Structure and dynamics of the cyanobacterial regulator SipA

The small, 78-residue long, regulator SipA interacts with the non-bleaching sensor histidine kinase (NblS). We have solved the solution structure of SipA on the basis of 990 nuclear Overhauser effect- (NOE-) derived distance constraints. The average pairwise root-mean-square deviation (RMSD) for the twenty best structures for the backbone residues, obtained by CYANA, was 1.35 ± 0.21 Å, and 1.90 ± 0.16 Å when all heavy atoms were considered (the target function of CYANA was 0.540 ± 0.08). The structure is that of a β-II class protein, basically formed by a five-stranded β-sheet composed of antiparallel strands following the arrangement: Gly6-Leu11 (β-strand 1), which packs against Leu66-Val69 (β-strand 5) on one side, and against Gly36-Thr42 (β-strand 2) on the other side; Trp50-Phe54 (β-strand 3); and Gly57-Leu60 (β-strand 4). The protein is highly mobile, as shown by measurements of R1, R2, NOE and ηxy relaxation parameters, with an average order parameter () of 0.70; this mobility encompasses movements in different time scales. We hypothesize that this high flexibility allows the interaction with other proteins (among them NblS), and it explains the large conformational stability of SipA.This research was funded by Consellería de Innovación, Universidades, Ciencia y Sociedad Digital (Generalitat Valenciana) [CIAICO 2021/0135 to ACA and JLN and PROMETEO/2020/012 to AM], Ministerio de Ciencia e Innovación (PID2020-118816GB-I00 to AC and PID2022-137201NB-I00 to AM) and by the European Commission NextGenerationEU fund (EU 2020/2094), through CSIC's Global Health Platform (PTI Salud Global) to AM

The cyanobacterial ribosomal-associated protein LrtA from Synechocystis sp. PCC 6803 is an oligomeric protein in solution with chameleonic sequence properties

The LrtA protein of Synechocystis sp. PCC 6803 intervenes in cyanobacterial post-stress survival and in stabilizing 70S ribosomal particles. It belongs to the hibernating promoting factor (HPF) family of proteins, involved in protein synthesis. In this work, we studied the conformational preferences and stability of isolated LrtA in solution. At physiological conditions, as shown by hydrodynamic techniques, LrtA was involved in a self-association equilibrium. As indicated by Nuclear Magnetic Resonance (NMR), circular dichroism (CD) and fluorescence, the protein acquired a folded, native-like conformation between pH 6.0 and 9.0. However, that conformation was not very stable, as suggested by thermal and chemical denaturations followed by CD and fluorescence. Theoretical studies of its highly-charged sequence suggest that LrtA had a Janus sequence, with a context-dependent fold. Our modelling and molecular dynamics (MD) simulations indicate that the protein adopted the same fold observed in other members of the HPF family ( - - - - - ) at its N-terminal region (residues 1–100), whereas the C terminus (residues 100–197) appeared disordered and collapsed, supporting the overall percentage of overall secondary structure obtained by CD deconvolution. Then, LrtA has a chameleonic sequence and it is the first member of the HPF family involved in a self-association equilibrium, when isolated in solution.Ministerio de Economía y Competitividad CTQ2015-64445-RMinisterio de Economía y Competitividad BIO2016-78020-RMinisterio de Economía y Competitividad FIS2014-52212-RMinisterio de Economía y Competitividad BIO2016-75634-PFundación Séneca 19353/PI/1

Efficacy of aldose reductase inhibitors is affected by oxidative stress induced under X-ray irradiation

Human aldose reductase (hAR, AKR1B1) has been explored as drug target since the 1980s for its implication in diabetic complications. An activated form of hAR was found in cells from diabetic patients, showing a reduced sensitivity to inhibitors in clinical trials, which may prevent its pharmacological use. Here we report the conversion of native hAR to its activated form by X-ray irradiation simulating oxidative stress conditions. Upon irradiation, the enzyme activity increases moderately and the potency of several hAR inhibitors decay before global protein radiation damage appears. The catalytic behavior of activated hAR is also reproduced as the KM increases dramatically while the kcat is not much affected. Consistently, the catalytic tetrad is not showing any modification. The only catalytically-relevant structural difference observed is the conversion of residue Cys298 to serine and alanine. A mechanism involving electron capture is suggested for the hAR activation. We propose that hAR inhibitors should not be designed against the native protein but against the activated form as obtained from X-ray irradiation. Furthermore, since the reactive species produced under irradiation conditions are the same as those produced under oxidative stress, the described irradiation method can be applied to other relevant proteins under oxidative stress environments.This work was started, and partly supported by a grant from the Spanish Nuclear Council (CSN)

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 hépatites 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_S 1109), 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. We are grateful to the Spanish Radiation Synchrotron Source (ALBA), Barcelona, Spain and the European Synchrotron Radiation Facility (ESRF), Grenoble, France, for the provision of time and staff assistance at XALOC (ALBA) and ID30B and ID23-2 (ESRF) beamlines during diffraction data collection. We thank María Carmen Salinas-García for her assistance in carrying out the crystallization screenings. We also thank Pilar González-García for helping us with the statistical analysis

Nurses' perceptions of aids and obstacles to the provision of optimal end of life care in ICU

Contains fulltext : 172380.pdf (publisher's version ) (Open Access

First 3-D structural evidence of a native-like intertwined dimer in the acylphosphatase family

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

Structural and Thermodynamic Analysis of HIV-1 Fusion Inhibition Using Small gp41 Mimetic Proteins

Development of effective inhibitors of the fusion between HIV-1 and the host cell membrane mediated by gp41 continues to be a grand challenge due to an incomplete understanding of the molecular and mechanistic details of the fusion process. We previously developed single-chain, chimeric proteins (named covNHR) that accurately mimic the N-heptad repeat (NHR) region of gp41 in a highly stable coiled-coil conformation. These molecules bind strongly to peptides derived from the gp41 C-heptad repeat (CHR) and are potent and broad HIV-1 inhibitors. Here, we investigated two covNHR variants differing in two mutations, V10E and Q123R (equivalent to V38E and Q40R in gp41 sequence) that reproduce the effect of HIV-1 mutations associated with resistance to fusion inhibitors, such as T20 (enfuvirtide). A detailed calorimetric analysis of the binding between the covNHR proteins and CHR peptides (C34 and T20) reveals drastic changes in affinity due to the mutations as a result of local changes in interactions at the site of T20 resistance. The crystallographic structure of the covNHR:C34 complex shows a virtually identical CHR–NHR binding interface to that of the post-fusion structure of gp41 and underlines an important role of buried interfacial water molecules in binding affinity and in development of resistance against CHR peptides. Despite the great difference in affinity, both covNHR variants demonstrate strong inhibitory activity for a wide variety of HIV-1 strains. These properties support the high potential of these covNHR proteins as new potent HIV-1 inhibitors. Our results may guide future inhibition approaches.Departamento de Química Física e Instituto de Biotecnología, Facultad de Ciencias, Universidad de Granada, 18071 Granada, SpainMinisterio de Economía y Competitividad (grants BIO2016-76640-R and BIO2016-78020-R) y Fondo Europeo para el Desarrollo Regional (FEDER) de la Unión EuropeaANRS and the Vaccine Research Institute for the Investissements d'Avenir program managed by the ANR under reference ANR-10-LABX-7