synthesis allows us to propose different mechanistic possibilities by which it generates the nucleophile for the synthesis reaction. This study underscores the selection of ribose nucleotides as second messengers and finds its roots in the old RNA world hypothesis.

Purification and preliminary characterization of four Rel homologues from pathogenic bacteria: Implications for species-specific inhibitor design.

Resistance to antibiotics is a serious concern to treat infectious diseases and also, for food preservation. Existing antibiotics generally inhibit enzymes participating in key bacterial processes, such as formation of cell wall, replication, transcription and translation. However, bacteria have rapidly evolved new mechanisms to combat these antibiotics and it hence becomes indispensable to identify newer targets and identify/design inhibitors against them. Another concern is that most antibiotics are broad spectrum; they largely bind and inhibit the active site of the target enzyme. Rel proteins, which synthesize (and hydrolyze) (p)ppGpp in response to a variety of stress encountered by bacteria, is a profitable target owing to its distinct absence in humans and an intricate regulation of the catalytic activities. Inactivation of (p)ppGpp synthesis by Rel, disables bacterial survival in Mycobacterium tuberculosis and Staphylococcus aureus, while inactivating the hydrolysis activity was lethal. The poor MIC values of the currently known Rel inhibitors present a distinct opportunity to develop better inhibitors and warrants a detailed structural characterization and understanding of the complex regulation in Rel proteins. It will open new avenues for the design of effective, species-specific inhibitors. In an attempt to identify unique sites for inhibitor design using structure-based approaches, we initiate a study of Rel homologues from four different pathogenic bacteria, in order to compare their attributes with well characterized Rel homologues. Here, we present cloning, over-expression, purification and preliminary characterization of these four homologues; and suggest similarities and differences that can be exploited for inhibitor design.

Mechanism of Nucleotidyltransfer Reaction and Role of Mg2+ Ion in Sugar Nucleotidyltransferases.

Sugar nucleotidyl transferases (SNTs) catalyze nucleotidyltransfer reactions to form sugar-nucleotides and pyrophosphate in the presence of two Mg2+ ions (Mg2+A and Mg2+B). We unveil the mechanism and free energetics of nucleotidyl transfer reaction in an SNT called GlmU through hybrid quantum mechanics-molecular mechanics molecular dynamics simulations and free energy calculations. The study identifies the roles of the active site residues and the Mg2+ ions in catalyzing the reaction. Of great significance, we are able to compare the free energy barrier for the reaction with that for the Mg2+-assisted release of the product (i.e., pyrophosphate) into the solution, shedding light on the general mechanistic and kinetic aspects of catalysis by SNTs.

Classification, characterization and structural analysis of sugar nucleotidylyltransferase family of enzymes.

Sugar Nucleotidyl Transferases (SNTs) constitute a large family of enzymes that play important metabolic roles. Earlier, for one such SNT, termed N-acetylglucosamine-1-phosphate uridyltransferase- GlmU, we had established that two magnesium ions - Mg2+A and Mg2+B - catalyze the sugar-nucleotidyl transfer reaction. Despite a common structural framework that SNTs share, we recognized key differences around the active-site based on the analysis of available structures. Based on these differences, we had classified SNTs into two major groups, Group - I & II; and further, variation in 'Mg2+A-stabilizing motifs' led us to sub-classify them into five distinct sub-groups. Since group specific conservation of 'Mg2+A-stabilizing motifs' was based only for 45 available structures, here we validate this via an exhaustive analysis of 1,42,025 protein sequences. Previously, we had hypothesized that a metal-ion-catalyzed mechanism would be operative in all SNTs. Here, we validate it biochemically and establish that Mg2+ is a strict requirement for nucleotidyl transfer reactions in every group or sub-group and that a common metal ion dependent mechanism operates in SNTs. Further, mutating Mg2+A coordinating residue in each sub-group led to abolished catalysis, indicating an important role for both of these residues and suggest that SNTs employ variations over 'a conserved catalytic mechanism mediated by Mg2+ ion(s)', to bring about functional diversity. This would constitute a comprehensive study to establish the catalytic mechanism across the family of SNTs.

Structural basis of noncanonical polyphenol oxidase activity in DLL-II: A lectin from Dolichos lablab.

