most recent results (See also our remaining publications
Other CBE issues...
The very first Acta Cryst. article by our predecessors...
Acta Cryst. (1948) 1, 3-4
About our origins...
Improved plants against climate change. Environmental damage, together with climate change, are driving the water-related crises we see around the world. Stress situations such as drought or the concomitant soil salinity affect crop productivity as they unbalance intracellular ion composition in plants. In these situations, and spontaneously, plants attempt to readjust ion homeostasis for normal growth. We have described the mechanism by which the regulatory protein kinase CIPK23 is specifically assembled to the AKT1 K+ channel to restore cell ion requirements. This information is central to produce novel crop varieties with improved performance in changing environment. The drawing shows the recognition and activation of the plant AKT1 potassium channel by the kinase CIPK23.
Plant Physiology (2020) February, in press (doi:10.1104/pp.19.01084)
BglX is a periplasmic glycoside hydrolase of the human pathogen Pseudomonas aeruginosa. Crystal structure revealed that BglX is a homodimer where each active site comprises catalytic residues provided by each monomer. Structural and in vitro analysis show that BglX performs catalysis on β -(1→ 2) and β -(1→ 3) saccharides while β -(1→ 4) muropeptides from the cell-wall peptidoglycan are not substrates. Several crystal structures of BglX were solved imitating the catalysis including structures of a mutant enzyme-derived Michaelis complex, two transition-state mimetics, and two enzyme−product complexes. The substrate pattern for BglX aligns with the [β -(1→ 2)-Glc]x and [β -(1→ 3)-Glc]x periplasmic osmoregulated periplasmic glucans, and possibly with the Psl exopolysaccharides, of P. aeruginosa . Both polysaccharides are implicated in biofilm formation. Accordingly, we show that inactivation of the bglX gene of P. aeruginosa PAO1 attenuates biofilm formation.
ACS Chemical Biology (2020) 15, 189-196 (doi:10.1021/acschembio.9b00754)
A team from the Spanish National Research Council (CSIC) and the University of Notre Dame (United States) has revealed the structure of a key machinery in the process of bacterial division. The conclusions, published in the latest issue of the journal Nature Communications, open the door to the design of a future drug capable of blocking this precise machinery, without which the bacteria become sensitive to the antibiotic effect. Virtually all bacterial species have specialized domains that recognize the bacterial wall (composed of peptidoglycan) at the time of division and allow the correct location in space and time of these proteins during the generation of the two daughter cells from the mother cell. Such is the case of SPOR domains, which are widely present in bacterial proteins that recognize cell-wall peptidoglycan strands stripped of the peptide stems (called "naked" glycans). This type of peptidoglycan is enriched in the septal ring as a product of catalysis by cell-wall amidases that participate in the separation of daughter cells during cell division. The authors document the binding of synthetic naked glycans to the SPOR domain of the RlpA lytic transglycosylase of Pseudomonas aeruginosa (SPOR-RlpA) by mass spectrometry and structural analysis, which demonstrate that, in fact, the presence of peptide stems in the peptidoglycan abrogates binding. The crystal structures of the SPOR domain at atomic resolution (1.2 Å), in the apo state and in complex with different synthetic glycans, provide insight into the molecular basis for recognition and delineate a conserved pattern in other SPOR domains. The biological and structural observations presented here are followed up by molecular-dynamics simulations and by exploration of the effect on binding of distinct peptidoglycan modifications. These results provide an explanation to an unresolved question since decades: how the SPOR domains, present in almost all bacteria and with very little sequence homology, can all recognize the same type of cell wall during bacterial division.
Nature Communications (2019) 10, Article number: 5567 (doi: 10.1038/s41467-019-13354-4)
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)
Lactose intolerance is a common digestive disorder that affects a large proportion of the adult human population, the severity of the symptoms being highly variable. Therefore, enzymes that can be used for the production of lactose-free milk and milk derivatives have acquired singular biotechnological importance. We have solved the Thermotoga maritima β-galactosidase cryo-EM structure at 2.0 Å resolution, revealing a novel domain at its C-terminus, which promotes a peculiar octameric arrangement that, in turn, affects its stability and specificity. Furthermore, we have described key features of the catalytic site that are crucial for interacting with large substrates and for transglycosylation reactions, thus enabling the use of the enzyme to produce galactooligosaccharides, prebiotic ingredients. The structural information obtained for the TmLac octamer allowed us to improve the immobilization strategies for this enzyme, achieving a 100% immobilization efficiency on cellulose and chitin substrates. This sets the groundwork for developing new versions of the enzyme with extended immobilization properties and enhanced substrate specificities.
ACS Chemical Biology (2020) 15, 179-188 (doi:10.1021/acschembio.9b00752)
The spectacular development that enzymatic engineering experienced in recent years has allowed the generation of highly effective biocatalysts for more sustainable processes and has enormous potential in many other therapeutic and diagnostic applications in biomedicine. In this sense, the de novo creation of active sites allows reaction rates close to diffusion limits, but also the generation of abiological sites operating on non-natural substrates. In this work we explored chemical and biochemical design, supported by computational and structural methods, to generate a plurizyme, a serine ester hydrolase with two active sites, one natural and one synthetic, in which the catalytic efficiency, specificity and stereoselectivity have been significantly improved. Furthermore, one of the two active sites has been subsequently transformed into a metal-complex chemocatalytic site for oxidation and Friedel–Crafts alkylation reactions, facilitating synergistic chemo- and biocatalysis in a dual, single protein. The work, a collaborative and multidisciplinar study done by researchers from Spain, UK, Austria and Switzerland, has given the first structural evidence of a plurizyme with two biological active sites, revealing the conformational changes observed at the artificially remodeled site, upon inhibitor binding. Moreover, we illuminate the novel chemocatalytic site arrangement at the molecular level. We propose this approach as a powerful tool to produce enhanced catalysts, combining biological and new-to-nature chemical transformations skills. This combination thereof may represent a valuable alternative to expand the joined use of enzymes and catalytic metals, particularly for cascade reactions.
Nature Catalysis (2019) (doi:10.1038/s41929-019-0394-4)
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) 63, e02637-18 (doi: 10.1128/AAC.02637-18)
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)
© CSIC. The text, data and information in these pages are available for personal use only. Therefore, permission of the CSIC is required for their publication, distribution or inclusion in any context accessible to third parties.