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The uneven worldwide vaccination coverage against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and emergence of variants escaping immunity call for broadly effective and easily deployable therapeutic agents. We have previously described the human single-chain scFv76 antibody, which recognizes SARS-CoV-2 Alpha, Beta, Gamma and Delta variants. We now show that scFv76 also neutralizes the infectivity and fusogenic activity of the Omicron BA.1 and BA.2 variants. Cryoelectron microscopy (cryo-EM) analysis reveals that scFv76 binds to a well-conserved SARS-CoV-2 spike epitope, providing the structural basis for its broad-spectrum activity. We demonstrate that nebulized scFv76 has therapeutic efficacy in a severe hACE2 transgenic mouse model of coronavirus disease 2019 (COVID-19) pneumonia, as shown by body weight and pulmonary viral load data. Counteraction of infection correlates with inhibition of lung inflammation, as observed by histopathology and expression of inflammatory cytokines and chemokines. Biomarkers of pulmonary endothelial damage were also significantly reduced in scFv76-treated mice. The results support use of nebulized scFv76 for COVID-19 induced by any SARS-CoV-2 variants that have emerged so far.

Immunoglobulin light chain amyloidosis (AL) is caused by the aberrant production of amyloidogenic light chains (LC) that accumulate as amyloid deposits in vital organs. Distinct LC sequences in each patient yield distinct amyloid structures. However different tissue microenvironments may also cause identical protein precursors to adopt distinct amyloid structures. To address the impact of the tissue environment on structural polymorphism of amyloids, we extracted fibrils from the kidney of an AL patient (AL55) whose cardiac amyloid structure was previously determined by our group. Here we show that the 4.0 Å resolution cryo-EM structure of the renal fibril is virtually identical to that reported for the cardiac fibril. These results provide the first structural evidence that LC amyloids independently deposited in different organs of the same AL patient share a common fold.

Amyloid fibrils are polymeric structures originating from aggregation of misfolded proteins. In vivo, proteolysis may modulate amyloidogenesis and fibril stability. In light chain (AL) amyloidosis, fragmented light chains (LCs) are abundant components of amyloid deposits; however, site and timing of proteolysis are debated. Identification of the N and C termini of LC fragments is instrumental to understanding involved processes and enzymes. We investigated the N and C terminome of the LC proteoforms in fibrils extracted from the hearts of two AL cardiomyopathy patients, using a proteomic approach based on derivatization of N- and C-terminal residues, followed by mapping of fragmentation sites on the structures of native and fibrillar relevant LCs. We provide the first high-specificity map of proteolytic cleavages in natural AL amyloid. Proteolysis occurs both on the LC variable and constant domains, generating a complex fragmentation pattern. The structural analysis indicates extensive remodeling by multiple proteases, largely taking place on poorly folded regions of the fibril surfaces. This study adds novel important knowledge on amyloid LC processing: although our data do not exclude that proteolysis of native LC dimers may destabilize their structure and favor fibril formation, the data show that LC deposition largely precedes the proteolytic events documentable in mature AL fibrils.

Aims Increased shedding of extracellular vesicles (EVs)—small, lipid bilayer-delimited particles with a role in paracrine signalling—has been associated with human pathologies, e.g. atherosclerosis, but whether this is true for cardiac diseases is unknown. Methods and results Here, we used the surface antigen CD172a as a specific marker of cardiomyocyte (CM)-derived EVs; the CM origin of CD172a+ EVs was supported by their content of cardiac-specific proteins and heart-enriched microRNAs. We found that patients with aortic stenosis, ischaemic heart disease, or cardiomyopathy had higher circulating CD172a+ cardiac EV counts than did healthy subjects. Cellular stress was a major determinant of EV release from CMs, with hypoxia increasing shedding in in vitro and in vivo experiments. At the functional level, EVs isolated from the supernatant of CMs derived from human-induced pluripotent stem cells and cultured in a hypoxic atmosphere elicited a positive inotropic response in unstressed CMs, an effect we found to be dependent on an increase in the number of EVs expressing ceramide on their surface. Of potential clinical relevance, aortic stenosis patients with the highest counts of circulating cardiac CD172a+ EVs had a more favourable prognosis for transcatheter aortic valve replacement than those with lower counts. Conclusion We identified circulating CD172a+ EVs as cardiac derived, showing their release and function and providing evidence for their prognostic potential in aortic stenosis patients.

