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15 páginas, 5 figuras ; Human carbamoyl phosphate synthetase (CPS1), a 1500-residue multidomain enzyme, catalyzes the first step of ammonia detoxification to urea requiring N-acetyl-L-glutamate (NAG) as essential activator to prevent ammonia/amino acids depletion. Here we present the crystal structures of CPS1 in the absence and in the presence of NAG, clarifying the on/off-switching of the urea cycle by NAG. By binding at the C-terminal domain of CPS1, NAG triggers long-range conformational changes affecting the two distant phosphorylation domains. These changes, concerted with the binding of nucleotides, result in a dramatic remodeling that stabilizes the catalytically competent conformation and the building of the ~35 Å-long tunnel that allows migration of the carbamate intermediate from its site of formation to the second phosphorylation site, where carbamoyl phosphate is produced. These structures allow rationalizing the effects of mutations found in patients with CPS1 deficiency (presenting hyperammonemia, mental retardation and even death), as exemplified here for some mutations. ; This work was supported by grants from The Fundación Alicia Koplowitz, the Valencian (PrometeoII/2014/029 to V.R) and Spanish Governments (BFU2011-30407 and BFU2014-58229-P to V.R; SAF2010-17933 to J.C; BFU2012-36827 to I.F.). The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under BioStruct-X (grant agreement Nº283570), within proposal 7687. ; Peer reviewed

The photooxygenation of model compounds containing the two enantiomers of naproxen (NPX) covalently linked to histidine (His), tryptophan (Trp) and tyrosine (Tyr) has been investigated by steady state irradiation, fluorescence spectroscopy and laser flash photolysis. The NPX–His systems presented the highest oxygen-mediated photoreactivity. Their fluorescence spectra matched that of isolated NPX and showed a clear quenching by oxygen, leading to a diminished production of the NPX triplet excited state ( 3 NPX*–His). Analysis of the NPX–His and NPX–Trp photolysates by UPLC-MS–MS revealed in both cases the formation of two photoproducts, arising from the reaction of singlet oxygen (1 O2) with the amino acid moiety. The most remarkable feature of NPX–Trp systems was a fast and stereoselective intramolecular fluorescence quenching, which prevented the efficient formation of 3 NPX*–Trp, thus explaining their lower reactivity towards photooxygenation. Finally, the NPX–Tyr systems were nearly unreactive and exhibited photophysical properties essentially coincident with those of the parent NPX. Overall, these results point to a type II photooxygenation mechanism, triggered by generation of 1 O2 from the 3 NPX* chromophore ; Financial support from the Spanish Government (CTQ2010-14882, JCI-2011-09926, Miguel Servet CP11/00154), from the EU (PCIG12-GA-2012-334257), from the Universitat Politecnica de Valencia (SP20120757) and from the Conselleria de Educacio, cultura i Esport (PROMETEOII/2013/005, GV/2013/051) is gratefully acknowledged. ; Vayá Pérez, I.; Andreu Ros, MI.; Jiménez Molero, MC.; Miranda Alonso, MÁ. (2014). Photooxygenation mechanisms in naproxen-amino acid linked systems. Photochemical & Photobiological Sciences Photochemical and Photobiological Sciences. 13:224-230. https://doi.org/10.1039/c3pp50252j ; S ; 224 ; 230 ; 13 ; L. I. Grossweiner and K. C.Smith, Photochemistry, in The Science of Photobiology, ed. K. C. Smith, Plenum Press, New York, 2nd edn, 1989, pp. 47–78 ; L. Pretali and A.Albini, in CRC ...

