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CBE = Cristalografía y Biología Estructural
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Acta Cryst. (1948) 1, 3-4
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Transpeptidases, members of the penicillin-binding protein (PBP) families, catalyze crosslinking of the bacterial cell wall. This transformation is critical for the survival of bacteria and it is the target of inhibition by β-lactam antibiotics. We report herein our structural insights into catalysis by the essential PBP2x of Streptococcus pneumoniae by disclosing a total of four X-ray structures, two computational models based on the crystal structures and molecular-dynamics simulations. The X-ray structures are for the apo PBP2x, the enzyme modified covalently in the active site by oxacillin (a penicillin antibiotic), the enzyme modified by oxacillin in the presence of a synthetic tetrasaccharide surrogate for the cell-wall peptidoglycan and a non-covalent complex of cefepime (a cephalosporin antibiotic) bound to the active site. A pre-requisite for catalysis by transpeptidases, including PBP2x, is the molecular recognition of nascent peptidoglycan strands, which harbor pentapeptide stems. We disclose that the recognition of nascent peptidoglycan by PBP2x takes place by complexation of one pentapeptide stem at an allosteric site located in the PASTA domains of this enzyme. This binding predisposes the third pentapeptide stem in the same nascent peptidoglycan strand to penetration into the active site for the turnover events. The complexation of the two pentapeptide stems in the same peptidoglycan strand is a recognition motif for the nascent peptidoglycan, critical for the cell-wall crosslinking reaction.
ACS Chemical Biology (2018) 13, 694–702 (doi:10.1021/acschembio.7b00817)
Abscisic acid (ABA) is the main phytohormone involved in the adaptive crop responses to drought. ABA signaling relies on the family of pyrabactin resistance 1/PYR1-like/regulatory components of ABA receptors (PYR/PYL/RCAR) receptors, which upon ABA binding form high-affinity ternary complexes with clade A protein phosphatases type 2C (PP2Cs) and inhibit them. Our current understanding on the structural mechanism of ABA signaling relies exclusively on crystallographic analyses performed with Arabidopsis thaliana ABA receptors. Although mechanistic insights obtained in Arabidopsis are usually translated to other plant species, studies in crops might overturn or update the dogma established in Arabidopsis. As a result from structural studies performed with Citrus sinensis (sweet orange) and Solanum lycopersicum (tomato) ABA receptors, we have identified a latch-closed gate-open ABA-bound intermediate that provides novel mechanistic insight on ABA signaling. This intermediate supports an ABA-dependent conformational transition for receptor activation where the equilibrium from this nonproductive intermediate to the productive form of the receptor is shifted by the PP2C. This highlights the role of the PP2C as necessary co-receptor to increase the ABA binding affinity by shifting this equilibrium toward the formation of the Receptor-ABA-PP2C ternary complex.
Molecular Plant (2017) 10, 1250–1253 (doi:10.1016/j.molp.2017.07.004)
The N-acetylglucosaminidase NagZ of Pseudomonas aeruginosa catalyzes the first cytoplasmic step in recycling of muropeptides, cell-wall-derived natural products. This reaction regulates gene expression for the β-lactam resistance enzyme, β-lactamase. The structural and functional aspects of catalysis by NagZ were investigated by a total of seven X-ray structures, three computational models based on the X-ray structures, molecular-dynamics simulations and mutagenesis. The structural insights came from the unbound state and complexes of NagZ with the substrate, products and a mimetic of the transient oxocarbenium species. The mechanism involves a histidine as acid/base catalyst, which is unique for glycosidases. The turnover process utilizes covalent modification of D244, requiring two transition-state species and is regulated by coordination with a zinc ion. The analysis provides a seamless continuum for the catalytic cycle, incorporating the large motions by loops that surround the active site.
