Acetylcholine is a central biological signal molecule present in all kingdoms of life. In humans, acetylcholine is the primary neurotransmitter of the peripheral nervous system; it mediates signal transmission at neuromuscular junctions. Here, we show that the opportunistic human pathogen Pseudomonas aeruginosa exhibits chemoattraction toward acetylcholine over a concentration range of 1 mM to 100 mM. The maximal magnitude of the response was superior to that of many other P. aeruginosa chemoeffectors. We demonstrate that this chemoattraction is mediated by the PctD (PA4633) chemoreceptor. Using microcalorimetry, we show that the PctD ligand-binding domain (LBD) binds acetylcholine with a equilibrium dissociation constant (KD) of 23 mM. It also binds choline and with lower affinity betaine. Highly sensitive responses to acetylcholine and choline, and less sensitive responses to betaine and L-carnitine, were observed in Escherichia coli expressing a chimeric receptor comprising the PctD-LBD fused to the Tar chemoreceptor signaling domain. We also identified the PacA (ECA_RS10935) chemoreceptor of the phytopathogen Pectobacterium atrosepticum, which binds choline and betaine but fails to recognize acetylcholine. To identify the molecular determinants for acetylcholine recognition, we report high-resolution structures of PctD-LBD (with bound acetylcholine and choline) and PacA-LBD (with bound betaine). We identified an amino acid motif in PctD-LBD that interacts with the acetylcholine tail. This motif is absent in PacA-LBD. Significant acetylcholine chemotaxis was also detected in the plant pathogens Agrobacterium tumefaciens and Dickeya solani. To the best of our knowledge, this is the first report of acetylcholine chemotaxis and extends the range of host signals perceived by bacterial chemoreceptors., This work was supported by grants PID2019-103972GA-I00 (to M.A.M.), PID2020-116261GB-I00 (to J.A.G.) and PID2020-112612GB-I00 (to T.K.) from the Spanish Ministry for Science and Innovation/Agencia Estatal de Investigación 10.13039/501100011033, grant P18-FR-1621 (to T.K.) from the Junta de Andalucía, by the Hessian Ministry of Higher Education, Research, and the Arts (HMWK)–LOEWE research cluster “Diffusible Signals,” subproject A1 (to V.S.), and a Peterson Group “Serving Hometown” Elites scholarship to W.X.
We thank Maria Rabyk for help with the hybrid receptor cloning. We are grateful to the European Synchrotron Radiation Facility (ESRF) for the provision of time through proposals MX2281 and MX2353 and to the staff at beamlines ID30B, ID23-1, and ID30A-3 and at the Xaloc beamline of the Alba Spanish synchrotron radiation source (Barcelona) for assistance during data collection.

Resumen del poster presentado en: XIII Reunión del Grupo de Microbiología Molecular de La SEM. Granada, 7-9 septiembre (2022), This work was supported by grants PID2019-103972GA-I00 (to M.A.M.), PID2020-116261GBI00 (to J.A.G.) and PID2020-112612GB-I00 (to T.K.) from the Spanish Ministry for Science and
Innovation/Agencia Estatal de Investigación 10.13039/501100011033, grant P18-FR-1621 (to T.K.) from the Junta de Andalucía.

14 pags., 8 figs., 1 tab., 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 (Asn146-Asp500-Asn498). 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 Å). 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., This work was supported by the Spanish Ministry of Science and Innovation/FEDER grants PID2020-116261GB-I00
(JAG) and RTI2018-097991-B-I00 (JLN), Secretarıa General de Universidades, Investigacion y Tecnologıa, Junta de
Andalucıa (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.

Bacteria have evolved a sophisticated array of signal transduction systems that allow them to adapt their physiology and metabolism to changing environmental conditions. Typically, these systems recognize signals through dedicated ligand binding domains (LBDs) to ultimately trigger a diversity of physiological responses. Nonetheless, an increasing number of reports reveal that signal transduction receptors also bind antagonists to inhibit responses mediated by agonists. The mechanisms by which antagonists block the downstream signaling cascade remain largely unknown. To advance our knowledge in this field, we used the LysR-type transcriptional regulator AdmX as a model. AdmX activates the expression of an antibiotic biosynthetic cluster in the rhizobacterium Serratia plymuthica. AdmX specifically recognizes the auxin phytohormone indole-3-acetic acid (IAA) and its biosynthetic intermediate indole-3-pyruvic acid (IPA) as signals. However, only IAA, but not IPA, was shown to regulate antibiotic production in S. plymuthica. Here, we report the high-resolution structures of the LBD of AdmX in complex with IAA and IPA. We found that IAA and IPA compete for binding to AdmX. Although IAA and IPA binding does not alter the oligomeric state of AdmX, IPA binding causes a higher degree of compactness in the protein structure. Molecular dynamics simulations revealed significant differences in the binding modes of IAA and IPA by AdmX, and the inspection of the three-dimensional structures evidenced differential agonist- and antagonist-mediated structural changes. Key residues for auxin binding were identified and an auxin recognition motif defined. Phylogenetic clustering supports the recent evolutionary emergence of this motif specifically in plant-associated enterobacteria., This study was supported through grants from the CSIC to M.A.M. (PIE-202040I003), from the Spanish Ministry for Science and Innovation/Agencia Estatal de Investigación 10.13039/501100011033 (PID2019-103972GA-I00 to M.A.M., grant PID2020-112612GB-I00 to T.K. and PID2020-116261GB-I00 to J.A.G.), the Junta de Andalucía (grant P18-FR-1621 to T.K.), by NIH Grant 1R35GM131760 (to I.B.Z.), and by the Academic Leadership Program Priority 2030 proposed by Federal State Autonomous Educational Institution of Higher Education I. M. Sechenov First Moscow State Medical University of the Ministry of Health of the Russian Federation (Sechenov University) to Y.B.P. A.O. acknowledges support by the Ministry of Science and Innovation (MCIN), the State Research Agency (AEI/10.13039/501100011033), the European Regional Development Fund (RTI2018–094393-BC21-MCIU/AEI/FEDER, UE), and the Seneca Foundation CARM, 20786/PI/18. We are grateful to the European Synchrotron Radiation Facility (ESRF) for the provision of time and to the staff at beamlines ID23-1, ID23-2, and ID30A-3 and at the Xaloc beamline of the ALBA Spanish synchrotron radiation source (Barcelona) for assistance during data collection.