Lectins known to possess an additional enzymatic function are called leczymes. Previous studies reported a unique polyphenol oxidase (PPO) activity in DLL-II-a leczyme from Dolichos lablab. DLL-II shares a high sequence and structural homology with DBL-another leczyme from Dolichos biflorus. Incidentally, DBL possesses lipoxygenase activity, but not the PPO activity. Legume lectins usually possess two metal-binding sites A and B. Although these sites are conserved in both DBL and DLL-II, site A in DLL-II is occupied by Mn2+ and site B by Ca2+ . In contrast, DLL-II binds Cu2+ and Ca2+ at sites A and B, respectively. Here, investigating the structural basis of PPO activity in DLL-II, we find that the PPO activity is only dependent on Cu2+ , but not Ca2+ ; and the lectin activity requires only Ca2+ . Further, our analysis suggests that an alternative mechanism of PPO reaction may be operative in DLL-II, which involves a mononuclear Cu2+ metal center; this is in contrast to the bi-nuclear Cu2+ metal center commonly observed in all PPOs. Importantly, structural and computational approaches employed here, we hypothesize possible PPO binding sites and the corresponding migration channels for accessing the active site.

Disrupting domain-domain interactions is indispensable for EngA-ribosome interactions.

EngA consists of two tandem GTPase-domains-GD1 and GD2-followed by a KH-domain. EngA was considered to be a 50S assembly factor since it was shown to bind 50S and its deletion leads to the accumulation of immature 45S ribosomal subunits. Subsequently, we demonstrated an additional ribosome bound state of EngA bound to 50S, 30S, and 70S. While the former (50S binding) is achieved upon GTP binding at both GD1 and GD2, the latter is formed upon GTP hydrolysis at GD1, which is believed to trigger a large conformational change in the protein. The present study brings out two key aspects of EngA regulation: First, that distinctly stabilized GD1-KH interfaces allows EngA to exist in different ribosome bound states, and second is the importance of these states to ribosome assembly. Our analyses suggest that distinct inter-domain (GD-KH) interfaces are stabilized by interactions arising from unique sets of motifs, conserved across EngA homologues, and seem to be mechanistically linked to GTP/GDP binding. By experimentally measuring binding affinities for several interface mutants, we show that disrupting the interface interactions is necessary to realize EngA-ribosome binding. These findings are also supported by a recent cryo-EM structure of EngA bound to 50S, wherein the GD1-KH interface is completely disrupted leading to an 'extended' or 'open state' of the protein. Overall, it appears that the transition of EngA from a 'closed state' with GD1-KH forming a tight interface, to an 'open state' mediates interaction with ribosomal subunits.

A salt-bridge stabilized C-terminal hook is critical for the dimerization of a Bowman Birk inhibitor.

Legume Bowman-Birk inhibitors (BBIs) that inhibit mammalian proteases exist as dimers in solution. The structural basis governing dimerization of HGI-III (horsegram seed BBI) was investigated. An intra-monomer salt bridge (D76-K71) stabilizes an atypical hook-like conformation at the C-terminus. We postulate that this hook, positions D75 to enable an inter-monomer salt-bridge D75(a)-K24(b), which results in dimerization. We verify this by K71A and D76A mutations of HGI-III. The mutants were both monomers, likely due to destabilization of the C-terminal hook. Dimerization was sustained in a double mutant K71D/D76K that was anticipated to form a similar hook critical for dimerization. Conversely, K24(b) that interacts with D75(a) of the loop is the specificity determining residue that interacts with trypsin to inhibit its activity. The inter-monomer salt bridge D75(a)-K24(b) must be disrupted for the inhibition of trypsin, requiring HGI-III to transition into a monomer. Size exclusion studies and a model of HGI-III-trypsin complex support this notion. Interestingly, isoforms of the inhibitor present in germinated seeds (HGGIs) are monomers; and most strikingly, the C-termini of these inhibitors are truncated with the loss the C-terminal hook critical for dimerization. The tendency of HGI-III to self-associate seems to relate to its physiological function of a storage protein.

Identification and characterization of a hitherto unknown nucleotide-binding domain and an intricate interdomain regulation in HflX-a ribosome binding GTPase.