Journal of the American Chemical Society (2017) 139, 6795–6798 (doi:10.1021/jacs.7b01626)
A complex link exists between cell-wall recycling/repair and the manifestation of resistance to β-lactam antibiotics in many Enterobacteriaceae and Pseudomonas aeruginosa. This process is mediated by specific cell-wall-derived muropeptide products. These muropeptides are internalized into the cytoplasm and bind to the transcriptional regulator AmpR, which controls the cytoplasmic events that lead to expression of β-lactamase, an antibiotic-resistance determinant. The effector-binding domain (EBD) of AmpR was crystallized and its structure solved to 2.2 Å resolution. The EBD crystallizes in a “closed” conformation, in contrast to the “open” structure required to bind the muropeptides. Structural issues of this ligand recognition are addressed by molecular dynamics simulations, which reveal significant differences among the complexes with the effector molecules. The EBD binds to the suppressor ligand UDP-N-acetyl-β-d-muramyl-l-Ala-γ-d-Glu-meso-DAP-d-Ala-d-Ala and binds to two activator muropeptides, N-acetyl-β-d-glucosamine-(1→4)-1,6-anhydro-N-acetyl-β-d-muramyl-l-Ala-γ-d-Glu-meso-DAP-d-Ala-d-Ala and 1,6-anhydro-N-acetyl-β-d-muramyl-l-Ala-γ-d-Glu-meso-DAP-d-Ala-d-Ala, as assessed by non-denaturing mass spectrometry. The EBD does not bind to 1,6-anhydro-N-acetyl-β-d-muramyl-l-Ala-γ- d-Glu-meso-DAP. This binding selectivity revises the dogma in the field.
Journal of the American Chemical Society (2017) 139, 1448–1451 (doi:10.1021/jacs.6b12819)
β-lactam antibiotics are currently the most used antibiotics. These antibiotics prevent bacterial cell wall formation, critical for bacterial survive. The cell wall contains the PG which is formed by alternated units of N-acetilglucosamine (NAG) and N-acetilmuramic acid (NAM) with peptide stems attached to NAM. These peptide stems are cross-linked creating a net. β-lactam antibiotics inhibit the transpeptidation process between peptides promoting the accumulation of aberrant long fragments. Pseudomonas aeruginosa attempts to repair this damage by the lytic transglycosilase Slt. In our work we have managed to solve the structure of the enzyme Slt to carry out the study of its catalytic mechanism getting several complexes with analogous of its natural substrate. Slt is able to degrade the PG through both endolytic (in the middle of chain) and exolytic (in one end) cut. Slt can accommodate the PG thanks to a long catalytic throat with up to 10 positions for NAG/NAM units together with certain key residues that interact with the peptide stems. These results disclose the details of bacterial response to the β-lactam antibiotic challenge.
Proceedings of the National Academy of Sciences, PNAS (2018) in press (doi:10.1073/pnas.1801298115)
Processes catalyzed by enzymes offer numerous advantages over chemical methods although in many occasions the stability of the biocatalysts becomes a serious concern. Traditionally, synthesis of nucleosides using poorly water-soluble purine bases, such as guanine, xanthine, or hypoxanthine, requires alkaline pH and/or high temperatures in order to solubilize the substrate. In this work, we demonstrate that the 2′-deoxyribosyltransferase from Leishmania mexicana (LmPDT) exhibits an unusually high activity and stability under alkaline conditions (pH 8–10) across a broad range of temperatures (30–70 °C) and ionic strengths (0–500 mM NaCl). Conversely, analysis of the crystal structure of LmPDT together with comparisons with hexameric, bacterial homologues revealed the importance of the relationships between the oligomeric state and the active site architecture within this family of enzymes. Moreover, molecular dynamics and docking approaches provided structural insights into the substrate-binding mode. Biochemical characterization of LmPDT identifies the enzyme as a type I NDT (PDT), exhibiting excellent activity, with specific activity values 100- and 4000-fold higher than the ones reported for other PDTs. Interestingly, LmPDT remained stable during 36 h at different pH values at 40 °C. In order to explore the potential of LmPDT as an industrial biocatalyst, enzymatic production of several natural and non-natural therapeutic nucleosides, such as vidarabine (ara A), didanosine (ddI), ddG, or 2′-fluoro-2′-deoxyguanosine, was carried out using poorly water-soluble purines. Noteworthy, this is the first time that the enzymatic synthesis of 2′-fluoro-2′-deoxyguanosine, ara G, and ara H by a 2′-deoxyribosyltransferase is reported.