NF-Y is a transcription factor (TF) comprising three subunits (NF-YA, NF-YB, NF-YC) that binds with high specificity to the CCAAT sequence, a widespread regulatory element in gene promoters of prosurvival, cell-cycle-promoting, and metabolic genes. Tumor cells undergo &ldquo, metabolic rewiring&rdquo, through overexpression of genes involved in such pathways, many of which are under NF-Y control. In addition, NF-YA appears to be overexpressed in many tumor types. Thus, limiting NF-Y activity may represent a desirable anti-cancer strategy, which is an ongoing field of research. With virtual-screening docking simulations on a library of pharmacologically active compounds, we identified suramin as a potential NF-Y inhibitor. We focused on suramin given its high water-solubility that is an important factor for in vitro testing, since NF-Y is sensitive to DMSO. By electrophoretic mobility shift assays (EMSA), isothermal titration calorimetry (ITC), STD NMR, X-ray crystallography, and molecular dynamics (MD) simulations, we showed that suramin binds to the histone fold domains (HFDs) of NF-Y, preventing DNA-binding. Our analyses, provide atomic-level detail on the interaction between suramin and NF-Y and reveal a region of the protein, nearby the suramin-binding site and poorly conserved in other HFD-containing TFs, that may represent a promising starting point for rational design of more specific and potent inhibitors with potential therapeutic applications.

d-Amino acids are the "wrong" enantiomers of amino acids as they are not used in proteins synthesis but evolved in selected functions. On this side, d-aspartate (d-Asp) plays several significant roles in mammals, especially as an agonist of N-methyl-d-aspartate receptors (NMDAR), and is involved in relevant diseases, such as schizophrenia and Alzheimer's disease. In vivo modulation of d-Asp levels represents an intriguing task to cope with such pathological states. As little is known about d-Asp synthesis, the only option for modulating the levels is via degradation, which is due to the flavoenzyme d-aspartate oxidase (DASPO). Here we present the first three-dimensional structure of a DASPO enzyme (from human) which belongs to the d-amino acid oxidase family. Notably, human DASPO differs from human d-amino acid oxidase (attributed to d-serine degradation, the main coagonist of NMDAR) showing peculiar structural features (a specific active site charge distribution), oligomeric state and kinetic mechanism, and a higher FAD affinity and activity. These results provide useful insights into the structure-function relationships of human DASPO: modulating its activity represents now a feasible novel therapeutic target.

NF-Y transcription factor comprises three subunits: NF-YA, NF-YB and NF-YC. NF-YB and NF-YC dimerize through their histone fold domain (HFD), which can bind DNA in a non-sequence-specific fashion while serving as a scaffold for NF-YA trimerization. Upon trimerization, NF-YA specifically recognizes the CCAAT box sequence on promoters and enhancers. In plants, each NF-Y subunit is encoded by several genes giving rise to hundreds of potential heterotrimeric combinations. In addition, plant NF-YBs and NF-YCs interact with other protein partners to recognize a plethora of genomic motifs, as the CCT protein family that binds CORE sites. The NF-Y subunit organization and its DNA-binding properties, together with the NF-Y HFD capacity to adapt different protein modules, represent plant-specific features that play a key role in development, growth and reproduction. Despite their relevance, these features are still poorly understood at the molecular level. Here, we present the structures of Arabidopsis and rice NF-YB/NF-YC dimers, and of an Arabidopsis NF-Y trimer in complex with the FT CCAAT box, together with biochemical data on NF-Y mutants. The dimeric structures identify the key residues for NF-Y HFD stabilization. The NF-Y/DNA structure and the mutation experiments shed light on HFD trimerization interface properties and the NF-YA sequence appetite for the bases flanking the CCAAT motif. These data explain the logic of plant NF-Y gene expansion: the trimerization adaptability and the flexible DNA-binding rules serve the scopes of accommodating the large number of NF-YAs, CCTs and possibly other NF-Y HFD binding partners and a diverse audience of genomic motifs.

