Phosphorylation is a key post-translational modification that alters the functional state of many proteins. The Escherichia coli toxin HipA, which phosphorylates glutamyl-tRNA synthetase and triggers bacterial persistence under stress, becomes inactivated upon autophosphorylation of Ser150. Interestingly, Ser150 is phosphorylation-incompetent in the crystal structure of HipA since it is deeply buried ("in-state"), although in the phosphorylated state it is solvent exposed ("out-state"). To be phosphorylated, a minor population of HipA must exist in the phosphorylation-competent "out-state" (solvent-exposed Ser150), not detected in the crystal structure of unphosphorylated HipA. Here we report a molten-globule-like intermediate of HipA at low urea (∼4 kcal/mol unstable than natively folded HipA). The intermediate is aggregation-prone, consistent with a solvent exposed Ser150 and its two flanking hydrophobic neighbors (Val/Ile) in the "out-state". Molecular dynamics simulations showed the HipA "in-out" pathway to contain multiple free energy minima with an increasing degree of Ser150 solvent exposure with the free energy difference between the "in-state" and the metastable exposed state(s) to be ∼2-2.5 kcal/mol, with unique sets of hydrogen bonds and salt bridges associated with the metastable loop conformations. Together, the data clearly identify the existence of a phosphorylation-competent metastable state of HipA. Our results not only suggest a mechanism of HipA autophosphorylation but also add to a number of recent reports on unrelated protein systems where the common proposed mechanism for phosphorylation of buried residues is their transient exposure even without phosphorylation.

The canonical function of glutamyl-tRNA synthetase (GluRS) is to glutamylate tRNA . Yet, not all bacterial GluRSs glutamylate tRNA ; many glutamylate both tRNA and tRNA , while some glutamylate only tRNA and not the cognate substrate tRNA . Understanding the basis of this unique tRNA -specificity is important. Mutational studies have hinted at hotspot residues, both on tRNA and GluRS, that play crucial roles in tRNA -specificity. But the underlying structural basis remains unexplored. Majority of biochemical studies related to tRNA -specificity have been performed on GluRS from Escherichia coli and other proteobacterial species. However, since the early crystal structures of GluRS and tRNA -bound GluRS were from non-proteobacterial species ( Thermus thermophilus ), the proteobacterial biochemical data have often been interpreted in the context of non-proteobacterial GluRS structures. Marked differences between proteo- and non-proteobacterial GluRSs have been demonstrated and therefore it is important that tRNA -specificity be understood vis-a-vis proteobacterial GluRS structures. Towards this goal we have solved the crystal structure of GluRS from E. coli . Using the solved structure and several other currently available proteo- and non-proteobacterial GluRS crystal structures, we have probed the structural basis of tRNA -specificity of bacterial GluRSs. Specifically, our analysis suggests a unique role played by a tRNA D-helix contacting loop of GluRS in modulation of tRNA -specificity. While earlier studies had identified functional hotspots on tRNA that controlled tRNA -specificity of GluRS, this is the first report of complementary signatures of tRNA -specificity in GluRS.

The canonical function of glutamyl-tRNA synthetase (GluRS) is to glutamylate tRNAGlu . Yet not all bacterial GluRSs glutamylate tRNAGlu ; many glutamylate both tRNAGlu and tRNAGln , while some glutamylate only tRNAGln and not the cognate substrate tRNAGlu . Understanding the basis of the unique specificity of tRNAGlx is important. Mutational studies have hinted at hotspot residues, both on tRNAGlx and GluRS, which play crucial roles in tRNAGlx -specificity. However, its underlying structural basis remains unexplored. The majority of biochemical studies related to tRNAGlx -specificity have been performed on GluRS from Escherichia coli and other proteobacterial species. However, since the early crystal structures of GluRS and tRNAGlu -bound GluRS were from non-proteobacterial species (Thermus thermophilus), proteobacterial biochemical data have often been interpreted in the context of non-proteobacterial GluRS structures. Marked differences between proteobacterial and non-proteobacterial GluRSs have been demonstrated; therefore, it is important to understand tRNAGlx -specificity vis-a-vis proteobacterial GluRS structures. To this end, we solved the crystal structure of a double mutant GluRS from E. coli. Using the solved structure and several other currently available proteo- and non-proteobacterial GluRS crystal structures, we probed the structural basis of the tRNAGlx -specificity of bacterial GluRSs. Specifically, our analyses suggest a unique role played by the tRNAGlx D-helix contacting loop of GluRS in the modulation of tRNAGln -specificity. While earlier studies have identified functional hotspots on tRNAGlx that control the tRNAGlx -specificity of GluRS, this is the first report of complementary signatures of tRNAGlx -specificity in GluRS.

