Shivendra Pratap

Active-Site Plasticity Is Essential to Carbapenem Hydrolysis by OXA-58 Class D β-Lactamase of Acinetobacter baumannii.

ABSTRACT Carbapenem-hydrolyzing class D β-lactamases (CHDLs) are a subgroup of class D β-lactamases, which are enzymes that hydrolyze β-lactams. They have attracted interest due to the emergence of multidrug-resistant Acinetobacter baumannii, which is not responsive to treatment with carbapenems, the usual antibiotics of choice for this bacterium. Unlike other class D β-lactamases, these enzymes efficiently hydrolyze carbapenem antibiotics. To explore the structural requirements for the catalysis of carbapenems by these enzymes, we determined the crystal structure of the OXA-58 CHDL of A. baumannii following acylation of its active-site serine by a 6α-hydroxymethyl penicillin derivative that is a structural mimetic for a carbapenem. In addition, several point mutation variants of the active site of OXA-58, as identified by the crystal structure analysis, were characterized kinetically. These combined studies confirm the mechanistic relevance of a hydrophobic bridge formed over the active site. This structural feature is suggested to stabilize the hydrolysis-productive acyl-enzyme species formed from the carbapenem substrates of this enzyme. Furthermore, our structural studies provide strong evidence that the hydroxyethyl group of carbapenems samples different orientations in the active sites of CHDLs, and the optimum orientation for catalysis depends on the topology of the active site allowing proper closure of the active site. We propose that CHDLs use the plasticity of the active site to drive the mechanism of carbapenem hydrolysis toward efficiency.

Crystallization, high-resolution data collection and preliminary crystallographic analysis of Aura virus capsid protease and its complex with dioxane

The C-terminal protease domain of capsid protein from Aura virus expressed in a bacterial expression system has been purified to homogeneity and crystallized. Crystals suitable for X-ray diffraction analysis were obtained by the vapour-diffusion method using 0.1 M bis-tris and polyethylene glycol monomethyl ether 2000. Crystals of the C-terminal protease domain of capsid protein in complex with dioxane were also produced and crystal data were obtained. Both crystals belonged to space group C2, with unit-cell parameters a = 79.6, b = 35.2, c = 49.5 Å. High-resolution data sets were collected to a resolution of 1.81 Å for the native protein and 1.98 Å for the complex. Preliminary crystallographic studies suggested the presence of a single molecule in the crystallographic asymmetric unit, with a solvent content of 38.5%.

Structure of Chorismate Mutase-like Domain of DAHPS from Bacillus subtilis Complexed with Novel Inhibitor Reveals Conformational Plasticity of Active Site

Abstract3-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.

New insights about pilus formation in gut-adapted Lactobacillus rhamnosus GG from the crystal structure of the SpaA backbone-pilin subunit

Thus far, all solved structures of pilin-proteins comprising sortase-assembled pili are from pathogenic genera and species. Here, we present the first crystal structure of a pilin subunit (SpaA) from a non-pathogen host (Lactobacillus rhamnosus GG). SpaA consists of two tandem CnaB-type domains, each with an isopeptide bond and E-box motif. Intriguingly, while the isopeptide bond in the N-terminal domain forms between lysine and asparagine, the one in the C-terminal domain atypically involves aspartate. We also solved crystal structures of mutant proteins where residues implicated in forming isopeptide bonds were replaced. Expectedly, the E-box-substituted E139A mutant lacks an isopeptide bond in the N-terminal domain. However, the C-terminal E269A substitution gave two structures; one of both domains with their isopeptide bonds present, and another of only the N-terminal domain, but with an unformed isopeptide bond and significant conformational changes. This latter crystal structure has never been observed for any other Gram-positive pilin. Notably, the C-terminal isopeptide bond still forms in D295N-substituted SpaA, irrespective of E269 being present or absent. Although E-box mutations affect SpaA proteolytic and thermal stability, a cumulative effect perturbing normal pilus polymerization was unobserved. A model showing the polymerized arrangement of SpaA within the SpaCBA pilus is proposed.