The unintentional crystallization of contaminant proteins in the place of target recombinant proteins is sporadically reported, despite the availability of stringent expression/purification protocols and of software for the detection of contaminants. Typically, the contaminant protein originates from the expression organism (for example Escherichia coli), but in rare circumstances contaminants from different sources have been reported. Here, a case of contamination from a Serratia bacterial strain that occurred while attempting to crystallize an unrelated protein from Burkholderia pseudomallei (overexpressed in E. coli) is presented. The contamination led to the unintended crystallization and structure analysis of a cyanate hydratase from a bacterial strain of the Serratia genus, an opportunistic enterobacterium that grows under conditions similar to those of E. coli and that is found in a variety of habitats, including the laboratory environment. In this context, the procedures that were adopted to identify the contaminant based on crystallographic data only are presented and the crystal structure of Serrata spp. cyanate hydratase is briefly discussed.

Meeting of Plant Molecular Biology 2020, online the 26 – 27 November 2020, The Food and Agriculture Organization of the United Nations warned that population growth will drive food needs up by 40% by 2050. One of the major threats to achieve the objective is the climate change. To overcome this problem, it is required the understanding of the mechanisms for plant adaptation to stresses such as drought, the concomitant soil salinity, floods or frosts. The primary consequence of these stresses is the unbalance of K+ ion homeostasis (Pardo; 2010; Cherel & Gaillard; 2019). To cope to this situation, plants regulate the activity of plant-specific voltage-gated K+ channels. The high-resolution structure of these channels will unravel the mechanisms underlying their regulation; so that it will be possible to identify hot-spots in the structure suitable for mutagenesis that would lead to channel variants with altered activity. We have determined the structure of the complex between the ankyrin domain of the cytosolic side of the inward-rectifier K channel AKT1 and the activating kinase CIPK23 and showed that this interaction regulates channel activation. Point mutations on this domain specifically alter binding to CIPK23, enhancing or impairing the ability of CIPK23 to regulate channel activity (Sanchez-Barrena et al, 2020). These modified versions will be effectively used to engineer plants with improved performance under abiotic stresses., This work was funded by Agencia Estatal de Investigación (AEI,Spain) and Fondo Europeo de Desarrollo Regional (FEDER, European Union) (BIO2017- 89523-R to A.A., BIO2015-70946-R to F.J.Q.,BIO2016-81957-REDT to J.M.P., and RTI2018- 094027-B-I00 to J.M.P.)

Summary The HCN1–4 channel family is responsible for the hyperpolarization-activated cation current If/Ih that controls automaticity in cardiac and neuronal pacemaker cells. We present cryoelectron microscopy (cryo-EM) structures of HCN4 in the presence or absence of bound cAMP, displaying the pore domain in closed and open conformations. Analysis of cAMP-bound and -unbound structures sheds light on how ligand-induced transitions in the channel cytosolic portion mediate the effect of cAMP on channel gating and highlights the regulatory role of a Mg2+ coordination site formed between the C-linker and the S4-S5 linker. Comparison of open/closed pore states shows that the cytosolic gate opens through concerted movements of the S5 and S6 transmembrane helices. Furthermore, in combination with molecular dynamics analyses, the open pore structures provide insights into the mechanisms of K+/Na+ permeation. Our results contribute mechanistic understanding on HCN channel gating, cyclic nucleotide-dependent modulation, and ion permeation., Graphical abstract, Highlights • HCN4 structure is shown in ligand-free and ligand-bound state • Pore domain is shown in closed and in open configuration • Permeability and selectivity mechanisms of HCN channels are uncovered • A metal ion coordination site functionally couples cytoplasmic and transmembrane domains, HCN4 channels underlie the pacemaker current that controls heart rate. Saponaro et al. report the structure of HCN4 with the pore in closed and in open configuration and provide information on ion permeability and selectivity. In HCN4, a metal ion coordination site functionally connects the C-linker to the S4-S5 linker.