Modeling the dynamic nature of protein-ligand binding with atomistic simulations is one of the main challenges in computational biophysics, with important implications in the drug design process. Although in the past few years hardware and software advances have significantly revamped the use of molecular simulations, we still lack a fast and accurate ab initio description of the binding mechanism in complex systems, available only for up-to-date techniques and requiring several hours or days of heavy computation. Such delay is one of the main limiting factors for a larger penetration of protein dynamics modeling in the pharmaceutical industry. Here we present a game-changing technology, opening up the way for fast reliable simulations of protein dynamics by combining an adaptive reinforcement learning procedure with Monte Carlo sampling in the frame of modern multi-core computational resources. We show remarkable performance in mapping the protein-ligand energy landscape, being able to reproduce the full binding mechanism in less than half an hour, or the active site induced fit in less than 5 minutes. We exemplify our method by studying diverse complex targets, including nuclear hormone receptors and GPCRs, demonstrating the potential of using the new adaptive technique in screening and lead optimization studies. ; We thank Drs Anders Hogner and Christoph Grebner, from AstraZeneca, and Jorge Estrada, from BSC, for fruitful discussions and feedback on the manuscript. We acknowledge the BSC-CRG-IRB Joint Research Program in Computational Biology. This work was supported by the CTQ2016-79138-R grant from the Spanish Government. D.L. acknowledges the support of SEV-2011-00067, awarded by the Spanish Government. ; Peer Reviewed ; Postprint (published version)

Modeling the dynamic nature of protein-ligand binding with atomistic simulations is one of the main challenges in computational biophysics, with important implications in the drug design process. Although in the past few years hardware and software advances have significantly revamped the use of molecular simulations, we still lack a fast and accurate ab initio description of the binding mechanism in complex systems, available only for up-to-date techniques and requiring several hours or days of heavy computation. Such delay is one of the main limiting factors for a larger penetration of protein dynamics modeling in the pharmaceutical industry. Here we present a game-changing technology, opening up the way for fast reliable simulations of protein dynamics by combining an adaptive reinforcement learning procedure with Monte Carlo sampling in the frame of modern multi-core computational resources. We show remarkable performance in mapping the protein-ligand energy landscape, being able to reproduce the full binding mechanism in less than half an hour, or the active site induced fit in less than 5 minutes. We exemplify our method by studying diverse complex targets, including nuclear hormone receptors and GPCRs, demonstrating the potential of using the new adaptive technique in screening and lead optimization studies. ; We thank Drs Anders Hogner and Christoph Grebner, from AstraZeneca, and Jorge Estrada, from BSC, for fruitful discussions and feedback on the manuscript. We acknowledge the BSC-CRG-IRB Joint Research Program in Computational Biology. This work was supported by the CTQ2016-79138-R grant from the Spanish Government. D.L. acknowledges the support of SEV-2011-00067, awarded by the Spanish Government. ; Peer Reviewed ; Postprint (published version)

Abstract
Polyzwitterions (PZs) are considered as model synthetic analogs of intrinsically disordered proteins. Based on this analogy, PZs in dilute aqueous solutions are expected to attain either globular (i.e. molten, compact) or random coil conformations. Addition of salt is expected to open these conformations. To the best of our knowledge, these hypotheses about conformations of PZs have never been verified. In this study, we test these hypotheses by studying effects of added salt [potassium bromide (KBr)] on gyration and hydrodynamic radii of poly(sulfobetaine methacrylate) in dilute aqueous solutions using dynamic light scattering and small-angle X-ray scattering, respectively. Effects of zwitteration are revealed by direct comparisons of the PZs with the polymers of the same backbone but containing (1) no explicit charges on side groups such as poly(2-dimethylaminoethyl methacrylate)s and (2) explicit cationic side groups with tertiary amino bromide pendants. Zeta-potential measurements, transmission electron microscopy, and ab initio molecular dynamics simulations reveal that the PZs acquire net positive charge in near salt-free conditions due to protonation but retain coiled conformations. Added KBr leads to nonmonotonic changes exhibiting an increase followed by a decrease in radius of gyration (and hydrodynamic radius), which are called antipolyelectrolyte and polyelectrolyte effects, respectively. Charge regulation and screening of charge–charge interactions are discussed in relation to the antipolyelectrolyte and polyelectrolyte effects, respectively, which highlight the importance of salt in affecting net charge and conformations of PZs.