Interaction between human positive coactivator 4 (PC4), an abundant nuclear protein, and the tumor suppressor protein p53 plays a crucial role in initiating apoptosis. In certain neurodegenerative diseases PC4 assisted-p53-dependent apoptosis may play a central role. Thus, disruption of p53-PC4 interaction may be a good drug target for certain disease pathologies. A p53-derived short peptide (AcPep) that binds the C-terminal domain of PC4 (C-PC4) is known to disrupt PC4-p53 interaction. To fully characterize its binding mode and binding site on PC4, we co-crystallized C-PC4 with the peptide and determined its structure. The crystal, despite exhibiting mass spectrometric signature of the peptide, lacked peptide electron density and showed a novel crystal lattice, when compared to C-PC4 crystals without the peptide. Using peptide-docked models of crystal lattices, corresponding to our structure and the peptide-devoid structure we show the origin of the novel crystal lattice to be dynamically bound peptide at the previously identified putative binding site. The weak binding is proposed to be due to the lack of the N-terminal domain of PC4 (N-PC4), which we experimentally show to be disordered with no effect on PC4 stability. Taking cue from the structure, virtual screening of ∼18.6 million small molecules from the ZINC15 database was performed, followed by toxicity and binding free energy filtering. The novel crystal lattice of C-PC4 in presence of the peptide, the role of the disordered N-PC4 and the high throughput identification of potent small molecules will allow a better understanding and control of p53-PC4 interaction.

The shikimate pathway is an attractive target for the development of herbicides and antimicrobial agents, as it is essential in plants, bacteria, and apicomplexan parasites but absent in humans. The enzymes of shikimate pathway are conserved among bacteria. Thus, the inhibitors of the shikimate pathway act on wide range of pathogens. We have identified that chlorogenic acid targets the enzymes of the shikimate pathway. The crystal structure of dehydroquinate synthase, the second enzyme of the pathway, in complex with chlorogenic acid and enzymatic inhibition studies explains the mechanism of inhibition of chlorogenic acid. These results suggest that chlorogenic acid has a good chemical scaffold and have important implications for its further development as a potent inhibitor of shikimate pathway enzymes.

The first committed step of the shikimate pathway is catalyzed by a metalloenzyme 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAH7PS), which exhibits vulnerability to the oxidative stress. DAH7PS undergoes inactivation in multiple ways in the presence of redox metal, H2O2, and superoxide. The molecular mechanism and susceptibility of its inactivation might differ in different organisms and are presently unclear. In the present work, we have cloned, expressed and purified a DAH7PS from Providencia alcalifaciens (PaDAH7PS). The oligomeric state and effect of redox metal treatment on its stability were analyzed through the size exclusion chromatography. The FTIR, MALDI-TOF/TOF-MS studies revealed that methionine residues were modified to methionine sulfoxide in PaDAH7PS. During oxidation, PaDAH7PS is altered into partially folded protein and unfolded states as determined by CD and Fluorescence studies. A significant loss in enzymatic activity of PaDAH7PS was determined and the formation of amorphous aggregates was visualized using AFM imaging and also confirmed by ThT binding based assay. This is the first report where we have shown a hexameric DAH7PS and the methionine residues of PaDAH7PS get oxidize in the presence of oxidative stress. The partially folded and unfolded oligomeric states with high β-content of PaDAH7PS might be the critical precursors for aggregation.

Lipopolysaccharide (LPS) is an important surface component and a potential virulence factor in the pathogenesis of Gram-negative bacteria. UDP-N-acetylglucosamine acyltransferase (LpxA) enzyme catalyzes the first reaction of LPS biosynthesis, reversible transfer of R-3-hydroxy-acyl moiety from donor R-3-hydroxy-acyl-acyl carrier protein to the 3' hydroxyl position of UDP-N-acetyl-glucosamine. LpxA enzyme's essentiality in bacterial survival and absence of any homologous protein in humans makes it a promising target for anti-bacterial drug development. Herein, we present the crystal structure of Moraxella catarrhalis LpxA (McLpxA). We propose that L171 is responsible for limiting the acyl chain length in McLpxA to 10C or 12C. The study reveals the plausible interactions between the highly conserved clusters of basic residues at the C-terminal end of McLpxA and acidic residues of acyl carrier protein (ACP). Furthermore, the crystal structure of McLpxA was used to screen potential inhibitors from NCI open database using various computational approaches viz. pharmacophore mapping, virtual screening and molecular docking. Molecules Mol212032, Mol609399 and Mol152546 showed best binding affinity with McLpxA among all screened molecules. These molecules mimic the substrate-LpxA binding interactions.