The recently determined crystal structures of the sequence-specific transcription factor NF-Y have illuminated the structural mechanism underlying transcription at the CCAAT box. NF-Y is a trimeric protein complex composed by the NF-YA, NF-YB, and NF-YC subunits. NF-YB and NF-YC contain a histone-like domain and assemble on a head-to-tail fashion to form a dimer, which provides the structural scaffold for the DNA sugar-phosphate backbone binding (mimicking the nucleosome H2A/H2B-DNA assembly) and for the interaction with NF-YA. The NF-YA subunit hosts two structurally extended α-helices; one is involved in NF-YB/NF-YC binding and the other inserts deeply into the DNA minor groove, providing exquisite sequence-specificity for recognition and binding of the CCAAT box. The analysis of these structural data is expected to serve as a powerful guide for future experiments aimed at understanding the role of post-translational modification at NF-Y regulation sites and to unravel the three-dimensional architecture of higher order complexes formed between NF-Y and other transcription factors that act synergistically for transcription activation. Moreover, these structures represent an excellent starting point to challenge the formation of a stable hybrid nucleosome between NF-Y and core histone proteins, and to rationalize the fine molecular details associated with the wide combinatorial association of plant NF-Y subunits. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani.

Plants acquire potassium (K+) ions for cell growth and movement via regulated diffusion through K(+)channels. Here, we present crystallographic and functional data showing that the K(+)inward rectifier KAT1 (K(+)Arabidopsis thaliana 1) channel is regulated by 14-3-3 proteins and further modulated by the phytotoxin fusicoccin, in analogy to the H+-ATPase. We identified a 14-3-3 mode III binding site at the very C terminus of KAT1 and cocrystallized it with tobacco [Nicotiana tabacum) 14-3-3 proteins to describe the protein complex at atomic detail. Validation of this interaction by electrophysiology shows that 14-3-3 binding augments KAT1 conductance by increasing the maximal current and by positively shifting the voltage dependency of gating. Fusicoccin potentiates the 14-3-3 effect on KAT1 activity by stabilizing their interaction. Crystal structure of the ternary complex reveals a noncanonical binding site for the toxin that adopts a novel conformation. The structural insights underscore the adaptability of fusicoccin, predicting more potential targets than so far anticipated. The data further advocate a common mechanism of regulation of the proton pump and a potassium channel, two essential elements in K(+)uptake in plant cells.

International audience; Molecular CellArticleCas9 Allosteric Inhibitionby the Anti-CRISPR Protein AcrIIA6Olivier Fuchsbauer,1,2,9Paolo Swuec,3,4,9Claire Zimberger,1,2Be ́atrice Amigues,1,2Se ́bastien Levesque,5Daniel Agudelo,5Alexis Duringer,5Antonio Chaves-Sanjuan,4Silvia Spinelli,1,2Genevie`ve M. Rousseau,6,7Minja Velimirovic,5Martino Bolognesi,3,4Alain Roussel,1,2Christian Cambillau,1,2Sylvain Moineau,6,7,8Yannick Doyon,5and Adeline Goulet1,2,10,*1Architecture et Fonction des Macromole ́cules Biologiques, Centre National de la Recherche Scientifique (CNRS), Campus de Luminy, Case932, 13288 Marseille Cedex 09, France2Architecture et Fonction des Macromole ́cules Biologiques, Aix-Marseille Universite ́, Campus de Luminy, Case 932, 13288 Marseille Cedex09, France3Dipartimento di Bioscienze, Universita`degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy4Centro di Ricerca Pediatrica Romeo ed Enrica Invernizzi, Universita`degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy5Centre Hospitalier Universitaire de Que ́bec–Universite ́Laval Research Center, Que ́bec City, QC G1V 4G2, Canada6De ́partement de biochimie, de microbiologie, et de bio-informatique, Faculte ́des sciences et de ge ́nie, Universite ́Laval, Que ́bec City, QC,G1V 0A6, Canada7Groupe de recherche en e ́cologie buccale, Faculte ́de me ́decine dentaire, Universite ́Laval, Que ́bec City, QC, G1V 0A6, Canada8Fe ́lix d’He ́relle Reference Center for Bacterial Viruses, Faculte ́de me ́decine dentaire, Universite ́Laval, Que ́bec City, QC, G1V 0A6, Canada9These authors contributed equally10Lead Contact*Correspondence:adeline.goulet@afmb.univ-mrs.frhttps://doi.org/10.1016/j.molcel.2019.09.012SUMMARYIn the arms race against bacteria, bacteriophageshave evolved diverse anti-CRISPR proteins (Acrs)that block CRISPR-Cas immunity. Acrs play key rolesin the molecular coevolution of bacteria with theirpredators, use a variety of mechanisms of action,and provide tools to regulate Cas-based genomemanipulation. Here, we present structural and func-tional analyses of AcrIIA6, an Acr from virulentphages, exploring its unique anti-CRISPR action.Our cryo-EM structures and functional data ofAcrIIA6 binding toStreptococcus thermophilusCas9 (St1Cas9) show that AcrIIA6 acts as an allo-steric inhibitor and induces St1Cas9 dimerization.AcrIIA6 reduces St1Cas9 binding affinity for DNAand prevents DNA binding within cells. The PAMand AcrIIA6 recognition sites are structurally closeand allosterically linked. Mechanistically, AcrIIA6 af-fects the St1Cas9 conformational dynamics associ-ated with PAM binding. Finally, we identify a naturalSt1Cas9 variant resistant to AcrIIA6 illustratingAcr-driven mutational escape and molecular diversi-fication of Cas9 proteins

