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 ß-II class protein, basically formed by a five-stranded ß-sheet composed of antiparallel strands following the arrangement: Gly6-Leu11 (ß-strand 1), which packs against Leu66-Val69 (ß-strand 5) on one side, and against Gly36-Thr42 (ß-strand 2) on the other side; Trp50-Phe54 (ß-strand 3); and Gly57-Leu60 (ß-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.

C-2 Thiophenyl Tryptophan Trimers Inhibit Cellular Entry of SARS-CoV-2 through Interaction with the Viral Spike (S) Protein.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, by infecting cells via the interaction of its spike protein (S) with the primary cell receptor angiotensin-converting enzyme (ACE2). To search for inhibitors of this key step in viral infection, we screened an in-house library of multivalent tryptophan derivatives. Using VSV-S pseudoparticles, we identified compound 2 as a potent entry inhibitor lacking cellular toxicity. Chemical optimization of 2 rendered compounds 63 and 65, which also potently inhibited genuine SARS-CoV-2 cell entry. Thermofluor and microscale thermophoresis studies revealed their binding to S and to its isolated receptor binding domain (RBD), interfering with the interaction with ACE2. High-resolution cryoelectron microscopy structure of S, free or bound to 2, shed light on cell entry inhibition mechanisms by these compounds. Overall, this work identifies and characterizes a new class of SARS-CoV-2 entry inhibitors with clear potential for preventing and/or fighting COVID-19.

Correction: The structural role of SARS-CoV-2 genetic background in the emergence and success of spike mutations: The case of the spike A222V mutation.

[This corrects the article DOI: 10.1371/journal.ppat.1010631.].

The structural role of SARS-CoV-2 genetic background in the emergence and success of spike mutations: The case of the spike A222V mutation.

The S:A222V point mutation, within the G clade, was characteristic of the 20E (EU1) SARS-CoV-2 variant identified in Spain in early summer 2020. This mutation has since reappeared in the Delta subvariant AY.4.2, raising questions about its specific effect on viral infection. We report combined serological, functional, structural and computational studies characterizing the impact of this mutation. Our results reveal that S:A222V promotes an increased RBD opening and slightly increases ACE2 binding as compared to the parent S:D614G clade. Finally, S:A222V does not reduce sera neutralization capacity, suggesting it does not affect vaccine effectiveness.

Structural basis for the alternating access mechanism of the cation diffusion facilitator YiiP.

YiiP is a dimeric antiporter from the cation diffusion facilitator family that uses the proton motive force to transport Zn2+ across bacterial membranes. Previous work defined the atomic structure of an outward-facing conformation, the location of several Zn2+ binding sites, and hydrophobic residues that appear to control access to the transport sites from the cytoplasm. A low-resolution cryo-EM structure revealed changes within the membrane domain that were associated with the alternating access mechanism for transport. In the current work, the resolution of this cryo-EM structure has been extended to 4.1 Å. Comparison with the X-ray structure defines the differences between inward-facing and outward-facing conformations at an atomic level. These differences include rocking and twisting of a four-helix bundle that harbors the Zn2+ transport site and controls its accessibility within each monomer. As previously noted, membrane domains are closely associated in the dimeric structure from cryo-EM but dramatically splayed apart in the X-ray structure. Cysteine crosslinking was used to constrain these membrane domains and to show that this large-scale splaying was not necessary for transport activity. Furthermore, dimer stability was not compromised by mutagenesis of elements in the cytoplasmic domain, suggesting that the extensive interface between membrane domains is a strong determinant of dimerization. As with other secondary transporters, this interface could provide a stable scaffold for movements of the four-helix bundle that confers alternating access of these ions to opposite sides of the membrane.

The transmembrane domain of the p75 neurotrophin receptor stimulates phosphorylation of the TrkB tyrosine kinase receptor.

The function of protein products generated from intramembraneous cleavage by the γ-secretase complex is not well defined. The γ-secretase complex is responsible for the cleavage of several transmembrane proteins, most notably the amyloid precursor protein that results in Aß, a transmembrane (TM) peptide. Another protein that undergoes very similar γ-secretase cleavage is the p75 neurotrophin receptor. However, the fate of the cleaved p75 TM domain is unknown. p75 neurotrophin receptor is highly expressed during early neuronal development and regulates survival and process formation of neurons. Here, we report that the p75 TM can stimulate the phosphorylation of TrkB (tyrosine kinase receptor B). In vitro phosphorylation experiments indicated that a peptide representing p75 TM increases TrkB phosphorylation in a dose- and time-dependent manner. Moreover, mutagenesis analyses revealed that a valine residue at position 264 in the rat p75 neurotrophin receptor is necessary for the ability of p75 TM to induce TrkB phosphorylation. Because this residue is just before the γ-secretase cleavage site, we then investigated whether the p75(αγ) peptide, which is a product of both α- and γ-cleavage events, could also induce TrkB phosphorylation. Experiments using TM domains from other receptors, EGFR and FGFR1, failed to stimulate TrkB phosphorylation. Co-immunoprecipitation and biochemical fractionation data suggested that p75 TM stimulates TrkB phosphorylation at the cell membrane. Altogether, our results suggest that TrkB activation by p75(αγ) peptide may be enhanced in situations where the levels of the p75 receptor are increased, such as during brain injury, Alzheimer's disease, and epilepsy.