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 C, with an apparent thermal melting temperature of 46 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., 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 Andalucía-Consejería de Transformación Económica, Industria, Conocimiento y Universidades grants P18-FR-3533 (LAC) and P12-FQM-790 (RCM), and from the University of Granada grant PPJI2017-1 (SMR).

About half of the known bacterial species perform chemotaxis that gains them access to sites that are optimal for growth and survival. The motility apparatus and chemotaxis signaling pathway impose a large energetic and metabolic burden on the cell. There is almost no limit to the type of chemoeffectors that are recognized by bacterial chemoreceptors. For example, they include hormones, neurotransmitters, quorum-sensing molecules, and inorganic ions. However, the vast majority of chemoeffectors appear to be of metabolic value. We review here the experimental evidence indicating that accessing nutrients is the main selective force that led to the evolution of chemotaxis., This work was supported by the Spanish Ministry for Science and Innovation/Agencia Estatal de Investigación 10.13039/501100011033 (grants PID2020-112612GB-I00 to TK, PID2019-
103972GA-I00 to MAM, and PID2020-116261GB-I00 to JAG), Consejo Superior de Investigaciones Científicas (CSIC) (grant 2023AEP002 to MAM), and the Junta de Andalucía (grant P18-FR-1621 to TK)

Microorganisms are exposed in their natural niches to a wide diversity of signal molecules. Specific detection of these signals results in alterations in microbial 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 regulatory 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., Junta de Andalucía, Grant/Award Number: P18-FR-1621; Spanish Ministry for Science and Innovation/Agencia Estatal de Investigación, Grant/Award Number: PID2019-103972GA-I00, PID2020-112612GB-I00, PID2020-116261GB-I00 and RYC2019-026481-I

Acylphosphatase (AcP, EC 3.6.1.7) is a small model protein conformed by a ferredoxin-like fold, profoundly studied to get insights into protein folding and aggregation processes. Numerous studies focused on the aggregation and/or amyloidogenic properties of AcPs suggest the importance of edge-β-strands in the process. In this work, we present the first crystallographic structure of Escherichia coli AcP (EcoAcP), showing notable differences with the only available NMR structure for this enzyme. EcoAcP is crystalised as an intertwined dimer formed by replacing a single C-terminal β-strand between two protomers, suggesting a flexible character of the C-terminal edge of EcoAcP. Despite numerous works where AcP from different sources have been used as a model system for protein aggregation, our domain-swapped EcoAcP structure is the first 3-D structural evidence of native-like aggregated species for any AcP reported to date, providing clues on molecular determinants unleashing aggregation., This work was supported by the Spanish Ministry of Science and Innovation/FEDER funds Grant PID2020-116261GB-I00/AEI/10.13039/501100011033 (JAG). SMR is grateful to the Andalusian Regional Government through the Endocrinology and Metabolism Group (CTS-202) and to University of Granada “Plan Propio de Investigación de la UGR” (PP2022.PP.18). ACA is grateful to Junta de Andalucía (PY20_00149). We are grateful to the European Synchrotron Radiation Facility (ESRF), Grenoble, France, for providing time through proposal MX2353 and MX2454, and the staff at ID30-A3 beamlines for assistance during data collection. We are also grateful to the Spanish Synchrotron Light Facility (ALBA), Barcelona, Spain, for providing time through proposals 2021085252 and 2022086950, and the staff at XALOC beamline for assistance during data collection. We deeply thank Ma Carmen López-Sánchez for technical assistance., Peer reviewed

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., This study was supported by projects PID2020-116261GB-I00 (J.A.G.), PID2020-118498GB-I00 (L.A.d.C.) and PDC2021-121135.100 (C.J.L.) of the Ministerio de Ciencia e Innovación, MCIN/AEI/10.13039/501100011033 and by projects P18-FR-3533 and A-FQM-340-UGR20 of the FEDER/Junta de Andalucía-Consejería de Transformación Económica, Industria, Conocimiento y Universidades (Spain)., Peer reviewed

Modified surfaces like siliconized glass are commonly used to support protein crystallization and facilitate obtaining crystals. Over the years, various surfaces have been proposed to decrease the energetic penalty required for consistent protein clustering, but scarce attention has been paid to the underlying mechanisms of interactions. Here, we propose self-assembled monolayers that are surfaces exposing fine-tuned moieties with a very regular topography and subnanometer roughness, as a tool to unveil the interaction between proteins and functionalized surfaces. We studied the crystallization of three model proteins having progressively narrower metastable zones, i.e., lysozyme, catalase, and proteinase K, on monolayers exposing thiol, methacrylate, and glycidyloxy groups. Thanks to comparable surface wettability, the induction or the inhibition of nucleation was readily attributed to the surface chemistry. For example, thiol groups strongly induced the nucleation of lysozyme thanks to electrostatic pairing, whereas methacrylate and glycidyloxy groups had an effect comparable to unfunctionalized glass. Overall, the action of surfaces led to differences in nucleation kinetics, crystal habit, and even crystal form. This approach can support the fundamental understanding of the interaction between protein macromolecules and specific chemical groups, which is crucial for many technological applications in the pharmaceutical and food industry., This work was supported by the Spanish Ministry of Science and Innovation/FEDER funds AEI/10.13039/501100011033 grant number PID2020-116261GB-I00 (J.A.G.).