Carotenoids are one of the largest classes among biological pigments. They are found both in free form (dissolved in hydrophobic environments), and bound in stoichiometric complexes with protein (carotenoproteins). The interaction between the carotenoid and the protein environment is determined by the molecular conformation and dynamic behavior of the cofactor. In this work, using molecular modeling methods, we analyzed the entire set of alternative conformational states of cyclic carotenoids. The result of the theoretical study is an energysorted catalog of alternative conformational states for biogenic cyclic carotenoids and their optical isomers. The conformational states characterized in this study can be used to correctly select initial conditions when solving problems of molecular modeling of carotenoproteins

Carotenoids are one of the largest classes among biological pigments. They are found both in free form (dissolved in hydrophobic environments), and bound in stoichiometric complexes with protein (carotenoproteins). The interaction between the carotenoid and the protein environment is determined by the molecular conformation and dynamic behavior of the cofactor. In this work, using molecular modeling methods, we analyzed the entire set of alternative conformational states of cyclic carotenoids. The result of the theoretical study is an energysorted catalog of alternative conformational states for biogenic cyclic carotenoids and their optical isomers. The conformational states characterized in this study can be used to correctly select initial conditions when solving problems of molecular modeling of carotenoproteins

Derivatives from biomass have attracted increasing interest in academic, social and political areas because of environmental problems caused by fossils fuel. Among these derivatives, stand out the polyalcohols, whose electrooxidation leads to value-added chemicals besides generating energy from fuel cells. In this work, the adsorption-desorption process of 4 solutions of polyalcohols on platinum (100), (110) and (111) surfaces will be investigated to compare the polyalcohol distributions and conformation on the different surfaces.

Metabotropic glutamate receptors (mGluRs) are dimeric G-protein-coupled receptors activated by the main excitatory neurotransmitter, L-glutamate. mGluR activation by agonists binding in the venus flytrap domain is regulated by positive (PAM) or negative (NAM) allosteric modulators binding to the 7-transmembrane domain (7TM). We report the cryo-electron microscopy structures of fully inactive and intermediate-active conformations of mGlu5 receptor bound to an antagonist and a NAM or an agonist and a PAM, respectively, as well as the crystal structure of the 7TM bound to a photoswitchable NAM. The agonist induces a large movement between the subunits, bringing the 7TMs together and stabilizing a 7TM conformation structurally similar to the inactive state. Using functional approaches, we demonstrate that the PAM stabilizes a 7TM active conformation independent of the conformational changes induced by agonists, representing an alternative mode of mGlu activation. These findings provide a structural basis for different mGluR activation modes. ; We would like to thank Joël Bockaert, Chris Tate, Philippe Marin, and Richard Henderson for careful reading and for their comments on the manuscript. We would like to thank SLS PSI XA06 microfocus beamline scientist (Villigen; Switzerland), MRC Laboratory Molecular Biology EM and computing core facilities (Cambridge, UK), and the platform of pharmacology Arpeges of the Institut de Génomique Fonctionnelle (Montpellier, France). This project was funded by the ATIP grant from CNRS. C.N. was funded by the Post-doctoral program from Montpellier University. A.E.B. was supported by the Labex EpiGenMed (program "Investissements d'avenir," ANR-10-LABX-12-01), L.B. was supported by Ligue Contre le Cancer. J.F.-I., X.G.-S., J.C., and A.L. were supported by Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación and ERDF-FEDER European Fund (project CTQ2017-89222-R) and by the Catalan government (2017 SGR 1604). J.-L.B. was supported by ANR (ANR-17-CE11-0011). J.-P.P. was supported by the Fondation Recherche Médicale (FRM team: DEQ20170326522), grants from ANR (DynaMuR2, ANR-18-CE11-0004; Lanthslider, ANR-17-CE11-0046). G.C. and K.R.V. were supported by Medical Research Council grant MC-U105184322. K.R.V. acknowledges SERB, India for the Ramanujan Fellowship (RJN-094/2017), DBT B-Life grant DBT/PR12422/MED/31/287/2014, and the support of the Department of Atomic Energy, Government of India, Government of India, under Project Identification No. RTI4006. G.L. was supported by ANR grants AT2R-TRAAK Bioanalgesics (ANR-17-CE18-0001), DynaMuR2 (ANR-18-CE11-0004), SWITCH-ON (ANR-20-CE11-0019), and Fondation Recherche Médicale (ING20121226326). ; Peer reviewed

