Hinge-shift mechanism as a protein design principle for the evolution of β-lactamases from substrate promiscuity to specificity

W.D.V.H. acknowledges support from National Institutes of Health (Grant: R01GM112077). S.B.O. acknowledges support from the Gordon and Betty Moore Foundations and National Science Foundation (Awards: 1715591 and 1901709). J.M.S.R. acknowledges support from Spanish Ministry of Economy and Competitiveness/FEDER Funds (Grants BIO2015-66426-R and RTI2018-097142-B-100) and the Human Frontier Science Program (Grant RGP0041/2017). V.A.R. acknowledges support from FEDER/Junta de Andalucia-Consejeria de Economia y Conocimiento (Grant E.FQM.113.UGR18). We would like to thank the beamline staff of ID30B of the ESRF (European Synchrotron Radiation Facility, Grenoble, France) for their assistance during data collection and the ESRF for the provision of time through proposals MX-2064.TEM-1 β-lactamase degrades β-lactam antibiotics with a strong preference for penicillins. Sequence reconstruction studies indicate that it evolved from ancestral enzymes that degraded a variety of β-lactam antibiotics with moderate efficiency. This generalist to specialist conversion involved more than 100 mutational changes, but conserved fold and catalytic residues, suggesting a role for dynamics in enzyme evolution. Here, we develop a conformational dynamics computational approach to rationally mold a protein flexibility profile on the basis of a hinge-shift mechanism. By deliberately weighting and altering the conformational dynamics of a putative Precambrian β-lactamase, we engineer enzyme specificity that mimics the modern TEM-1 β-lactamase with only 21 amino acid replacements. Our conformational dynamics design thus re-enacts the evolutionary process and provides a rational allosteric approach for manipulating function while conserving the enzyme active site.United States Department of Health & Human Services National Institutes of Health (NIH) - USA R01GM112077Gordon and Betty Moore FoundationsNational Science Foundation (NSF) 1715591 1901709Spanish Ministry of Economy and Competitiveness/FEDER Funds BIO2015-66426-R RTI2018-097142-B-100Human Frontier Science Program RGP0041/2017FEDER/Junta de Andalucia-Consejeria de Economia y Conocimiento E.FQM.113.UGR1

McCLEC, a robust and stable enzymatic based microreactor platform

A microfluidic chip for cross-linked enzyme crystals (McCLEC) is presented and demonstrated to be a stable, reusable and robust biocatalyst-based device with very promising biotechnological applications. The cost-effective microfluidic platform allows in situ crystallization, cross-linking and enzymatic reaction assays on a single device. A large number of enzymatic reuses of the McCLEC platform were achieved and a comparative analysis is shown illustrating the efficiency of the process and its storage stability for more than one year.This work has been partly funded by the MICINN (Spain) projects BIO2010-16800 (JAG) and BIO2012-34937 (SMR), the European Commission (Contract No. 317916) under the LiPhos project (AL and IRR), “Factoría Española de Cristalización” Consolider-Ingenio 2010 (JAG and MCM) and EDRF Funds (JAG and AL). MCM thanks the Consejo Superior de Investigaciones Científicas (CSIC, Spain) for a JAE predoc fellowship

The emerging role of auxins as bacterial signal molecules: Potential biotechnological applications

This study was supported through grants from the Spanish Ministry for Science and Innovation/Agencia Estatal de Investigación 10.13039/501100011033 (PID2019-103972GA-I00 to M.A.M., PID2020- 112612GB-I00 to T.K. and PID2020-116261GB-I00 to J.A.G.) and the Junta de Andalucía (grant P18- FR-1621 to T.K.). A.R. was supported by the Ramon y Cajal R&D&i Programme (RYC2019- 026481-I) from the Spanish Ministry for Science and Innovation/Agencia Estatal de Investigación 10.13039/501100011033 y FSE ‘El FSE invierte en tu futuro’.Microorganisms are exposed in their natural niches to a wide diversity of sig- nal molecules. Specific detection of these signals results in alterations in mi- crobial metabolism and physiology. Auxins like indole-3-acetic acid are key phytohormones that regulate plant growth and development. Nonetheless, auxin biosynthesis is not restricted to plants but is ubiquitous in all kingdoms of life. This wide phylogenetic distribution of auxins production, together with the diversity of regulated cellular processes, have made auxins key intra- and inter-kingdom signal molecules in life modulating, for example microbial physiology, metabolism and virulence. Despite their increasing importance as global signal molecules, the mechanisms by which auxins perform their regu- latory functions in microorganisms are largely unknown. In this article, we outline recent research that has advanced our knowledge of the mechanisms of bacterial auxin perception. We also highlight the potential applications of this research in aspects such as antibiotic production, biosensor design, plant microbiome engineering and antivirulence therapies.Spanish Ministry for Science and Innovation/Agencia Estatal de Investigacion PID2020-112612GB-I00, PID2020-116261GB-I00, RYC2019- 026481-IJunta de Andalucia P18-FR-1621, PID2019-103972GA-I0