The PDZ domains are modular domains that recognise short linear C-terminal sequences in proteins that organise the formation of complex multi-component assemblies. We have crystallised the third PDZ domain of the neuronal postsynaptic density-95 protein (PSD95-PDZ3) at mildly acidic pH conditions and obtained up to four polymorphs. Thus, below pH 4.0, the protein crystallised into prism-shaped crystals that belonged to the trigonal space group P312. In contrast, above this pH value, the crystals’ shape changes to long needles belonging to the monoclinic P2 and two different orthorhombic packings of the P222 space group. In addition, all the polymorphs share the main crystallographic interface, where the sidechain of the Asp332 imitates the binding of the C-terminal moiety to the canonical binding motif. Furthermore, we have analysed how changes in the ionisation state of some specific residues might be critical for crystallising the different polymorphs. The analysis of these polymorphs provides clues on the relevance of specific protein-protein interactions in protein crystallisation. However, these structures allow dissecting those electrostatic interactions that play a role in the conformation adopted by some residues and the extra-domain components upon binding C-terminal sequences., This research was funded by the Junta de Andalucía and FEDER, grant numbers UAL18-BIO-B005-B and PY20_00149 (A.C.A.). J.A.G. is supported by project PID2020-116261GB-I00 from the Spanish Ministry for Science and Innovation (AEI 10.13039/501100011033).

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 protein-mediated 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., This work was supported by Human Frontier Science Program grant RGP0041/2017 (J.M.S.R. and E.A.G.), National Science Foundation grant 2032315 (E.A.G.), Department of Defense grant MURI W911NF-16-1-0372 (E.A.G.), National Institutes of Health grant R01AR069137 (E.A.G.), Spanish Ministry of Science and Innovation/ FEDER Funds grant PID2021-124534OB-100 (J.M.S.R.), and grant PID2020-116261GB-I00 (J.A.G.)., Peer reviewed

XXX simposio del Grupo Especializado de Cristalografía y Crecimiento Cristalino - GE3C, Supported the Spanish Ministry for Science and Innovation/Agencia Estatal de Investigación 10.13039/501100011033, grant PID2020-116261GB-I.

Resumen de la comunicación oral presentada en: XXIX Congreso de la Sociedad Española de Microbiología SEM. Microorganismos: un universo en continua evolución. Burgos, España. 25-28 junio (2023), Polyethylene terephthalate (PET) is a versatile, and low-cost polyester with low reactivity properties that has become a key element in modern society. It is used in a wide variety of applications, such as bottles, packaging, and textile fibers. However, the lack of an effective recycling system has led to an alarming accumulation of PET and other plastics in landfills and oceans, posing a significant threat to the environment and human health (1). The main goal of this work is to develop an optimal
biocatalyst for the efficient biodegradation of PET into its monomers (terephthalic acid (TPA) and ethylene glycol (EG)). The development of such a biocatalyst could offer a viable and environmentally friendly alternative to existing recycling processes that are either less specific or more contaminant.
Our approach involved the combination of two engineering strategies. First, we used Ancestral Sequence Reconstruction (ARS) to infer the protein sequence of a hypothetical ancient hydrolase, using the modern sequence of PET hydrolase from Ideonella sakaiensis (IsPETase) as a reference (2). This strategy allowed us to generate a new set of functional enzymes that retain the spatial structure of IsPETase, yet exhibit significant amino acid sequence changes. The resulting enzymes showed
increased robustness and improved stability properties (3). After a preliminary characterization, the putative ancestral enzyme with most promising features was selected as a parental template in a directed evolution campaign to improve its PET-degrading activity. This strategy required the development of a reliable High-Through Put Screening (HTPS) protocol to detect significant improvements among mutants and select the most suitable candidate for industrial applications.
The combination of both strategies allowed us to identify regions that have been highly conserved across evolution, as well as regions where novel mutations could potentially enhance PET-hydrolyzing activity., 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., grant
PID2020-112612GBI00 to T.K. and PID2020-116261GB-I00 to J.A.G.), the Junta de Andalucía (grant P18-FR-1621 to T.K.) and by NIH Grant 1R35GM131760 (to I.B.Z.)

β-xylosidases (4-β-d-xylan xylohydrolase, E.C. 3.2.1.37) are glycoside hydrolases (GH) catalyzing the hydrolysis of (1→4)-β-d-xylans, allowing for the removal of β-d-xylose residues from its non-reducing termini. Together with other xylan-degrading enzymes, β-xylosidases are involved in the enzymatic hydrolysis of lignocellulosic biomass, making them highly valuable in the biotechnological field. Whereas different GH families are deeply characterized from a structural point of view, the GH52 family has been barely described. In this work, we report the 2.25 Å resolution structure of Geobacillus stearothermophilus CECT43 XynB2, providing the second structural characterization for this GH family. A plausible dynamic loop closing the entrance of the catalytic cleft is proposed based on the comparison of the available GH52 structures, suggesting the relevance of a dimeric structure for members of this family. The glycone specificity at the −1 site for GH52 and GH116 members is also explained by our structural studies., This research was funded by the Spanish Ministry of Science and Innovation/FEDER funds Grant PID2020-116261GB-I00/AEI/10.13039/501100011033 (JAG), by the European Regional Development Fund Andalucía 2014–2020 Grant UAL18-CTS-B032-A (FRV) and by the Own Research and Transfer Plan 2020 of the University of Almeria Grant PPUENTE2020/006 (FJLHV)., Peer reviewed

