most recent results (See also our remaining publications
In collaboration with BRUKER Española S.A., the Spanish Society of Biochemistry and Molecular Biology (SEBBM) has awarded Dr Antonio Chaves-Sanjuán with the “José Tormo” Prize for his work published in PNAS in 2017 and performed at Dr MJ Sánchez-Barrena Lab from the Dept. of Crystallography and Structural Biology, Institute “Rocasolano”. This prize gives recognition to the best piece of work performed by a young researcher in a Spanish laboratory, and published during 2016-2017 in any of the areas related to Structural Biology. Dr Chaves-Sanjuán will give a conference entitled Las fenotiazinas regulan la función sináptica interfiriendo en la formación del complejo NCS-1/Ric8a: Un nuevo enfoque para el síndrome X frágil at the XL Congress of the SEBBM, that will be held in Barcelona (23rd-26th October 2017).
Our colleague, Dr MJ Sánchez-Barrena, contracted researcher at Institute “Rocasolano”, has been awarded with a very competitive “Leonardo” fellowship from the BBVA Foundation. She will be able to continue her research on synapse alterations (contact among neurons) that are present, among others, in pathologies such as Alzheimer´s, dementia or Huntington disease. Her research is aimed to design molecules that are able to restore these contacts, avoiding memory loss and improving cognitive ability in earlier stages of the disease.
ABC press release
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)
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)
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.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.
Biochimica et Biophysica Acta (2017) 1865, 1227-1236 (doi:10.1016/j.bbapap.2017.07.007)
Journal of the American Chemical Society (2017) 139, 2102-2110 (doi:10.1021/jacs.6b12565)
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