Enolpyruvate transferase MurAAA149E, identified during adaptation of Enterococcus faecium to daptomycin, increases stability of MurAA-MurG interaction.

Daptomycin (DAP) is an antibiotic frequently used as a drug of last resort against vancomycin-resistant enterococci. One of the major challenges when using DAP against vancomycin-resistant enterococci is the emergence of resistance, which is mediated by the cell-envelope stress system LiaFSR. Indeed, inhibition of LiaFSR signaling has been suggested as a strategy to "resensitize" enterococci to DAP. In the absence of LiaFSR, alternative pathways mediating DAP resistance have been identified, including adaptive mutations in the enolpyruvate transferase MurAA (MurAAA149E), which catalyzes the first committed step in peptidoglycan biosynthesis; however, how these mutations confer resistance is unclear. Here, we investigated the biochemical basis for MurAAA149E-mediated adaptation to DAP to determine whether such an alternative pathway would undermine the potential efficacy of therapies that target the LiaFSR pathway. We found cells expressing MurAAA149E had increased susceptibility to glycoside hydrolases, consistent with decreased cell wall integrity. Furthermore, structure-function studies of MurAA and MurAAA149E using X-ray crystallography and biochemical analyses indicated only a modest decrease in MurAAA149E activity, but a 16-fold increase in affinity for MurG, which performs the last intracellular step of peptidoglycan synthesis. Exposure to DAP leads to mislocalization of cell division proteins including MurG. In Bacillus subtilis, MurAA and MurG colocalize at division septa and, thus, we propose MurAAA149E may contribute to DAP nonsusceptibility by increasing the stability of MurAA-MurG interactions to reduce DAP-induced mislocalization of these essential protein complexes.

Biochemical and structural basis for Moraxella catarrhalis enoyl-acyl carrier protein reductase (FabI) inhibition by triclosan and estradiol.

The enoyl-acyl carrier protein reductase (ENR) is an established drug target and catalyzes the last reduction step of the fatty acid elongation cycle. Here, we report the crystal structures of FabI from Moraxella catarrhalis (McFabI) in the apo form, binary complex with NAD+ and ternary complex with NAD + -triclosan (TCL) determined at 2.36, 2.12 and 2.22 Å resolutions, respectively. The comparative study of these three structures revealed three different conformational states for the substrate-binding loop (SBL), including an unstructured intermediate, a structured intermediate and a closed conformation in the apo, binary and ternary complex forms, respectively; indicating the flexibility of SBL during the ligand binding. Virtual screening has suggested that estradiol cypionate may be a potential inhibitor of McFabI. Subsequently, estradiol (EST), the natural form of estradiol cypionate, was assessed for its FabI-binding and -inhibition properties. In vitro studies demonstrated that TCL and EST bind to McFabI with high affinity (KD = 0.038 ± 0.004 and 5 ± 0.06 µM respectively) and inhibit its activity (Ki = 62.93 ± 3.95 nM and 25.97 ± 1.93 µM respectively) and suppress the growth of M. catarrhalis. These findings reveal that TCL and EST inhibit the McFabI activity and thereby affect cell growth. This study suggests that estradiol may be exploited as a novel scaffold for the designing and development of more potential FabI inhibitors.

Structural characterization and in-silico analysis of Momordica charantia 7S globulin for stability and ACE inhibition.

Momordica charantia (Mc) seeds are widely used edible crop with high nutritional quality. The food and pharmaceutical industries use it as a natural anti-oxygenic agent. Herein, a ~52 kDa protein, which is a major part of seed proteome has been purified, biochemically characterized and structure has been determined. MALDI-ESI-MS identified peptide fragments and contig-deduced sequence suggested the protein to be homologous to 7S globulins. The crystal structure shows that protein has a bicupin fold similar to 7S globulins and the electron density for a copper and acetate ligand were observed in the C-terminal barrel domain. In silico study reveals that a tripeptide (VFK) from Mc7S possess a higher binding affinity for angiotensin converting enzyme (ACE) than already reported drug Lisinopril (LPR). The protein is a glycoprotein and highly stable under varying thermal and pH conditions due to its secondary structures. The DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) assay showed the protein to have an anti-oxygenic nature and can aid in scavenging free radical from sample. The protein can assist to enhance the nutritional and functional value of food by acting as a food antioxidant. Further, characterization of Mc7S required which might add in importance of Mc7S as antioxidant, anti-diabetic and anti-hypertensive.