Peptide assembly is attractive not only to develop biotechnological tools and smart nanomaterials, but also to treat pathologies in new ways. This Review focuses on recent progress made in the exciting area pertaining the interaction between peptide assemblies and proteins. Earlier works aimed to identify proteins able to bind peptide assemblies for therapeutics’ delivery and biocompatible scaffolds. Recent advancements cover more applications that go beyond tissue regeneration and biomaterials. On one hand, self-assembling peptides interacting with proteins can inhibit pathological amyloid fibrillation, or boost the immune response to vaccines. On the other, they have been exploited to promote protein crystallization, also for therapeutics’ delivery. As research advances in this exciting area, it opens the way towards a qualitative leap in the clinical translation of supramolecular medicinal chemistry to creatively tackle unmet challenges of modern disease treatment and prevention., The authors acknowledge Francesca Prochilo and Elisa Prelli at the University of Trieste for administrative assistance. This study was supported by FEDER/Junta de Andalucía-Consejería de Transformación Económica, Industria, Conocimiento y Universidades (Spain) projects P18-FR-3533 and A-FQM-340-UGR20. J.A.G. is supported by the Spanish Ministry of Science and Innovation/FEDER funds grant PID2020-116261GB-I00/AEI/10.13039/501100011033. M.C.M.T. acknowledges grant PRE2018-083773 funded by MCIN/AEI/10.13039/501100011033 and FSE “El FSE invierte en tu futuro”, Spain. Open Access Funding provided by Università degli Studi di Trieste within the CRUI-CARE Agreement. Open Access funding provided by Università degli Studi di Trieste within the CRUI-CARE Agreement.

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., This study was supported by project PID2020-118498GB-I00, PID2020-116261GB-I00 and PID2020-115372RB-I00 funded by MCIN/AEI/ 10.13039/501100011033/ and PID2019-106947RA-C22 funded by MCIN/AEI/ 10.13039/501100011033/FEDER “Una manera de hacer Europa”, Spain, and by FEDER/Junta de Andalucía-Consejería de Transformación Económica, Industria, Conocimiento y Universidades (Spain) projects A-FQM-340-UGR20, P18-FR-3533, P18-RT-1653, B-TEP-026-UGR18.

Accurate characterization of protein–protein interactions is essential for understanding and controlling protein crystallization and phase transitions in a wide range of multidisciplinary fields, from fundamental and materials research to various pharma and industrial applications. This study introduces an innovative use of Fluorescence Lifetime Imaging Microscopy (FLIM) with the non-conjugated molecular rotor Sulforhodamine-B (SRB) for the characterization and comprehension of protein interactions, offering a new, efficient method for screening protein phase transition conditions. As models of study, non-specific intermolecular interactions in Lysozyme and Bovine Serum Albumin (BSA) solutions were investigated under various precipitant agents (salts, polymers) and pH conditions. FLIM analyses reveal a strong correlation between SRB fluorescence lifetime and the transition from repulsive to attractive protein interaction state, with a pronounced increase of the lifetime around the solubility point, enabling robust qualitative solubility predictions. Complementary SAXS analysis confirms that SRB acts uniquely as a probe for protein interactions without influencing the system state. Further time-resolved FLIM analysis on SRB lifetime populations indicates that the sensitivity of the fluorescence lifetime to protein interactions is driven by variation in probe-protein interaction affinity upon screening of the protein surface charges. These findings and the proposed methodology are of high interest particularly for structural biology and applications reliant on protein crystallization, including the development of novel biomaterials., This work was supported by the projects ANR 2021 TITANS, France, and LP-to-CRYST ETI 2024 of Toulouse INP University, France. YK has received financial support through a PhD grant from French Ministry of higher education, France. Data collection were performed at the XALOC beamline of the Spanish Synchrotron radiation source, ALBA (Barcelona), Spain with the help of the ALBA staff. JAG is supported by the Spanish Ministry of Science and Innovation/FEDER funds Grant PID2020-116261GB-I00, AEI/10.13039/501100011033, Spain.

Lysozyme (Lyz) and cross-linked Lyz crystals (CLLC) are among the proteins and templates used in the bioinspired synthesis of subnanometer-sized gold nanoclusters (AuNCs) utilized in various biological applications as biosensors, bioimaging agents, and recyclable catalyst. While previous studies have explored the formation and stabilization of AuNCs in the presence of Lyz, little is known about the enzymatic activity of Lyz after AuNCs stabilization in both its free form (AuNC-Lyz) and within CLLC (AuNC-CLLC). In this study, we conducted a comprehensive analysis of AuNC-Lyz, including hydrodynamic characteristics, using multiwavelength fluorescence analytical ultracentrifugation and enzymatic activity assays. Results show that AuNC-Lyz retains bactericidal performance at pH 4.0 and exhibits enhanced hydrolytic activity compared with Lyz alone. Additionally, AuNC-Lyz showed peroxidase-like performance due to the catalytically active surface of AuNCs providing the bifunctional catalytic capabilities of AuNC-Lyz. In contrast, fluorescent-based hydrolytic activity assays of AuNC-CLLC revealed that morphological variations in CLLC affect the catalytic performance. The AuNC-CLLC enzyme activity depends on the size of the solvent channels and Lyz conformation within CLLC. As a consequence of the occluded solvent channels and limited breathing (in/out diffusion) of CLLC, AuNC-CLLC displayed a diminishing hydrolytic performance., We acknowledge the Konstanz Research School of Chemical Biology (KoRS-CB) and Deutscher Akademischer Austauschdienst (DAAD) for financial support. O.A. thanks Dr. Paavo Bergmann from the Bioimaging Center, University of Konstanz for helping with the dehydration and embedding of the AuNCs incorporated protein crystals. J.W. acknowledges funding by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)\u2500Project-ID 416229255\u2500SFB 1411. J.A.G. was supported by the Spanish Ministry of Science and Innovation/FEDER funds Grant PID2020- 116261GB-I00/AEI/10.13039/501100011033., Peer reviewed