Characterization of isoflavonoids as inhibitors of β-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from Moraxella catarrhalis : Kinetics, spectroscopic, thermodynamics and in silico studies

BACKGROUD β-hydroxyacyl-acyl carrier protein dehydratase (FabZ) is an essential component of type II fatty acid biosynthesis (FAS II) pathway in bacteria. It performs dehydration of β-hydroxyacyl-ACP to trans-2-acyl-ACP in the elongation cycle of the FAS II pathway. FabZ is ubiquitously expressed and has uniform distribution, which makes FabZ an excellent target for developing novel drugs against pathogenic bacteria. METHODS We focused on the biochemical and biophysical characterization of FabZ from drug-resistant pathogen Moraxella catarrhalis (McFabZ). More importantly, we have identified and characterized new inhibitors against McFabZ using biochemical, biophysical and in silico based studies. RESULTS We have identified three isoflavones (daidzein, biochanin A and genistein) as novel inhibitors against McFabZ. Mode of inhibition of these compounds is competitive with IC50 values lie in the range of 6.85μΜ to 27.7μΜ. Conformational changes observed in secondary and tertiary structure marked by a decrease in the helical and the sheet content in McFabZ structure upon inhibitors binding. In addition, thermodynamic data suggest that biochanin A has a strong binding affinity for McFabZ as compare to daidzein and genistein. Molecular docking studies have revealed that these inhibitors are interacting with the active site of McFabZ and making contacts with catalytic and substrate binding tunnel residues. CONCLUSION AND GENERAL SIGNIFICANCE Three new inhibitors against McFabZ have been identified and characterized. These biochemical and biophysical findings lead to the identification of chemical scaffolds, which can lead to broad-spectrum antimicrobial drugs targeted against FabZ, and modification to existing FabZ inhibitors to improve affinity and potency.

Biophysical and in silico interaction studies of aporphine alkaloids with Malonyl-CoA: ACP transacylase (FabD) from drug resistant Moraxella catarrhalis

Malonyl-CoA:acyl carrier protein transacylase (FabD), being an essential enzyme of the FAS II pathway, is an attractive target for developing broad-spectrum antibiotics. It performs initiation reaction to form malonyl-ACP, which is a key building block in fatty acid biosynthesis. In this study, we have characterized the FabD from drug-resistant pathogen Moraxella catarrhalis (McFabD). More importantly, we have shown the binding of McFabD with three new compounds from the class of aporphine alkaloids. ITC based binding studies have shown that apomorphine is binding to McFabD with a stronger affinity (KD = 4.87 μM) as compared to boldine (KD = 7.19 μM) and magnoflorine (KD = 11.7 μM). The possible mechanism of fluorescence quenching is found to be static with Kq values higher than 1010, which was associated with the ground state complex formation of aporphine alkaloids with McFabD. Conformational changes observed in the secondary and tertiary structure marked by the loss of helical content during the course of interactions. Molecular docking based studies have predicted the binding mode of aporphine alkaloids and it is found that these compounds are interacting in a similar fashion as known inhibitor corytuberine is interacting with McFabD. The analysis of docking poses have revealed that His 210, Leu102, Gln19, Ser101 and Arg 126 are critical residues, which may play important role in binding. The growth inhibition assay has shown that apomorphine has better MIC value (4-8 μg/ml) against Moraxella catarrhalis as compared to boldine and magnoflorine. Therefore, the current study suggests that aporphine alkaloids can act as antibacterial agents and possible target of these compounds could be FabD enzyme from the FAS II pathway, and apomorphine scaffold will be more suitable among these compounds for potential development of antibacterial agents.

The inhibitory and binding studies of methyl-sulfone hydroxamate based inhibitors against LpxC from drug resistant Moraxella catarrhalis using biophysical, biochemical and in silico approaches

Several reported potential compounds against UDP-3-O-(R-3-Hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) have shown large variation in the potency and efficacy. The differential susceptibility and selective binding of these inhibitors against LpxC are still unexplored. In the present work, we have characterized LpxC from Moraxella catarrhalis (McLpxC) and investigated its binding with potent inhibitors LpxC-2 and LpxC-4 using biochemical, biophysical and in silico approaches. The circular dichroism studies have revealed the changes in the secondary and tertiary structure of McLpxC upon inhibitors binding. The fluorescence quenching mechanism was found to be static with kq > 1010 suggesting the ground state complex formation between the McLpxC and inhibitors. Altogether spectroscopic findings suggest that the interaction of LpxC-4 and LpxC-2 caused conformational changes marked by the loss of α-helical content in McLpxC. In ITC based studies, both inhibitors have shown comparable binding affinities (KD = ~10.0 μΜ), and their interactions were exothermically driven by enthalpy change. The docking studies have shown the possibility of two binding sites in McLpxC for these inhibitors with similar binding energies (~10.0 kcal mol-1). Thus, the present study significantly suggests that further optimization and utilization of molecules based on this scaffold will be helpful in designing the new antimicrobial agents targeting LpxC.