Protection of Catalytic Cofactors by Polypeptides as a Driver for the Emergence of Primordial Enzymes

Enzymes catalyze the chemical reactions of life. For nearly half of known enzymes, catalysis requires the binding of small molecules known as cofactors. Polypeptide-cofactor complexes likely formed at a primordial stage and became starting points for the evolution of many efficient enzymes. Yet, evolution has no foresight so the driver for the primordial complex formation is unknown. Here, we use a resurrected ancestral TIM-barrel protein to identify one potential driver. Heme binding at a flexible region of the ancestral structure yields a peroxidation catalyst with enhanced efficiency when compared to free heme. This enhancement, however, does not arise from proteinmediated promotion of catalysis. Rather, it reflects the protection of bound heme from common degradation processes and a resulting longer lifetime and higher effective concentration for the catalyst. Protection of catalytic cofactors by polypeptides emerges as a general mechanism to enhance catalysis and may have plausibly benefited primordial polypeptide-cofactor associations.Human Frontier Science Program grant RGP0041/2017National Science Foundation grant 2032315Department of Defense grant MURI W911NF-16-1-0372National Institutes of Health grant R01AR069137Spanish Ministry of Science and Innovation/ FEDER Funds grant PID2021-124534OB-100Grant PID2020-116261GB-I0

Novel chimeric proteins mimicking SARS-CoV-2 spike epitopes with broad inhibitory activity.

SARS-CoV-2 spike (S) protein mediates virus attachment to the cells and fusion between viral and cell membranes. Membrane fusion is driven by mutual interaction between the highly conserved heptad-repeat regions 1 and 2 (HR1 and HR2) of the S2 subunit of the spike. For this reason, these S2 regions are interesting therapeutic targets for COVID-19. Although HR1 and HR2 have been described as transiently exposed during the fusion process, no significant antibody responses against these S2 regions have been reported. Here we designed chimeric proteins that imitate highly stable HR1 helical trimers and strongly bind to HR2. The proteins have broad inhibitory activity against WT B.1 and BA.1 viruses. Sera from COVID-19 convalescent donors showed significant levels of reactive antibodies (IgG and IgA) against the HR1 mimetic proteins, whereas these antibody responses were absent in sera from uninfected donors. Moreover, both inhibitory activity and antigenicity of the proteins correlate positively with their structural stability but not with the number of amino acid changes in their HR1 sequences, indicating a conformational and conserved nature of the involved epitopes. Our results reveal previously undetected spike epitopes that may guide the design of new robust COVID-19 vaccines and therapies.This work was supported by grants CV20.26565 from the Consejería de Economía y Conocimiento, Junta de Andalucía (Spain), PID2019.107515RB.C21 from the Spanish State Research Agency (SRA/10.13039/501100011033), and co-funded by ERDF/ESF, “A way to make Europe”/“Investing in your future. The work performed in C.M.’s laboratory was supported by grants from ANRS (Agence Nationale de Recherches sur le SIDA et les h´epatites virales), the Investissements d’Avenir program managed by the ANR under reference ANR-10-LABX-77 and EHVA (No. 681032, Horizon 2020). Work in S.B.’s laboratory was supported by grants from the Agence Nationale de la Recherche (ANR) (ANR-11-LABX-0070_TRANSPLANTEX), the INSERM (UMR_S1109), the Institut Universitaire de France (IUF), all the University of Strasbourg (IDEX UNISTRA), the European Regional Development Fund (European Union) INTERREG V program (project no. 3.2 TRIDIAG) and MSD-Avenir grant AUTOGEN