Purines and their derivatives control intracellular energy homeostasis and nucleotide synthesis, and act as signaling molecules. Here, we combine structural and sequence information to define a purine-binding motif that is present in sensor domains of thousands of bacterial receptors that modulate motility, gene expression, metabolism, and second-messenger turnover. Microcalorimetric titrations of selected sensor domains validate their ability to specifically bind purine derivatives, and evolutionary analyses indicate that purine sensors share a common ancestor with amino-acid receptors. Furthermore, we provide experimental evidence of physiological relevance of purine sensing in a second-messenger signaling system that modulates c-di-GMP levels., This work was supported by the Spanish Ministry for Science, Innovation and Universities/Agencia Estatal de Investigación https://doi.org/10.13039/501100011033 (grants PID2020-112612GB-I00 to T.K., PID2019-103972GA-I00 to M.A.M. and PID2020-116261GB-I00 to J.A.G.), the Junta de Andalucía (grant P18-FR-1621 to T.K.), CSIC (grant 2023AEP002 to M.A.M.) and the NIH (grant 1R35GM131760 to I.B.Z.). We thank Mª Carmen López-Sánchez and Raquel Vázquez Santiago for technical assistance. We are grateful to the Spanish Synchrotron Light Facility (Barcelona, Spain) and the European Synchrotron Radiation Facility (Grenoble, France) for the provision of time (proposals MX1830 and Mx1938) and thank the beamline staff for their assistance with data collection.

A crystallogenesis study of human transthyretin using the counter-diffusion method is described as an alternative to conventional convective vapour diffusion and batch approaches for protein crystallization. The X-ray diffraction results show systematic trends that exhibit unique patterns of crystallization and high crystal quality as well as a remarkable degree of coherence within extended crystal rods that wholly fill the capillaries used. Preliminary neutron diffraction data have been recorded from a number of these samples, validating the feasibility of this methodology for neutron crystallography., C. De'Ath was funded during the period of this work by the award of trainee placements from both the ILL and the ESRF as well as co-funding from the Erasmus+ programme of the European Union. M. F. Oliva was funded by a grant from the Human Frontier Science Programme (HFSP) to H. Chapman, V. T. Forsyth, R. P. Millane and N. Seeman (grant reference RGP0010/2017). J. A. Gavira is supported by the Spanish Ministry of Science and Innovation/FEDER funds (grant PID2020-116261GB-I00). V. T. Forsyth acknowledges support from the Swedish Research Council (Vetenskapsrådet) under grant 2022-01104. V. T. Forsyth also acknowledges support from the LMK Foundation (reference 147418). This work used the platforms of the Grenoble Instruct-ERIC center (ISBG; UAR 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-0005-02) and GRAL, financed within the Université Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003)., Peer reviewed

Transfer of chirality, or transmission of asymmetric information from one system to another, plays an essential role in fundamental biological and chemical processes and, therefore, is essential for life. This phenomenon also holds immense potential in spintronics in the context of chirality-induced spin selectivity (CISS). In the CISS, the spatial arrangement of chiral molecules influences the spin state of electrons during the charge-transfer processes. Transfer of chirality from chiral molecules to an achiral material in a hybrid environment enables induction of spin polarization in the achiral material, thus vastly expanding the library of CISS-active electronic materials. Such “induced” CISS signals could have different responses compared to pure chiral molecules because the electronic properties of the achiral material come into play in the former case. In addition, multiple chiral sources can be used, which can have a nontrivial contribution to the induced CISS effect and can act either synergistically or antagonistically. This opens the way to achieving tunability of the CISS signals via chemical means. Earlier, such a chirality-transfer phenomenon and the resulting induced CISS effect were demonstrated in a hybrid system containing carbon nanotubes (CNTs) functionalized with a chiral agent (Fmoc-diphenylalanine l/d). In this context, we extend this result by investigating the role of an additional chiral moiety (l-lysozyme enzyme crystals) in this system. Here, the chiral crystal surrounds the chiral-functionalized CNTs, and we show that synergistic interactions result in more efficient chirality transfer, resulting in nontrivial changes in the CISS effect. This manifests in the form of (a) a stronger CISS signal compared to only one single chiral agent, (b) nonmonotonic temperature dependence and sign reversal of the CISS signal, and (c) persistence of the CISS signal at higher temperatures. Hybrid chiral materials with multiple chiral sources could, therefore, offer intricate control of the CISS signal via modification of its constituents, which is not possible in homogeneous chiral systems with single chiral sources., This study was supported by grants PID2023-150318NB-I00 and PID2020-116261GB-I00 funded by MCIN/AEI/10.13 039/50110001103, Spain, and the Project P18-FR-3533 funded by FEDER/Junta de Andaluci\u0301a-Consejeri\u0301a de Transformacio\u0301n Econo\u0301mica, Industria, Conocimiento y Universidades (Spain). S.P. acknowledges support from Natural Resources Canada (project RES0064735), New Frontiers in Research Fund-Exploration (project NFRFE-2019-01298), and Natural Sciences and Engineering Research Council (NSERC) Canada (project RGPIN-2018-05127). Funding for open access charge: Universidad de Granada / CBUA.