Plant growth largely depends on the maintenance of adequate intracellular levels of potassium (K(+)). The families of 10 Calcineurin B-Like (CBL) calcium sensors and 26 CBL-Interacting Protein Kinases (CIPKs) of Arabidopsis (Arabidopsis thaliana) decode the calcium signals elicited by environmental inputs to regulate different ion channels and transporters involved in the control of K(+) fluxes by phosphorylation-dependent and -independent events. However, the detailed molecular mechanisms governing target specificity require investigation. Here, we show that the physical interaction between CIPK23 and the noncanonical ankyrin domain in the cytosolic side of the inward-rectifier K(+) channel AKT1 regulates kinase docking and channel activation. Point mutations on this domain specifically alter binding to CIPK23, enhancing or impairing the ability of CIPK23 to regulate channel activity. Our data demonstrate the relevance of this protein–protein interaction that contributes to the formation of a complex between CIPK23/CBL1 and AKT1 in the membrane for the proper regulation of K(+) transport.

IWPMB, Glasgow, UK, July 7-12, 2019, Plant growth depends on the maintenance of the adequate intracellular levels of potassium ion (K+). Different abiotic stresses threaten this homeostasis, thus, the characterization of the molecules involved in the regulation of cytoplasmic K+ concentration is important as they could be targeted for plant crop improvement. The families of 10 Calcineurin B like (CBL) calcium sensors and 26 CBL-Interacting Protein Kinases (CIPK) decode the calcium signals elicited by stress and regulate different ion channels and transporters involved in the control of K+ fluxes in plants. Nevertheless, the detailed molecular mechanisms balancing specificity and versatility to the system require investigation. Here, we show that the cytosolic domain of t he inwardrectifier K+ channel (AKT1) harbors a non-canonical ankyrin domain which is not essential for channel function but serves as a docking site for its regulator, the CIPK23 protein kinase. Mutations on this domain impair kinase channel association. This interaction is not strong enough to ensure signal specificity per se but should contribute to the formation of a multivalent complex between CIPK23/CBL1 and AKT1 in the membrane vicinity for the proper regulation of K+ transport.

Protein-protein interactions (PPIs) are known to play an essential role between the neuronal calcium sensor 1 (NCS-1) and the guanine exchange factor Ric8a to regulate synapse function, emerging as a druggable interface for synaptopathies such as the fragile X syndrome (FXS). Recently, the phenothiazine FD44 has been identified as an inhibitor of this PPI, decreasing the abnormally high synapse number and enhancing associative learning in a FXS animal model. Here, we have integrated advanced experimental and computational studies to obtain important structural insights into Drosophila NCS-1/FD44 recognition to understand the basis of its affinity and specificity and generate improved PPI regulators. This has allowed the identification of a new small drug-like molecule, IGS-1.76, which efficiently inhibits the human NCS-1/Ric8a complex with improved binding potency. The crystal structure of the Drosophila NCS-1/IGS-1.76 complex demonstrates that the new inhibitor, although chemically different from FD44, shares the same mechanism of action and constitutes a new hit candidate for FXS.