A role for HflX in 50S-biogenesis was suggested based on its similarity to other GTPases involved in this process. It possesses a G-domain, flanked by uncharacterized N- and C-terminal domains. Intriguingly, Escherichia coli HflX was shown to hydrolyze both GTP and adenosine triphosphate (ATP), and it was unclear whether G-domain alone would explain ATP hydrolysis too. Here, based on structural bioinformatics analysis, we suspected the possible existence of an additional nucleotide-binding domain (ND1) at the N-terminus. Biochemical studies affirm that this domain is capable of hydrolyzing ATP and GTP. Surprisingly, not only ND1 but also the G-domain (ND2) can hydrolyze GTP and ATP too. Further; we recognize that ND1 and ND2 influence each other's hydrolysis activities via two salt bridges, i.e. E29-R257 and Q28-N207. It appears that the salt bridges are important in clamping the two NTPase domains together; disrupting these unfastens ND1 and ND2 and invokes domain movements. Kinetic studies suggest an important but complex regulation of the hydrolysis activities of ND1 and ND2. Overall, we identify, two separate nucleotide-binding domains possessing both ATP and GTP hydrolysis activities, coupled with an intricate inter-domain regulation for Escherichia coli HflX.

Mutational analysis of the ribosome assembly GTPase RbgA provides insight into ribosome interaction and ribosome-stimulated GTPase activation.

Ribosome biogenesis GTPase A protein (RbgA) is an essential GTPase required for the biogenesis of the 50S subunit in Bacillus subtilis. Homologs of RbgA are widely distributed in bacteria and eukaryotes and are implicated in ribosome assembly in the mitochondria, chloroplast and cytoplasm. Cells depleted of RbgA accumulate an immature large subunit that is missing key ribosomal proteins. RbgA, unlike many members of the Ras superfamily of GTPases, lacks a defined catalytic residue for carrying out guanosine triphosphate (GTP) hydrolysis. To probe RbgA function in ribosome assembly, we used a combined bioinformatics, genetic and biochemical approach. We identified a RNA-binding domain within the C-terminus of RbgA that is structurally similar to AmiR-NasR Transcription Anti-termination Regulator (ANTAR) domains, which are known to bind structured RNA. Mutation of key residues in the ANTAR domain altered RbgA association with the ribosome. We identified a putative catalytic residue within a highly conserved region of RbgA, His9, which is contained within a similar PGH motif found in elongation factor Tu (EF-Tu) that is required for GTP hydrolysis on interaction with the ribosome. Finally, our results support a model in which the GTPase activity of RbgA directly participates in the maturation of the large subunit rather than solely promoting dissociation of RbgA from the 50S subunit.

Structural basis unifying diverse GTP hydrolysis mechanisms.

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.

Substrate-bound crystal structures reveal features unique to Mycobacterium tuberculosis N-acetyl-glucosamine 1-phosphate uridyltransferase and a catalytic mechanism for acetyl transfer.

N-acetyl-glucosamine-1-phosphate uridyltransferase (GlmU), a bifunctional enzyme involved in bacterial cell wall synthesis is exclusive to prokaryotes. GlmU, now recognized as a promising target to develop new antibacterial drugs, catalyzes two key reactions: acetyl transfer and uridyl transfer at two independent domains. Hitherto, we identified GlmU from Mycobacterium tuberculosis (GlmU(Mtb)) to be unique in possessing a 30-residue extension at the C terminus. Here, we present the crystal structures of GlmU(Mtb) in complex with substrates/products bound at the acetyltransferase active site. Analysis of these and mutational data, allow us to infer a catalytic mechanism operative in GlmU(Mtb). In this S(N)2 reaction, His-374 and Asn-397 act as catalytic residues by enhancing the nucleophilicity of the attacking amino group of glucosamine 1-phosphate. Ser-416 and Trp-460 provide important interactions for substrate binding. A short helix at the C-terminal extension uniquely found in mycobacterial GlmU provides the highly conserved Trp-460 for substrate binding. Importantly, the structures reveal an uncommon mode of acetyl-CoA binding in GlmU(Mtb); we term this the U conformation, which is distinct from the L conformation seen in the available non-mycobacterial GlmU structures. Residues, likely determining U/L conformation, were identified, and their importance was evaluated. In addition, we identified that the primary site for PknB-mediated phosphorylation is Thr-418, near the acetyltransferase active site. Down-regulation of acetyltransferase activity upon Thr-418 phosphorylation is rationalized by the structures presented here. Overall, this work provides an insight into substrate recognition, catalytic mechanism for acetyl transfer, and features unique to GlmU(Mtb), which may be exploited for the development of inhibitors specific to GlmU.