Supramolecular gels are unique materials formed through the self-assembly of molecular building blocks, typically low molecular weight gelators (LMWGs), driven by non-covalent interactions. The process of crystallization within supramolecular gels has broadened the scope of the traditional gel-phase crystallization technique offering the possibility of obtaining crystals of higher quality and size. The broad structural diversity of LMWGs allows crystallization in multiple organic and aqueous solvents, favouring screening and optimization processes and the possibility to search for novel polymorphic forms. These supramolecular gels have been used for the crystallization of inorganic, small organic compounds of pharmaceutical interest, and proteins. Results have shown that these gels are not only able to produce crystals of high quality but also to influence polymorphism and physicochemical properties of the crystals, giving rise to crystals with potential new bio- and technological applications. Thus, understanding the principles of crystallization in supramolecular gels is essential for tailoring their properties and applications, ranging from drug delivery systems to composite crystals with tunable stability properties. In this review, we summarize the use of LMWG-based supramolecular gels as media to grow single crystals of a broad range of compounds., We are grateful for funding to the European Commission (HORIZON-MSCA-PF-2022, project 101110492), to the Ministerio de Ciencia, Innovación y Universidades (Spain) MCIN/AEI/10.13039/501100011033 (PID2020-118498GB-I00 and PID2020-116261GB-I00), to the FEDER/Junta de Andalucia-Consejeria de Transformación Económica, Industria, Conocimiento y Universidades (Spain) (P18-FR-3533), and to the Engineering and Physical Research Council (EP/S035877/1). Funding for open access charge: Universidad de Granada/CBUA.

Bacterial receptors feed into multiple signal transduction pathways that regulate a variety of cellular processes including gene expression, second messenger levels, and motility. Receptors are typically activated by signal binding to ligand-binding domains (LBDs). Cache domains are omnipresent LBDs found in bacteria, archaea, and eukaryotes, including humans. They form the predominant family of extracytosolic bacterial LBDs and were identified in all major receptor types. Cache domains are composed of either a single (sCache) or a double (dCache) structural module. The functional relevance of bimodular LBDs remains poorly understood. Here, we identify the PacF chemoreceptor in the phytopathogen Pectobacterium atrosepticum that recognizes formate at the membrane-distal module of its dCache domain, triggering chemoattraction. We further demonstrate that a family of formate-specific sCache domains has evolved from a dCache domain, exemplified by PacF, by losing the membrane-proximal module. By solving high-resolution structures of two family members in complex with formate, we show that the molecular basis for formate binding at sCache and dCache domains is highly similar, despite their low sequence identity. The apparent loss of the membrane-proximal module may be related to the observation that dCache domains bind ligands typically at the membrane-distal module, whereas studies have failed to find ligands bound in the membrane-proximal module. This work advances our understanding of signal sensing in bacterial receptors and suggests that evolution by reducing complexity may be a route for shaping diversity., This study was supported by Grants from the Spanish Ministry for Science and Innovation/Agencia Estatal de Investigación 10.13039/501100011033 (Grants PID2020-112612 GB-I00 and PID2023-146216NB-I00 to T.K., PID2019-103972GA-I00 and PID2023-146281NB-I00 to M.A.M., and PID2020-116261 GB-I00 to J.A.G.), the Consejo Superior de Investigaciones Científicas (Grant 2024AEP062 to T.K.), the Junta de Andalucía (Grant P18-FR-1621 to T.K.), and the NIH (Grant R35GM131760 to I.B.Z.). We thank Raquel Vázquez Santiago for technical support., Peer reviewed

Silica gels act as nucleation inhibitors and have been used to grow large protein crystals in convection-free environments. However, a large amount of protein is required to overcome the inhibition effect, and chances of successful crystallization are limited, hampering its potential benefits. In the present study, we propose the substitution of silanol groups with methylated additives to increase the hydrophobicity of the gel network,
decrease the interaction between proteins and gel fibers, and tune the inhibition effect of silica gels. We observed an increased hen egg white lysozyme (HEWL) nucleation density in gels bearing a higher number of methyl groups. We used the counter-diffusion crystallization technique for our proof of concept since it does not require a fine adjustment of the supersaturation. We then moved to batch crystallization for maintaining constant supersaturation conditions in order to have comparative results. We were able to grow HEWL crystals with tailored sizes depending on the amount of hydrophobic moieties’ substitution. The modification of the gel reduced the amount of protein required to induce nucleation. This effect was attributed to the decreased adsorption of protein
macromolecules on gel fibers carrying hydrophobic groups. This simple chemical modification approach may expand the use of silica gels, traditionally seen as protein nucleation inhibitors, to produce new crystalline composite materials., This work was supported by the Spanish Ministry of Science, Innovation and Agencia Estatal de Investigación 10.13039/501,100,011,033 grant PID2020-116261GB-I00 (to J.A.G)., Peer reviewed

N-Acyl-D-amino acid deacylases (EC 3.5.1.81, also known as D-acylases) have been studied for decades for their utility in the kinetic resolution of N-acetyl-D,L-amino acids (NAAs) due to a marked stereospecificity. In conjunction with an N-succinyl-amino acid racemase (NSAR), they impulse the dynamic kinetic resolution (DKR) of different NAAs until the corresponding enantiomerically pure D-amino acids. Besides the clear interest in this enzyme cascade, the application of D-acylase/NSAR tandems has been only briefly described outside the industrial field. In this work, we revisit D-acylases for the DKR of NAAs, reporting the characterisation of two new recombinant D-acylases belonging to Bordetella petrii and Klebsiella pneumoniae. The enzymes were successfully coupled with the recombinant NSAR from Geobacillus stearothermophilus for the biosynthesis of D-methionine or D-aminobutyric acid. We also carried out the structural characterisation of the D-acylase from Klebsiella pneumoniae (KleDacyl), providing the second experimental 3-D structure of a member of this family of enzymes. The structural model shows a highly dynamic character of this amidohydrolase superfamily member, supplying a snapshot of an open conformation of the enzyme most likely preceding substrate entrance into the catalytic cleft. Our results confirm for the first time the importance of an α/β mobile domain in the substrate specificity of D-acylases (region 282–341 in KleDacyl), opening up new strategies for structural-based protein engineering strategies., unding: This work was supported by Ministerio de Ciencia e Innovación, Grant PID2020-116261GB-I00/AEI/10.13039/5011000110, PID2020-116261GB-I00. Consejería de Universidad, Investigación e Innovación and ERDF Andalusia Program 2021-2027, C-EXP-120-UGR23. ERDF Andalusia Program 2021-2027. We are grateful to the Spanish Synchrotron Light Facility (ALBA), Barcelona, Spain, for providing time through proposals 2021085252, 2022086950 and 2023087670, and the staff at XALOC beamline for assistance during data collection. We are also grateful to the European Synchrotron Radiation Facility (ESRF), Grenoble, France, for providing time through proposal MX2353 and MX2454, and the staff at ID30-A3 beamline for assistance during data collection. We deeply thank M\u00AA Carmen López-Sánchez for technical assistance. This research was funded by the Spanish Ministry of Science and Innovation/FEDER funds Grant PID2020-116261GB-I00/AEI/10.13039/501100011033 (JAG). S.M.R. is thankful for Grant C-EXP-120-UGR23 by Consejería de Universidad, Investigación e Innovación and ERDF Andalusia Program 2021-2027., Peer reviewed