The protein complex formed by the Ca2+ sensor neuronal calcium sensor 1 (NCS-1) and the guanine exchange factor protein Ric8a coregulates synapse number and probability of neurotransmitter release, emerging as a potential therapeutic target for diseases affecting synapses, such as fragile X syndrome (FXS), the most common heritable autism disorder. Using crystallographic data and the virtual screening of a chemical library, we identified a set of heterocyclic small molecules as potential inhibitors of the NCS-1/Ric8a interaction. The aminophenothiazine FD44 interferes with NCS-1/Ric8a binding, and it restores normal synapse number and associative learning in a Drosophila FXS model. The synaptic effects elicited by FD44 feeding are consistent with the genetic manipulation of NCS-1. The crystal structure of NCS-1 bound to FD44 and the structure- function studies performed with structurally close analogs explain the FD44 specificity and the mechanism of inhibition, in which the small molecule stabilizes a mobile C-Terminal helix inside a hydrophobic crevice of NCS-1 to impede Ric8a interaction. Our study shows the drugability of the NCS-1/Ric8a interface and uncovers a suitable region in NCS-1 for development of additional drugs of potential use on FXS and related synaptic disorders.

The NF-Y transcription factor is a heterotrimer formed by evolutionarily conserved subunits: NF-YA, NF-YB, and NF-YC. NF-YB and NF-YC harbor a histone fold domain (HFD), structurally similar to that of nucleosome core histones, and form a tight dimer (Romier et al., 2003). NF-YA binds to the NF-YB/NF-YC dimer and provides exquisite sequence specificity for recognizing and binding the CCAAT box (Huber et al., 2012; Nardini et al., 2013), an important DNA regulatory element of all eukaryotes (Dolfini et al., 2009).

2 pags. -- 31st European Crystallographic Meeting, Oviedo, Spain 22-27 August

10 páginas.-- 6 figuras.-- 77 referencias.-- This article contains supporting information online at http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1407610111/-/DCSupplemental, Data deposition: The atomic coordinates have been deposited in the Protein Data Bank (PDB), www.pdb.org (PDB ID codes 4CZT, 4CZU, and 4D28)., © 2014, National Academy of Sciences. All rights reserved. Plant cells have developed specific protective molecular machinery against environmental stresses. The family of CBL-interacting protein kinases (CIPK) and their interacting activators, the calcium sensors calcineurin B-like (CBLs), work together to decode calcium signals elicited by stress situations. The molecular basis of biological activation of CIPKs relies on the calcium-dependent interaction of a self-inhibitory NAF motif with a particular CBL, the phosphorylation of the activation loop by upstream kinases, and the subsequent phosphorylation of the CBL by the CIPK. We present the crystal structures of the NAF-truncated and pseudophosphorylated kinase domains of CIPK23 and CIPK24/SOS2. In addition, we provide biochemical data showing that although CIPK23 is intrinsically inactive and requires an external stimulation, CIPK24/SOS2 displays basal activity. This data correlates well with the observed conformation of the respective activation loops: Although the loop of CIPK23 is folded into a well-ordered structure that blocks the active site access to substrates, the loop of CIPK24/SOS2 protrudes out of the active site and allows catalysis. These structures together with biochemical and biophysical data show that CIPK kinase activity necessarily requires the coordinated releases of the activation loop from the active site and of the NAF motif from the nucleotide-binding site. Taken all together, we postulate the basis for a conserved calcium-dependent NAF-mediated regulation of CIPKs and a variable regulation by upstream kinases., A.A. thanks Dr. Douglas Vinson Laurents for critical reading of the manuscript, and the European Syncrotron Radiation Facility (beamlines ID14-4 and ID23-2) and PETRAIII (beamline P13, BIOSTRUCTX_3100.5) for the access to the synchrotron radiation source. This work was funded by Ministerio de Economía y Competitividad Grants BFU2011-25384 and CSD2006-00015 (to A.A.), BIO2011-28184-C02-02 (toM.J.S.-B.), and BIO2012-36533 (to F.J.Q.), which was cofinanced by the European Regional Development Fund, and Comunidad de Madrid Grant S2010/BMD-2457 (to A.A.). A.C.-S. is supported by a Formación de Personal Investigador Predoctoral Fellowship, and M.J.S.-B. is supported by Ramón y Cajal Contract RYC-2008-03449 from MINECO.