Crystal structure of chikungunya virus nsP2 cysteine protease reveals a putative flexible loop blocking its active site

Chikungunya virus (CHIKV), a mosquito-borne pathogenic alphavirus is a growing public health threat. No vaccines or antiviral drug is currently available in the market for chikungunya treatment. nsP2pro, the viral cysteine protease, carries out an essential function of nonstructural polyprotein processing and forms four nonstructural proteins (nsPs) that makes the replication complex, hence constitute a promising drug target. In this study, crystal structure of nsP2pro has been determined at 2.59 Å, which reveals that the protein consists of two subdomains: an N-terminal protease subdomain and a C-terminal methyltransferase subdomain. Structural comparison of CHIKV nsP2pro with structures of other alphavirus nsP2 advances that the substrate binding cleft is present at the interface of two subdomains. Additionally, structure insights revealed that access to the active site and substrate binding cleft is blocked by a flexible interdomain loop in CHIKV nsP2pro. This loop contains His548, the catalytic residue, and Trp549 and Asn547, the residues predicted to bind substrate. Interestingly, mutation of Asn547 leads to three-fold increase in Km confirming that Asn547 plays important role in substrate binding and recognition. This study presents the detailed molecular analysis and signifies the substrate specificity residues of CHIKV nsP2pro, which will be beneficial for structure-based drug design and optimization of CHIKV protease inhibitors.

Bent conformation of a backbone pilin N-terminal domain supports a three-stage pilus assembly mechanism

Effective colonization of host cells by some Gram-positive bacteria often involves using lengthy, adhesive macromolecular structures called sortase-dependent pili. Among commensals, the gut-adapted Lactobacillus rhamnosus GG strain encodes the operons for two varieties of these pili (SpaCBA and SpaFED), with each structure consisting of backbone, tip, and basal pilin subunits. Although the tertiary structure was recently solved for the backbone subunit (SpaA) of the SpaCBA pilus, no structural information exists for its counterpart in the SpaFED pilus. Here, we report several crystal structures for the SpaD backbone pilin, two of which capture the N-terminal domain in either the closed (linear) or open (bent) conformation. To our knowledge, this is the first observation of the bent conformation in Gram-positive pilin structures. Based on this bent conformation, we suggest a three-stage model, which we call the expose-ligate-seal mechanism, for the docking and assembly of backbone pilins into the sortase-dependent pilus.Priyanka Chaurasia et al. report crystal structures of the SpaD backbone pilin from a gut-adapted bacteria, Lactobacillus rhamnosus. The observed bent conformation of the N-terminal domain has not been seen in other Gram-positive pilin structures.

Biochemical and biophysical characterization of 1,4-naphthoquinone as a dual inhibitor of two key enzymes of type II fatty acid biosynthesis from Moraxella catarrhalis

The fatty acid biosynthesis (FAS II) is a vital process in bacteria and regarded as an attractive pathway for the development of potential antimicrobial agents. In this study, we report 1,4-naphthoquinone (NPQ) as a dual inhibitor of two key enzymes of FAS II pathway, namely FabD (Malonyl-CoA:ACP transacylase) and FabZ (β-hydroxyacyl-ACP dehydratase). Mode of inhibition of NPQ was found to be non-competitive for both enzymes with IC50 of 26.67 μΜ and 23.18 μΜ against McFabZ and McFabD respectively. Conformational changes in secondary and tertiary structures marked by the loss of helical contents were observed in both enzymes upon NPQ binding. The fluorescence quenching was found to be static with a stable ground state complex formation. ITC based studies have shown that NPQ is binding to McFabZ with a stronger affinity (~1.5×) as compared to McFabD. Molecular docking studies have found that NPQ interacts with key residues of both McFabD (Ser209, Arg126, and Leu102) and McFabZ (His74 and Tyr112) enzymes. Both complexes have shown the structural stability during the 20 ns run of molecular dynamics based simulations. Altogether, the present study suggests that NPQ scaffold can be exploited as a multi-targeted inhibitor of FAS II pathway, and these biochemical and biophysical findings will further help in the development of potent antibacterial agents targeting FAS II pathway.