This research was funded by the Spanish Ministry of Science and Innovation/FEDER funds Grant PID2020-116261GB-I00/AEI/10.13039/501100011033 (JAG), by the European Regional Development Fund Andalucía 2014–2020 Grant UAL18-CTS-B032-A (FRV) and by the Own Research and Transfer Plan 2020 of the University of Almeria Grant PPUENTE2020/006 (FJLHV).
European Synchrotron Radiation Facility (ESRF), Grenoble, France, proposal MX2353ESRF, Peer reviewed

Description of methods used for collection/generation of data: Single Crystal X-ray diffraction (MX) data collection at synchrotron source i.e. ESRF beam line .
Methods for processing the data: Diffraction data from the latter radiation source were used for structuredetermination. Data were indexed and integrated with XDS, and scaled and reduced with AIMLESS of the CCP4 program suite. Phases were determined by molecular replacement using Molrep and rather refined with Refmac and phenix.refine of the PHENIX suite., This work was supported by the Spanish Ministry of Science and Innovation/FEDER funds Grant PID2020-116261GB-I00/AEI/10.13039/501100011033 (JAG). SMR is grateful to the Andalusian Regional Government through the Endocrinology and Metabolism Group (CTS-202) and to University of Granada “Plan Propio de Investigación de la UGR” (PP2022.PP.18). ACA is grateful to Junta de Andalucía (PY20_00149).
European Synchrotron Radiation Facility (ESRF), Grenoble, France, proposal MX2353 and MX2454 and Spanish Synchrotron Light Facility (ALBA), Barcelona, Spain, proposal 022086950, Peer reviewed

Description of methods used for collection/generation of data: Single Crystal X-ray diffraction (MX) data collection at synchrotron source i.e. ESRF beam line .
Methods for processing the data: Diffraction data from the latter radiation source were used for structuredetermination. Data were indexed and integrated with XDS, and scaled and reduced with AIMLESS of the CCP4 program suite. Phases were determined by molecular replacement using Molrep and rather refined with Refmac and phenix.refine of the PHENIX suite., This work was supported by the Spanish Ministry for Science, Innovation and Universities/Agencia Estatal de Investigación https://doi.org/10. 13039/501100011033 (grants PID2020-112612GB-I00 to T.K., PID2019- 103972GA-I00 to M.A.M. and PID2020-116261GB-I00 to J.A.G.), the Junta de Andalucía (grant P18-FR-1621 to T.K.), CSIC (grant 2023AEP002 to M.A.M.) and the NIH (grant 1R35GM131760 to I.B.Z.).
Spanish synchrotron ALBA at beamline XALOC., Peer reviewed

Interactions within the plant holobiont involve a wide diversity of plant- and microbial-derived signals. Bacterial perception of these signals allows directed movement along chemical gradients through chemotaxis, a process that is an important determinant for plant colonization and infection. The quaternary amine acetylcholine is primarily known for its function as a neurotransmitter in mammalian nervous systems, but is emerging as a signal molecule that regulates plant growth, development, and stress resistance. Here, we report that plant pathogenic bacteria of the genera Agrobacterium and Dickeya exhibit strong chemotaxis to acetylcholine. These responses are mediated in Agrobacterium fabrum, Dickeya solani, and Dickeya dadantii by the dCache_1-type ligand binding domain (LBD) containing chemoreceptors AtuA, MkcA, and DdaA, respectively, which recognize acetylcholine with affinities between 19 and 91 µM. These chemoreceptors also recognize additional quaternary amines such as choline, L-carnitine, and betaine. The high-resolution structure of MkcA-LBD of D. solani was solved in complex with choline, which allowed the identification of the molecular determinants of choline and acetylcholine recognition in this chemoreceptor. Functionally, DdaA was found to contribute to competitive colonization fitness in planta. Acetylcholine serves as a nutritional source for A. fabrum, but not for D. solani and D. dadantii, and provides osmoprotective effects in all three phytopathogenic species. Plant-associated bacteria exhibit chemotaxis toward a diversity of key plant signals, and their ability to sense acetylcholine may represent an adaptive strategy to ensure efficient plant infection and manage osmotic stress. Our findings suggest potential co-evolutionary interactions between plants and their associated microbiomes., This work was supported by the Spanish Ministry of Science, Innovation and Universities/Agencia Estatal de Investigación 10.13039/501100011033 and FEDER-EU (grants PID2019\u2013103972GA-I00 and PID2023-146281NB-I00 to M.A.M., PID2020\u2013116261GB-I00 to J.A.G, and PID2021\u2013125673OB-I00 to E.L.S) and through a grant from the CSIC (grant 2023AEP002 to M.A.M.)., Peer reviewed