On the Quality of Protein Crystals Grown under Diffusion Mass-transport Controlled Regime (I)

It has been previously shown that the diffraction quality of protein crystals strongly depends on mass transport during their growth. In fact, several studies support the idea that the higher the contribution of the diffusion during mass transport, the better the diffraction quality of the crystals. In this work, we have compared the crystal quality of two model (thaumatin and insulin) and two target (HBII and HBII-III) proteins grown by two different methods to reduce/eliminate convective mass transport: crystal growth in agarose gels and crystal growth in solution under microgravity. In both cases, we used identical counterdiffusion crystallization setups and the same data collection protocols. Additionally, critical parameters such as reactor geometry, stock batches of proteins and other chemicals, temperature, and duration of the experiments were carefully monitored. The diffraction datasets have been analyzed using a principal component analysis (PCA) to determine possible trends in quality indicators. The relevant indicators show that, for the purpose of structural crystallography, there are no obvious differences between crystals grown under reduced convective flow in space and convection-free conditions in agarose gel, indicating that the key factor contributing to crystal quality is the reduced convection environment and not how this reduced convection is achieved. This means that the possible detrimental effect on crystal quality due to the incorporation of gel fibers into the protein crystals is insignificant compared to the positive impact of an optimal convection-free environment provided by gels. Moreover, our results confirm that the counterdiffusion technique optimizes protein crystal quality and validates both environments in order to deliver high quality protein crystals, although other considerations, such as protein/gel interactions, must be considered when defining the optimal crystallization setup.This study was supported by projects ESP2005-23831-E and ESP2007-29071-E (Spanish Ministry of Education and Science) and BIO2016-74875-P (JAG) (MINECO), Spain co-funded by the Fondo Europeo de Desarrollo Regional, FEDER funds, European Union

Structural insights into choline-O-sulfatase reveal the molecular determinants for ligand binding

This work was supported by the Spanish Ministry of Science and Innovation/FEDER grants PID2020-116261GB-I00 (JAG) and RTI2018-097991-B-I00 (JLN), Secretaria General de Universidades, Investigacion y Tecnologia, Junta de Andalucia (PY20-00149 and UAL18-BIO-B005-B; ACA) and the University of Granada (grant PPJI2017-1; SMR). Funding for open access charge: Universidad de Granada/CBUA.Choline-O-sulfatase (COSe; EC 3.1.6.6) is a member of the alkaline phosphatase (AP) superfamily, and its natural function is to hydrolyze choline-O-sulfate into choline and sulfate. Despite its natural function, the major interest in this enzyme resides in the landmark catalytic/substrate promiscuity of sulfatases, which has led to attention in the biotechnological field due to their potential in protein engineering. In this work, an in-depth structural analysis of wild-type Sinorhizobium (Ensifer) meliloti COSe (SmeCOSe) and its C54S active-site mutant is reported. The binding mode of this AP superfamily member to both products of the reaction (sulfate and choline) and to a substrate-like compound are shown for the first time. The structures further confirm the importance of the C-terminal extension of the enzyme in becoming part of the active site and participating in enzyme activity through dynamic intra-subunit and inter-subunit hydrogen bonds (Asn146A–Asp500B–Asn498B). These residues act as the ‘gatekeeper’ responsible for the open/closed conformations of the enzyme, in addition to assisting in ligand binding through the rearrangement of Leu499 (with a movement of approximately 5 A ° ). Trp129 and His145 clamp the quaternary ammonium moiety of choline and also connect the catalytic cleft to the C-terminus of an adjacent protomer. The structural information reported here contrasts with the proposed role of conformational dynamics in promoting the enzymatic catalytic proficiency of an enzyme.Spanish Government European Commission PID2020-116261GB-I00 RTI2018-097991-B-I00Secretaria General de UniversidadesJunta de Andalucia PY20-00149 UAL18-BIO-B005-BUniversity of Granada PPJI2017-

A New L-Proline Amide Hydrolase with Potential Application within the Amidase Process