3-deoxy-D-arabino-heptulosonate-7-phosphate-synthase (DAHPS) is the first enzyme of the shikimate pathway and is responsible for the synthesis of aromatic amino acids in microorganisms. This pathway is an attractive target for antimicrobial drugs. In Bacillus subtilis, the N-terminal domain of the bifunctional DAHPS enzyme belongs to an AroQ class of chorismate mutase and is functionally homologous to the downstream AroH class chorismate mutase. This is the first structure of chorismate mutase, AroQ (BsCM_2) enzyme from Bacillus subtilis in complex with citrate and chlorogenic acid at 1.9 Å and 1.8 Å resolution, respectively. This work provides the structural basis of ligand binding into the active site of AroQ class of chorismate mutase, while accompanied by the conformational flexibility of active site loop. Molecular dynamics results showed that helix H2′ undergoes uncoiling at the first turn and increases the mobility of loop L1′. The side chains of Arg45, Phe46, Arg52 and Lys76 undergo conformational changes, which may play an important role in DAHPS regulation by the formation of the domain-domain interface. Additionally, binding studies showed that the chlorogenic acid binds to BsCM_2 with a higher affinity than chorismate. These biochemical and structural findings could lead to the development of novel antimicrobial drugs.

The glycosyl hydrolase 18 (GH18) family consists of active chitinases as well as chitinase like lectins/proteins (CLPs). The CLPs share significant sequence and structural similarities with active chitinases, however, do not display chitinase activity. Some of these proteins are reported to have specific functions and carbohydrate binding property. In the present study, we report a novel chitinase like lectin (TCLL) from Tamarindus indica. The crystal structures of native TCLL and its complex with N-acetyl glucosamine were determined. Similar to the other CLPs of the GH18 members, TCLL lacks chitinase activity due to mutations of key active site residues. Comparison of TCLL with chitinases and other chitin binding CLPs shows that TCLL has substitution of some chitin binding site residues and more open binding cleft due to major differences in the loop region. Interestingly, the biochemical studies suggest that TCLL is an N-acetyl glucosamine specific chi-lectin, which is further confirmed by the complex structure of TCLL with N-acetyl glucosamine complex. TCLL has two distinct N-acetyl glucosamine binding sites S1 and S2 that contain similar polar residues, although interaction pattern with N-acetyl glucosamine varies extensively among them. Moreover, TCLL structure depicts that how plants utilize existing structural scaffolds ingenuously to attain new functions. To date, this is the first structural investigation of a chi-lectin from plants that explore novel carbohydrate binding sites other than chitin binding groove observed in GH18 family members. Consequently, TCLL structure confers evidence for evolutionary link of lectins with chitinases.

The shikimate pathway is found in microorganisms, fungi, plants and also in several apicomplexan parasites. This metabolic pathway consists of seven enzymes and converts the primary metabolites phosphoenolpyruvate and erythrose-4-phosphate to chorismate, the last common precursor for the three aromatic amino acids Phe, Tyr, and Trp and other aromatic compounds. The significance of targeting the enzymes of this pathway as selective targets for anti microbial drug design involves the fact that they are essential for microbes but absent in humans.In present scenario, the emergence of multi-drug resistance in pathogenic bacteria and herbicide resistance in weeds is of great clinical and agro-economical concern. Therefore in this review, we did the comparative sequence and three-dimensional structure analysis of these enzymes from various microorganisms and plants for structure-function analysis, motif search, common structural signatures of active site and elucidation of regulation mechanisms. Also, the available structures of five shikimate pathway enzymes from M. tuberculosis, a dreadful microorganism, which causes 1.5 million deaths per year, have been comparatively analyzed with other reported homologous structures. To get the structural insight of remaining two shikimate pathway enzymes (dehydroquinate synthase and shikimate-5-dehydrogenase) of M. tuberculosis we did molecular modeling to find out key active site residues. These studies can further be proven helpful in designing novel structure based antimicrobial drugs.

Bunyaviridae family consists of vector borne lethal viruses, stands out as the largest virus family with its 350 members. One such virus of this family, Crimean-Congo hemorrhagic fever virus (CCHFV) is transmitted through bites of ixodid ticks or by direct contact with blood from infected animals. Crimean-Congo haemorrhagic fever (CCHF) is a severe disease in humans which is endemic in large parts of the world with a high mortality rate. This virus could also be used as a bioterrorism agent due to its human-to-human transmission with no specific therapy. The pathogenicity factor of CCHFV is unexplored due to the lack of animal models. CCHFV, being an RNA virus, is able to mutate rapidly hence preventing the development of effective therapy against it. Till now ribavarin is the only available drug for supportive treatment but has many side-effects. New technologies like RNA interference have emerged as a solution for epidemics of CCHF. RNAi is a sequence specific approach, has been used successfully against different pathogens. This review focuses on designing and application of RNAi with emphasis on the role of bioinformatics for the anti CCHFV therapeutic development strategy.