The ability of bacteria to adapt to changing environmental conditions largely depends on transmembrane receptors that sense signal molecules and generate responses such as chemotaxis, changes in gene expression, or alterations in second-messenger levels. Although these receptors differ significantly in function, they share a common mode of activation that involves signal molecule interaction with sensor domains. A major challenge in microbiology lies in the limited knowledge of ligands that stimulate receptors. Here, we review recent advances in this field, including the occurrence of multi-modular sensor domains, the identification of co-component signal transduction systems, evidence for sensor domain evolution from transporters, and the use of binding pocket sequence motifs to identify sensor domain ligands., This work was supported by the Spanish Ministry of Science and Innovation/FEDER funds Grant PID2020-116261GB-I00/AEI/10.13039/501100011033 (JAG). SMR is grateful to the Andalusian Regional Government through the Endocrinology and Metabolism Group (CTS-202) and to University of Granada “Plan Propio de Investigación de la UGR” (PP2022.PP.18). ACA is grateful to Junta de Andalucía (PY20_00149). We are grateful to the European Synchrotron Radiation Facility (ESRF), Grenoble, France, for providing time through proposal MX2353 and MX2454, and the staff at ID30-A3 beamlines for assistance during data collection. We are also grateful to the Spanish Synchrotron Light Facility (ALBA), Barcelona, Spain, for providing time through proposals 2021085252 and 2022086950, and the staff at XALOC beamline for assistance during data collection. We deeply thank Ma Carmen López-Sánchez for technical assistance.

A microfluidic enzymatic sensor based on cross-linked crystals of an ancestral β-lactamase (βLa-CLECs) was developed for the detection of β-lactam antibiotics under continuous flow conditions. The sensor is presented in a modular configuration, consisting of a separated microprobe for enzymatic catalysis, allowing for precise enzyme loading, and a photonic lab-on-a-chip (PhLoC) platform for detection by in-line spectrophotometric measurements. The exceptional thermal stability of the reconstructed Precambrian β-lactamase made it suitable for the steps required in device fabrication and carrier-free immobilization treatment, while its inherent promiscuity, i.e., its capability to degrade a variety of lactam antibiotics, including man-made third-generation antibiotics, broadens its potential application. Full catalytic activity of the ancestral enzyme was retained after immobilization, and inhibition by antibiotics such as ampicillin and sulbactam was detected at concentrations as low as parts per billion. These results support the use of ancient enzymes as stable and responsive biorecognition elements in cost-effective, high-throughput analytical systems targeting environmental pollutants and pharmaceutical compounds., This work was partly funded by the Spanish Ministry of Science, Innovation and Agencia Estatal de Investigación http://dx.doi.org/10.13039/501100011033 grant PID2020-116261GB-I00 (JAG), the Institut National Politechnique de Toulouse ETI 2025 program (project InCySe) (IRR), and Junta de Andalucia-Consejeria de Universidad, Investigación e Innovación (PAIDI 2020) P21.00112 (VAR). PMG acknowledges the support of the CSIC through the JAE program (2025)., Peer reviewed

This study was supported by Grants from the Spanish Ministry for Science and Innovation/Agencia Estatal de Investigación 10.13039/ 501100011033 (Grants PID2020- 112612 GB- I00 and PID2023- 146216NB- I00 to T.K., PID2019- 103972GA- I00 and PID2023- 146281NB- I00 to M.A.M., and PID2020- 116261 GB- I00 to J.A.G.), the Consejo Superior de Investigaciones Científicas (Grant 2024AEP062 to T.K.), the Junta de Andalucía (Grant P18- FR- 1621 to T.K.), and the NIH (Grant R35GM131760 to I.B.Z.).
Data collection at beamline XALOC of the Spanish synchrotron radiation source ALBA was carried out under project 2022086950., Peer reviewed

This study was supported by Grants from the Spanish Ministry for Science and Innovation/Agencia Estatal de Investigación 10.13039/ 501100011033 (Grants PID2020- 112612 GB- I00 and PID2023- 146216NB- I00 to T.K., PID2019- 103972GA- I00 and PID2023- 146281NB- I00 to M.A.M., and PID2020- 116261 GB- I00 to J.A.G.), the Consejo Superior de Investigaciones Científicas (Grant 2024AEP062 to T.K.), the Junta de Andalucía (Grant P18- FR- 1621 to T.K.), and the NIH (Grant R35GM131760 to I.B.Z.).
Data collection at beamline XALOC of the Spanish synchrotron radiation source ALBA was carried out under project 2022086950., Peer reviewed

This research was funded by the Spanish Ministry of Science and Innovation/FEDER funds Grant PID2020- 116261GB- I00/AEI/10.13039/501100011033 (JAG). S.M.R. is thankful for Grant C- EXP- 120- UGR23 by Consejería de Universidad, Investigación e Innovación and ERDF Andalusia Program 2021- 2027.
Data collection at beamline XALOC of the Spanish synchrotron radiation source ALBA was carried out under project 2023087670., Peer reviewed

This research was funded by the Spanish Ministry of Science and Innovation/FEDER funds Grant PID2020- 116261GB- I00/AEI/10.13039/501100011033 (JAG). S.M.R. is thankful for Grant C- EXP- 120- UGR23 by Consejería de Universidad, Investigación e Innovación and ERDF Andalusia Program 2021- 2027.
Data collection at beamline XALOC of the Spanish synchrotron radiation source ALBA was carried out under project 2023087670., Peer reviewed

This work was supported by the Spanish Ministry of Science, Innovation and Universities/Agencia Estatal de Investigación 10.13039/ 501100011033 and FEDER-EU (grants PID2019–103972GA-I00 and PID2023-146281NB-I00 to M.A.M., PID2020–116261GB-I00 to J.A.G, and PID2021–125673OB-I00 to E.L.S) and through a grant from the CSIC (grant 2023AEP002 to M.A.M.).
Data collection at beamline XALOC of the Spanish synchrotron radiation source ALBA was carried out under project 202308767., Peer reviewed

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 (Asn146(A)-Asp500(B)-Asn498(B)). 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 angstrom). 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.

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.