This research was supported by the Spanish Ministry of Science and Innovation/FEDER funds grant PID2020-116261GB-I00/AEI/10.13039/501100011033 (JAG), from the FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades grants P18-FR-3533 (LAC) and P12-FQM-790 (RCM), and from the University of Granada grant PPJI2017-1 (SMR).L-proline amide hydrolase (PAH, EC 3.5.1.101) is a barely described enzyme belonging to the peptidase S33 family, and is highly similar to prolyl aminopeptidases (PAP, EC. 3.4.11.5). Besides being an S-stereoselective character towards piperidine-based carboxamides, this enzyme also hydrolyses different L-amino acid amides, turning it into a potential biocatalyst within the Amidase Process. In this work, we report the characterization of L-proline amide hydrolase from Pseudomonas syringae (PsyPAH) together with the first X-ray structure for this class of L-amino acid amidases. Recombinant PsyPAH showed optimal conditions at pH 7.0 and 35 degrees C, with an apparent thermal melting temperature of 46 degrees C. The enzyme behaved as a monomer at the optimal pH. The L-enantioselective hydrolytic activity towards different canonical and non-canonical amino-acid amides was confirmed. Structural analysis suggests key residues in the enzymatic activity.Spanish GovernmentEuropean Commission PID2020-116261GB-I00/AEI/10.13039/501100011033FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades P18-FR-3533 P12-FQM-790University of Granada PPJI2017-1 CTS-20

Magnetite Mineralization inside Cross-Linked Protein Crystals

Crystallization in confined spaces is a widespread process in nature that also has important implications for the stability and durability of many man-made materials. It has been reported that confinement can alter essential crystallization events, such as nucleation and growth and, thus, have an impact on crystal size, polymorphism, morphology, and stability. Therefore, the study of nucleation in confined spaces can help us understand similar events that occur in nature, such as biomineralization, design new methods to control crystallization, and expand our knowledge in the field of crystallography. Although the fundamental interest is clear, basic models at the laboratory scale are scarce mainly due to the difficulty in obtaining well-defined confined spaces allowing a simultaneous study of the mineralization process outside and inside the cavities. Herein, we have studied magnetite precipitation in the channels of cross-linked protein crystals (CLPCs) with different channel pore sizes, as a model of crystallization in confined spaces. Our results show that nucleation of an Fe-rich phase occurs inside the protein channels in all cases, but, by a combination of chemical and physical effects, the channel diameter of CLPCs exerted a precise control on the size and stability of those Fe-rich nanoparticles. The small diameters of protein channels restrain the growth of metastable intermediates to around 2 nm and stabilize them over time. At larger pore diameters, recrystallization of the Fe-rich precursors into more stable phases was observed. This study highlights the impact that crystallization in confined spaces can have on the physicochemical properties of the resulting crystals and shows that CLPCs can be interesting substrates to study this process.Ministry of Science and Innovation, Spain (MICINN) PID2020-116261GB-I00 PID2020-118498GB-I00 PDC2021-121135.100FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades (Spain) A-FQM-340-UGR20 UCE-PP2016-0

Lysozyme crystallization in hydrogel media under ultrasound irradiation

Sonocrystallization implies the application of ultrasound radiation to control the nucleation and crystal growth depending on the actuation time and intensity. Its application allows to induce nucleation at lower supersaturations than required under standard conditions. Although extended in inorganic and organic crystallization, it has been scarcely explored in protein crystallization. Now, that industrial protein crystallization is gaining momentum, the interest on new ways to control protein nucleation and crystal growth is advancing. In this work we present the development of a novel ultrasound bioreactor to study its influence on protein crystallization in agarose gel. Gel media minimize convention currents and sedimentation, favoring a more homogeneous and stable conditions to study the effect of an externally generated low energy ultrasonic irradiation on protein crystallization avoiding other undesired effects such as temperature increase, introduction of surfaces which induce nucleation, destructive cavitation phenomena, etc. In-depth statistical analysis of the results has shown that the impact of ultrasound in gel media on crystal size populations are statistically significant and reproducible.MCIN/AEI PID2020-118498GB-I00 PID2020-116261GB-I00 PID2020-115372RB-I00 MCIN/AEI/FEDER "Una manera de hacer Europa", Spain PID2019-106947RA-C22FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades (Spain) A-FQM-340-UGR20 P18-FR-3533 P18-RT-1653 BTEP-026-UGR1