Applied Microbiology and Biotechnology (2017) 101, 7187–7200 (doi:10.1007/s00253-017-8450-y)
Statistics from structural genomics initiatives reveal that around 50–55% of the expressed, non-membrane proteins cannot be purified and therefore structurally characterized due to solubility problems, which emphasized protein solubility as one of the most serious concerns in structural biology projects. Lactobacillus plantarum CECT 748T produces an aggregation-prone glycosidase (LpBgl) that we crystallized previously. However, this result could not be reproduced due to protein instability and therefore further high-resolution structural analyses of LpBgl were impeded. The obtained crystals of LpBgl diffracted up to 2.48 Å resolution and permitted to solve the structure of the enzyme. Analysis of the active site revealed a pocket for phosphate-binding with an uncommon architecture, where a phosphate molecule is tightly bound suggesting the recognition of 6-phosphoryl sugars. In agreement with this observation, we showed that LpBgl exhibited 6-phospho-β-glucosidase activity. Combination of structural and mass spectrometry results revealed the formation of dimethyl arsenic adducts on the solvent exposed cysteine residues Cys211 and Cys292. Remarkably, the double mutant Cys211Ser/Cys292Ser resulted stable in solution at high concentrations indicating that the marginal solubility of LpBgl can be ascribed specifically to these two cysteine residues. The 2.30 Å crystal structure of this double mutant showed no disorder around the newly incorporated serine residues and also loop rearrangements within the phosphate-binding site. Notably, LpBgl could be prepared at high yield by proteolytic digestion of the fusion protein LSLt-LpBgl, which raises important questions about potential hysteretic processes upon its initial production as an enzyme fused to a solubility enhancer.
Biochimica et Biophysica Acta (2017) 1865, 1227-1236 (doi:10.1016/j.bbapap.2017.07.007)
The mechanism of the β-lactam antibacterials is the functionally irreversible acylation of the enzymes that catalyze the cross-linking steps in the biosynthesis of their peptidoglycan cell wall. The Gram-positive pathogen Staphylococcus aureus uses one primary resistance mechanism. An enzyme, called penicillin-binding protein 2a (PBP2a), is brought into this biosynthetic pathway to complete the cross-linking. PBP2a effectively discriminates against the β- lactam antibiotics as potential inhibitors, and in favor of the peptidoglycan substrate. The basis for this discrimination is an allosteric site, distal from the active site, that when properly occupied concomitantly opens the gatekeeper residues within the active site and realigns the conformation of key residues to permit catalysis. We address the molecular basis of this regulation using crystallographic studies augmented by computational analyses. The crystal structures of three β-lactams (oxacillin, cefepime, ceftazidime) complexes with PBP2a, each with the β- lactam in the allosteric site, defined (with preceding PBP2a structures) as the “open” or “partially open” PBP2a states. A particular loop motion adjacent to the active site is identified as the driving force for the active-site conformational change that accompanies active-site opening. Correlation of this loop motion to effector binding at the allosteric site, in order to identify the signaling pathway, was accomplished computationally in reference to the known “closed” apo-PBP2a X-ray crystal structure state. This correlation enabled the computational simulation of the structures coinciding with initial peptidoglycan substrate binding to PBP2a, acyl enzyme formation, and acyl transfer to a second peptidoglycan substrate to attain cross-linking. These studies offer important insights into the structural bases for allosteric site-to-active site communication and for β-lactam mimicry of the peptidoglycan substrates, as foundational to the mechanistic understanding of emerging PBP2a resistance mutations.
Journal of the American Chemical Society (2017) 139, 2102-2110 (doi:10.1021/jacs.6b12565)
The protein complex formed by the Ca2+ sensor NCS-1 and the Guanyl Exchange Factor Ric8a co-regulates synapse number and probability of neurotransmitter release, emerging as a potential therapeutic target for diseases affecting synapses such as Fragile X syndrome (FXS), the most common heritable autism disorder. Using crystallographic data and the virtual screening of a chemical library, we identified a set of heterocyclic small molecules as potential inhibitors of the NCS-1/Ric8a interaction. The aminophenothiazine FD44 prevents the formation of the NCS-1/Ric8a complex, it reduces the aberrant excess of synapse number to normal levels and improves associative learning in a Drosophila FXS model. The high-resolution crystal structure of NCS-1 bound to FD44 and the structure-function studies performed with structurally close but inactive analogues explain the FD44 specificity and the mechanism of inhibition. We found that FD44 is an allosteric inhibitor that stabilizes NCS-1 in a conformation that is incompatible with Ric8a binding, which explains how a small molecule can inhibit such a big and complex protein-protein interaction surface. Our study demonstrates the druggability of the NCS-1/Ric8a interface and uncovers a suitable region in NCS-1 to develop additional drugs for the treatment of FXS and related synaptic disorders.
Proceedings of the National Academy of Sciences, PNAS (2017) 114, E999–E1008 (doi:10.1073/pnas.1611089114)
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