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The Grupo Especializado de Cristalografía y Crecimiento Cristalino (GE3C) has awarded the 2017 'Xavier Solans’ Prize to Dr. Teresa Domínguez-Gil Velasco (a former PhD student by Dr. Juan A. Hermoso Domíguez) for her excellent article Activation by Allostery in Cell-Wall Remodeling by a Modular Membrane-Bound Lytic Transglycosylase from Pseudomonas aeruginosa, published in Structure (2016) 24, 1729-1741. This award recognizes the work of young researchers who perform their research in Spain in the field of crystallography and crystal growth.
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) in press (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)
Design of generic methods aimed at the oriented attachment of proteins at the interfacial environment of magnetic nanoparticles currently represents an active field of research. With this in mind, we have prepared and characterized agarose-coated maghemite nanoparticles to setup a platform for the attachment of recombinant proteins fused to the β-trefoil lectin domain LSL150, a small protein that combines fusion tag properties with agarose-binding capacity. Analysis of the agarose-coated nanoparticles by dynamic light scattering, Fourier transform infrared spectroscopy and thermogravimetric studies shows that decoupling particle formation from agarose coating provides better results in terms of coating efficiency and particle size distribution. LSL150 interacts with these agarose-coated nanoparticles exclusively through the recognition of the sugars of the polymer, forming highly stable complexes, which in turn can be dissociated ad hoc with the competing sugar lactose. Characterization of the complexes formed with the fusion proteins LSL-EGFP (LSL-tagged enhanced green fluorescent protein from Aquorea victoria) and LSL-BTL2 (LSL-tagged lipase from Geobacillus thermocatenolatus) provided evidences supporting a topologically oriented binding of these molecules to the interface of the agarose-coated nanoparticles. This is consistent with the marked polarity of the β-trefoil structure where the sugar-binding sites and the N- and C-terminus ends are at opposed sides. In summary, LSL150 displays topological and functional features expected from a generic molecular adaptor for the oriented attachment of proteins at the interface of agarose-coated nanoparticles.
Bioconjugate Chemistry (2016) 27, 2734−2743 (doi:10.1021/acs.bioconjchem.6b00504)
Journal of Biological Chemistry (2016) (doi:10.1074/jbc.M116.747527)
Pseudomonas aeruginosa is a human pathogen that causes pneumonia, bloodstream infections, urinary tract infections, and surgical site infections. Strains of P. aeruginosa have been found to be broadly resistant to antibiotics such us aminoglycosides, cephalosporins, fluoroquinolones, and carbapenems. Recycling of the cell wall in bacteria is linked to antibiotic resistance and virulence mechanisms. Lytic transglycosylases (LTs) initiate the cell-wall recycling processes by cleaving crosslinked and uncrosslinking glycan strands. 3D structure of LT SltB3 has four domains arranged in a unique annular conformation. The structures of SltB3 in complex with a substrate analog and with a reaction-product analog together with the study of the reaction catalyzed by this enzyme using liquid chromatography and high-resolution mass-spectrometric analyses (LC/MS/MS) revealed that SltB3 is an exolytic enzyme that recognizes a minimum of four sugars in its substrate but can process a substrate comprised of a peptidoglycan of 20 sugars. Interestingly, the high-resolution structures of the SltB3 complexes provided indications on how the peptidoglycan and its products of turnover would span the opening of the annulus during catalysis. The analysis reveals that polymeric linear peptidoglycan substrate threads through the opening of the annulus of the enzyme.
ACS Chemical Biology (2016) 11, 1525-1531 (doi:10.1021/acschembio.6b00194)
Invertases, or β-fructofuranosidases, are biotechnologically important enzymes secreted by many fungi showing wide applications in the food and pharmaceutical industries. Besides their hydrolytic function, they catalyze the synthesis of short-chain fructooligosaccharides (FOS), prebiotics that selectively stimulate growth of lactobacilli and bifidobacteria in the digestive tract, contributing to the prevention of cardiovascular diseases, colon cancer or osteoporosis. XdINV is a highly glycosylated, bimodular enzyme producing neo-FOS having enhanced prebiotic effect as compared to the common 1F-FOS series. Our study reveals that N-linked glycans and an insert at the C-terminus mediate the formation of an unusual dimer that shapes a peculiar active site able to house branched substrates. Analysis of complexes allowed mapping the catalytic pocket and point to the role of a flexible loop in discriminating substrate specificity. The plasticity of its active site makes the enzyme a valuable and flexible biocatalyst to produce novel bioconjugates.
Journal of Biological Chemistry (2016) 291, 6843-6857 (doi:10.1074/jbc.M115.708495)
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)
The human pathogen Streptococcus pneumoniae is decorated with a special class of surface-proteins known as choline-binding proteins (CBPs) attached to choline (Cho) moieties from cell-wall teichoic acids (TA). By a combination of X-ray crystallography, NMR, molecular dynamics techniques and in vivo virulence and phagocytosis studies, we provide structural information of choline-binding protein L (CbpL) and demonstrate its impact on pneumococcal pathogenesis and immune evasion.
Scientific Reports (2016) 6, art. 38094 (doi:10.1038/srep38094)
The bacterial cell wall is an elastic polymer that defines the shape of the bacterium and prevents cell lysis under high osmotic pressure. Since the cell wall is a unique part of the bacteria it becomes very interesting target. Lytic transglycosylases (LTs) cleave the non-hydrolytic fragmentation of the β-1,4-glycosidic bond between the main components of peptidoglycan (PG). The mechanism by which LTs catalyse the fragmentation is unique. MltF is a modular LT from P. aeruginosa, the three-dimensional structure confirms that the enzyme is organized in two modules, the regulatory domain and the catalytic domain. Occupancy of the regulatory module by a PG-derived-muropeptide causes a dramatic conformational change, which opens the active site for catalysis. The richness of the structural information on MltF reveals this unique regulatory mechanism and provides a foundation for further inquiry in understanding of its contribution toward the complex orchestration of the structure of the peptidoglycan of the P. aeruginosa cell wall.
Structure (2016) 24, 1729-1741 (doi:10.1016/j.str.2016.07.019)
A new family of scaffold proteins orchestrates plant response to environmental stresses. Drought and salinity are the major threats to crop productivity at a worldwide scale. A fundamental portion of the plant response to these environmental stresses occurs at the cell membrane, where the molecular machinery to preserve cell turgor and the appropriate balance of intracellular ions is found. The C2-domain ABA-related (CAR) family of proteins contributes to these processes by delivering the regulatory proteins controlling this machinery from other cell compartments to the cell membrane. Our analysis provides an explanation on how CAR proteins specifically reach a particular membrane place to develop their function and trigger the plant defense mechanism against stress.
Proceedings of the National Academy of Sciences, PNAS (2016) 113, E396-E405 (doi:10.1073/pnas.1512779113)
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