14 páginas, 7 figuras, 1 tabla. Contiene material suplementario. ; Bacillus phages use a communication system, termed "arbitrium," to coordinate lysis-lysogeny decisions. Arbitrium communication is mediated by the production and secretion of a hexapeptide (AimP) during lytic cycle. Once internalized, AimP reduces the expression of the negative regulator of lysogeny, AimX, by binding to the transcription factor, AimR, promoting lysogeny. We have elucidated the crystal structures of AimR from the Bacillus subtilis SPbeta phage in its apo form, bound to its DNA operator and in complex with AimP. AimR presents intrinsic plasticity, sharing structural features with the RRNPP quorum-sensing family. Remarkably, AimR binds to an unusual operator with a long spacer that interacts nonspecifically with the receptor TPR domain, while the HTH domain canonically recognizes two inverted repeats. AimP stabilizes a compact conformation of AimR that approximates the DNA-recognition helices, preventing AimR binding to the aimX promoter region. Our results establish the molecular basis of the arbitrium communication system. ; This work was supported by grant BIO2016-78571-P from the Ministerio de Economia y Competitividad (Spain) to A.M. and grants MR/M003876/1 and MR/S00940X/1 from the Medical Research Council (UK), BB/S003835/1 from the Biotechnology and Biological Sciences Research Council (BBSRC, UK), Wellcome Trust 201531/Z/16/Z, and ERC-ADG-2014 Proposal n 670932 Dut-signal from European Union to J.R.P. X-ray diffraction data collection was supported by Diamond Light Source Block Allocation Group (BAG) proposal MX14739 and MX20229, and Spanish Synchrotron Radiation Facility ALBA proposal 2017072262 and 2018072901. ; Peer reviewed

AbstractThe pressure response of crystalline trans‐cinnamic acid is studied by means of Raman spectroscopy up to 6 GPa. Pressure application causes the reversible shift of all the observed Raman peaks to higher frequencies and changes in their relative intensities, with the intermolecular vibrational modes being by far more sensitive to pressurization compared to the intramolecular ones. The present high‐pressure Raman data indicate the structural stability of the trans‐cinnamic acid crystal and molecular conformation up to the highest pressure attained in the experiments, the importance of the hydrogen bonding, as well as the considerable strengthening of the intermolecular interactions at elevated pressures.

We report on the interplay between the short-range order of molecules in the liquid phase of 1,1,2,2-tetrachloro-1,2-difluoroethane and the possible molecular conformations, trans and gauche. Two complementary approaches have been used to get a comprehensive picture: analysis of neutron-diffraction data by a Bayesian fit algorithm and a molecular dynamics simulation. The results of both show that the population of trans and gauche conformers in the liquid state can only correspond to the gauche conformer being more stable than the trans conformer. Distinct conformer geometries induce distinct molecular short-range orders around them, suggesting that a deep intra- and intermolecular interaction coupling is energetically favoring one of the conformers by reducing the total molecular free energy. © 2011 American Physical Society. ; This work was supported by the Spanish Ministry of Science and Innovation (Grant No. FIS2008-00837) and by the Government of Catalonia (Grant No. 2009SGR-1251). ; Peer Reviewed

