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The structure of the human Phenylalanine hydroxylase (PAH), responsible for phenylketonuria, the most frequent congenital metabolic disease, disclosed!!!
Phenylalanine hydroxylase (PAH) is a key enzyme in the catabolism of phenylalanine, and mutations in this enzyme cause phenylketonuria (PKU), a genetic disorder that leads to brain damage and mental retardation if untreated. Some patients benefit from supplementation with a synthetic formulation of the cofactor tetrahydrobiopterin (BH4) that partly acts as a pharmacological chaperone. An international team lead by Rocasolano Institute and University of Bergen (Norway) has been able to determine the atomic structure of PAH. The results are published in the journal Proceedings of the National Academy of Sciences (PNAS). In this work we present the first structures of full-length human PAH (hPAH) both unbound and complexed with BH4 in the pre-catalytic state. The BH4-bound state is physiologically relevant, keeping PAH stable and in a pre-catalytic state at low L-Phe concentration. Furthermore, a synthetic form of BH4 (Kuvan®) is the only drug-based therapy for a subset of phenylketonuria patients. We found two tetramer conformations in the same crystal, depending on the active site occupation by BH4, which aid to understand the stabilization by BH4 and the allosteric mechanisms in PAH. The structure also reveals the increased mobility of human- compared with rat PAH, in line with an increased predisposition to disease-associated mutations in human. Two decades after the first partial structures of human phenylalanine hydroxylase (PAH) were published, the results of a long-term and successful collaboration between researchers at CSIC and CNIO in Spain, and the University of Bergen in Norway can finally be presented: full-length structures of this very important metabolic enzyme.
Proceedings of the National Academy of Sciences, PNAS (2019) 116, 11229-11234 (doi:10.1073/pnas.1902639116)
Streptococcus pneumoniae is a leading killer of children and immunocompromised individuals. S. pneumoniae has become increasingly resistant to major antibiotics, and therefore the development of new antibiotic strategies is desperately needed. Targeting bacterial cell division is one such strategy, specifically targeting essential proteins for the synthesis and breakdown of peptidoglycan. Across multiple species of bacteria, the protein FtsX is a cell division protein involved in the regulation of peptidoglycan hydrolases. FtsX represents a large group of ABC-transporter like proteins that function as ‘mechanotransmitters’, proteins that relay signals from inside the cell to the outside. In a collaborative international (Spain, Norway and USA) effort leaded by IQFR and University Indiana Bloomington we present the first structural characterization of the large extracellular loop of FtsX from the human opportunistic pathogen Streptococcus pneumoniae. We show the direct interaction between the peptidoglycan hydrolase PcsB and FtsX, and that this interaction is essential for cell viability. As such, FtsX represents an attractive, conserved target for the development of new classes of antibiotics.
mBio (2019) (doi:10.1128/mBio.02622-18)
Journal of Medicinal Chemistry (2018) (doi:10.1021/acs.jmedchem.8b00088)
A promising NCS-1/Ric8a protein-protein interaction (PPI) stabilizer with therapeutic potential in Alzheimer´s disease has been disclosed!!! The work, product of a colaborative and multidisciplinar study done by researchers from IQFR, CIB, IRYCIS and UCM, has revealed an innovative strategy to improve synapse function in neurodegenerative diseases. Synapse function regulation depends on the correct interaction between the Ca2+ sensor NCS-1 and the guanine exchange factor Ric8a. Therefore, the interface of this protein-protein complex is a target for the design of therapeutic molecules that may constitute an innovative treatment for synaptopathies, a group of neuronal diseases where synapse number is disregulated. Previously, we published in PNAS how the inhibition of the NCS-1/Ric8a complex with a small phenothiazine reduces the abnormally high synapse number and enhances associative learning in a FXS animal model. In this new work, we have demonstrated that the stabilization of the protein complex with an acylhydrazone produces the opposite effect, re-stablishes synapse number and improves neuronal function in an Alzheimer´s disease model. The crystal structure of NCS-1 in complex with the bioactive molecule explains its mechanism of action at atomic level and suggests how to improve the eficacy of these regulatory molecules.
Nature Communications (2019) 10, article number: 2798 (doi:10.1038/s41467-019-10627-w)
The quinazolinones are a new class of antibacterials with in vivo efficacy against methicillin resistant Staphylococcus aureus (MRSA). The quinazolinones target cell-wall biosynthesis and have a unique mechanism of action by binding to the allosteric site of penicillin-binding protein (PBP)2a. The combination of the quinazolinone with the commercial piperacillin-tazobactam showed bactericidal synergy. We demonstrated the efficacy of the triple-drug combination in a mouse MRSA neutropenic thigh-infection model. The proposed mechanism for the synergistic activity in MRSA involves inhibition of the β-lactamase by tazobactam, which protects piperacillin from hydrolysis, which can then inhibit its target PBP2a. Furthermore, the quinazolinone binds to the allosteric site of PBP2a triggering the allosteric response. This leads to the opening of the active site, which in turn binds another molecule of piperacillin. The collective effect is the impairment of cell-wall biosynthesis, with bactericidal consequence. Two crystal structures for complexes of the antibiotics with PBP2a provide support for the proposed mechanism of action.
Antimicrob. Agents Chemother. (2019) In press (doi: 10.1128/AAC.02637-18)
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) 115, 4393-4398 (doi:10.1073/pnas.1801298115)
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.
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