Characterizing a novel CMK-EngA fusion protein from Bifidobacterium: Implications for inter-domain regulation.

EngA is an essential and unique bacterial GTPase involved in ribosome biogenesis. The essentiality and species-specific variations among EngA homologues make the protein a potential target for future drug development. In this aspect, it is important to understand the variations of EngA among probiotic organisms and non-probiotic bacteria to understand species specificity. The search for variations among EngA homologues revealed a unique variant, exclusively found in Bifidobacterium and a few Actinobacteria species. Bifidobacterium possesses a multifunctional fusion protein, wherein EngA is fused with an N-terminal CMK (Cytidylate Monophosphate Kinase) domain. The resulting protein is therefore a large (70kDa size) with 3 consecutive P-loops and a 50 amino acid long linker connecting the EngA and CMK domains. EngA is known to regulate ribosome biogenesis via nucleotide-dependent conformational changes. The additional domain may introduce further intricate regulation in ribosome biogenesis or participate in newer biological processes. This study is the first attempt to characterise this novel class of bacterial EngA found in the Genus of Bifidobacteria.

Deciphering the catalytic machinery in a universally conserved ribosome binding ATPase YchF.

YchF, a universally conserved protein, hitherto thought to be a GTPase, was shown to be an ATPase based on structural and biochemical studies on hOLA1, a human ortholog of YchF. However, the cellular role of YchF is unclear. Based on the presence of a RNA binding domain in this protein and significant homology to ribosome binding Obg family GTPases, we examined its ability to associate with the ribosome. Here, we show that Escherichia coli YchF binds the 50S and 70S ribosomal particles in a nucleotide independent manner and it hydrolyzes ATP utilizing a potassium dependent mechanism. A potassium mediated acceleration of hydrolysis activity was thus far known for a few GTPases. Like these, YchF too conserves the structural features required for K(+) coordination, making it a unique ribosome binding ATPase utilizing a similar mechanism. Furthermore, we show that Lys78 is an important determinant of the potassium dependent ATPase activity.

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.

Rational design of hyperstable antibacterial peptides for food preservation.

We describe the design of peptides with properties like thermostability, pH stability, and antibacterial activity against a few bacterial food pathogens. Insights obtained from classical structure-function analysis of natural peptides and their mutants through antimicrobial and enzymatic assays are used to rationally develop a set of peptides. pH and thermostability assays were performed to demonstrate robust antimicrobial activity post-treatment with high temperatures and at wide pH ranges. We have also investigated the mode of action of these hyperstable peptides using membrane permeability assays, electron microscopy, and molecular dynamics simulations. Notably, through mutational studies, we show that these peptides elicit their antibacterial action via both membrane destabilization and inhibition of intracellular trypsin-the two functions attributable to separate peptide segments. Finally, toxicity studies and food preservation assays demonstrate the safety and efficacy of the designed peptides for food preservation. Overall, the study provides a general 'blueprint' for the development of stable antimicrobial peptides (AMPs). Insights obtained from this work may also be combined with combinatorial methods in high-throughput studies for future development of antimicrobials for various applications.

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.

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.

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.

Structure of N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) from Mycobacterium tuberculosis in a cubic space group.

GlmU is a bifunctional enzyme that catalyzes the final two steps in the biosynthesis of UDP-GlcNAc. Crystals of GlmU from Mycobacterium tuberculosis obtained using ammonium sulfate as a precipitant diffracted poorly (to 3.4 A resolution) and displayed an unusually high solvent content (>80%) with sparse crystal packing that resulted in large solvent channels. With one molecule per asymmetric unit, the monomers from three neighbouring asymmetric units related by the crystal threefold formed a biological trimer. Although this is the first report of the structure of GlmU determined in a cubic crystal form, the trimeric arrangement here is similar to that observed for other GlmU structures determined in hexagonal (H3, H32, P6(3)22) space groups.