29 p.-6 fig.-2 tab.-7 fig. supl.-2 tab. supl.-1 graph. abst. ; Siglec-10 is an inhibitory I-type lectin selectively recognizing sialoglycans exposed on cell surfaces, involved in several patho-physiological processes. The key role Siglec-10 plays in the regulation of immune cell functions has made it a potential target for the development of immunotherapeutics against a broad range of diseases. However, the crystal structure of the protein has not been resolved for the time being and the atomic description of Siglec-10 interactions with complex glycans has not been previously unraveled. We present here the first insights of the molecular mechanisms regulating the interaction between Siglec-10 and naturally occurring sialoglycans. We used combined spectroscopic, computational and biophysical approaches to dissect glycans' epitope mapping and conformation upon binding in order to afford a description of the 3D complexes. Our outcomes provide a structural perspective for the rational design and development of high-affinity ligands to control the receptor functionality. ; This study was supported by the project ''GLYTUNES'' funded by MIUR Progetti di Ricerca di Rilevante Interesse Nazionale (PRIN 2017) (2017XZ2ZBK, 2019–2022) to A.S.; by progetto POR SATIN and Progetto POR CampaniaOncoterapia to A.M.; by the European Commission (H2020-MSCA- 814102-SWEET CROSSTALK project) to A.M., R.M., and A.S. This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program under grant agreement No 851356 to R.M. FSE,PON Ricerca e Innovazione 2014–2020, Azione I.1 ''Dottorati Innovativi con caratterizzazione Industriale'' is acknowledged for funding the PhD grant to R.E.F. Grants by the Spanish Ministry of Science MICINN (CTQ2017-88353-R and fellowship BES 2015–071588 to J.G.-C.) and Wellcome Trust 103744/Z/14/Z to P.R.C. are acknowledged. ; Peer reviewed

Molecular beacons (MBs) are oligonucleotide probes with a hairpin-like structure that are typically labelled at the 5′ and 3′ ends with a fluorophore and a quencher dye, respectively. The conformation of the MB acts as a switch for fluorescence emission. When the fluorophore is in close proximity to the quencher, fluorescence emission cannot be detected, meaning that the switch is in an OFF state. However, if the MB structure is modified, separating the fluorophore from the quencher, the switch turns ON allowing fluorescence emission. This property has been extensively used for a wide variety of applications including real-time PCR reactions, study of protein-DNA interactions, and identification of conformational changes in RNA structures. Here, we describe a protocol based on the MB technology to measure the RNA unfolding capacities of the CspA RNA chaperone from Staphylococcus aureus. This method, with slight variations, may also be applied for testing the activity of other RNA chaperones, RNA helicases, or ribonucleases. ; This work was supported by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (Grant Agreement No. 646869) and the Spanish Ministry of Economy and Competitiveness (BFU2014-56698-P). C.J.C. was supported by a predoctoral contract from the Public University of Navarre (UPNA), Spain.

Molecular beacons (MBs) are oligonucleotide probes with a hairpin-like structure that are typically labelled at the 5′ and 3′ ends with a fluorophore and a quencher dye, respectively. The conformation of the MB acts as a switch for fluorescence emission. When the fluorophore is in close proximity to the quencher, fluorescence emission cannot be detected, meaning that the switch is in an OFF state. However, if the MB structure is modified, separating the fluorophore from the quencher, the switch turns ON allowing fluorescence emission. This property has been extensively used for a wide variety of applications including real-time PCR reactions, study of protein-DNA interactions, and identification of conformational changes in RNA structures. Here, we describe a protocol based on the MB technology to measure the RNA unfolding capacities of the CspA RNA chaperone from Staphylococcus aureus. This method, with slight variations, may also be applied for testing the activity of other RNA chaperones, RNA helicases, or ribonucleases. ; This work was supported by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (Grant Agreement No. 646869) and the Spanish Ministry of Economy and Competitiveness (BFU2014-56698-P). C.J.C. was supported by a predoctoral contract from the Public University of Navarre (UPNA), Spain.