Crystal structure of the atypically adhesive SpaB basal pilus subunit: Mechanistic insights about its incorporation in lactobacillar SpaCBA pili.

To successfully colonize a host or environment, certain genera and species of Gram-positive bacteria have evolved to utilize the so-called sortase-dependent pilus, a long multi-subunit and non-flagellar surface adhesin. One example of this is Lactobacillus rhamnosus GG, a gut-adapted probiotic strain that produces SpaCBA pili. These structures are covalent hetero-oligomers built from three types of pilin subunit, each with a specific location and function (i.e., backbone SpaA for length, tip SpaC for adhesion, and basal SpaB for anchoring). Functionally, the SpaCBA pilus exhibits a promiscuous affinity for components on intestinal surfaces (e.g., mucus, collagen, and epithelial cells), which is largely attributed to the SpaC subunit. Then again, the basal SpaB pilin, in addition to acting as the terminal subunit during pilus assembly, displays an out of character mucoadhesive function. To address the structural basis of this unusual dual functionality, we reveal the 2.39 â€‹Å resolution crystal structure of SpaB. SpaB consists of one immunoglobulin-like CnaB domain and contains a putative intermolecular isopeptide bond-linking lysine and internal isopeptide bond-asparagine in an FPKN pilin motif within the C-terminal end. Remarkably, we found that a C-terminal stretch of positively charged lysine and arginine residues likely accounts for the atypical mucoadhesiveness of SpaB. Although harboring an autocatalytic triad of residues for a potential internal isopeptide interaction, the SpaB crystal structure lacked the visible electron density for intact bond formation, yet its presence was subsequently confirmed by mass spectral analysis. Finally, we propose a structural model that captures the exclusive basal positioning of SpaB in the SpaCBA pilus.

A SUMOylation-dependent switch of RAB7 governs intracellular life and pathogenesis of Salmonella Typhimurium.

Salmonella Typhimurium is an intracellular pathogen that causes gastroenteritis in humans. Aided by a battery of effector proteins, S. Typhimurium resides intracellularly in a specialized vesicle, called the Salmonella-containing vacuole (SCV) that utilizes the host endocytic vesicular transport pathway (VTP). Here, we probed the possible role of SUMOylation, a post-translation modification pathway, in SCV biology. Proteome analysis by complex mass-spectrometry (MS/MS) revealed a dramatically altered SUMO-proteome (SUMOylome) in S. Typhimurium-infected cells. RAB7, a component of VTP, was key among several crucial proteins identified in our study. Detailed MS/MS assays, in vitro SUMOylation assays and structural docking analysis revealed SUMOylation of RAB7 (RAB7A) specifically at lysine 175. A SUMOylation-deficient RAB7 mutant (RAB7K175R) displayed longer half-life, was beneficial to SCV dynamics and functionally deficient. Collectively, the data revealed that RAB7 SUMOylation blockade by S. Typhimurium ensures availability of long-lived but functionally compromised RAB7, which was beneficial to the pathogen. Overall, this SUMOylation-dependent switch of RAB7 controlled by S. Typhimurium is an unexpected mode of VTP pathway regulation, and unveils a mechanism of broad interest well beyond Salmonella-host crosstalk. This article has an associated First Person interview with the first author of the paper.

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 (ß-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.

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.

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.

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.

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

BACKGROUD: ß-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 ß-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.

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

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.

Acyl chain preference and inhibitor identification of Moraxella catarrhalis LpxA: Insight through crystal structure and computational studies.

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.

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.

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

Carbapenem-hydrolyzing class D ß-lactamases (CHDLs) are a subgroup of class D ß-lactamases, which are enzymes that hydrolyze ß-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 ß-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%.