Insights into the mechanism of activation of the phosphorylation-independent response regulator NblR. Role of residues Cys69 and Cys96.

Cyanobacteria respond to environmental stress conditions by adjusting their photosynthesis machinery. In Synechococcus sp. PCC 7942, phycobilisome degradation and other acclimation responses after nutrient or high light stress require activation by the phosphorylation-independent response regulator NblR. Structural modelling of its receiver domain suggested a role for Cys69 and Cys96 on activation of NblR. Here, we investigate this hypothesis by engineering Cys to Ala substitutions. In vivo and in vitro analyses indicated that mutations Cys69Ala and/or Cys96Ala have a minor impact on NblR function, structure, size, or oligomerization state of the protein, and that Cys69 and Cys96 do not seem to form disulphide bridges. Our results argue against the predicted involvement of Cys69 and Cys96 on NblR activation by redox sensing.

Environmental control of phosphorylation pathways in a branched two-component system.

NblS, the most conserved histidine kinase in cyanobacteria, regulates photosynthesis and acclimatization to a variety of environmental conditions. We used in silico, in vivo and in vitro approaches to identify RpaB and SrrA as the cognate response regulators of NblS and to characterize relevant interactions between components of this signalling system. While genetic analysis showed the importance of the NblS to RpaB phosphorylation branch for culture viability in Synechococcus elongatus PCC 7942, in vitro assays indicated a strong preference for NblS to phosphorylate SrrA. This apparent discrepancy can be explained by environmental insulation of the RpaB pathway, achieved by RpaB-dependent repression of srrA under standard, low light culture conditions. After a strong but transient increase in srrA expression upon high light exposure, negative regulation of srrA and other high light inducible genes takes place, suggesting cooperation between pathways under environmental conditions in which both RpaB and SrrA are present. Complex regulatory interactions between RpaB and SrrA, two response regulators with a common evolutionary origin that are controlled by a single histidine kinase, are thus emerging. Our results provide a paradigm for regulatory interactions between response regulators in a branched two-component system.

The regulatory factor SipA is a highly stable beta-II class protein with a SH3 fold.

The small regulator SipA, interacts with the ATP-binding domain of non-bleaching sensor histidine kinase (NblS), the most conserved histidine kinase in cyanobacteria. NblS regulates photosynthesis and acclimation to a variety of environmental conditions. We show here that SipA is a highly stable protein in a wide pH range, with a thermal denaturation midpoint of 345 K. Circular dichroism and 1D 1H NMR spectroscopies, as well as modelling, suggest that SipA is a beta-II class protein, with short strands followed by turns and long random-coil polypeptide patches, matching the SH3 fold. The experimentally determined m-value and the heat capacity change upon thermal unfolding (DeltaCp) closely agreed with the corresponding theoretical values predicted from the structural model, further supporting its accuracy.

Phosphorylation-independent activation of the atypical response regulator NblR.

Cyanobacteria respond to environmental stress conditions by adjusting their photosynthesis machinery. In Synechococcus sp. PCC 7942, phycobilisome degradation and other acclimation responses after nutrient or high-light stress require activation by the orphan response regulator NblR, a member of the OmpR/PhoB family. Although NblR contains a putative phosphorylatable residue (Asp57), it lacks other conserved residues required to chelate the Mg(2+) necessary for aspartic acid phosphorylation or to transduce the phosphorylation signal. In close agreement with these features, NblR was not phosphorylated in vitro by the low-molecular-mass phosphate donor acetyl phosphate and mutation of Asp57 to Ala had no impact on previously characterized NblR functions in Synechococcus. On the other hand, in vitro and in vivo assays show that the default state of NblR is monomeric, suggesting that, despite input differences, NblR activation could involve the same general mechanism of activation by dimerization present in known members of the OmpR/PhoB family. Structural and functional data indicate that the receiver domain of NblR shares similarities with other phosphorylation-independent response regulators such as FrzS and HP1043. To acknowledge the peculiarities of these atypical 'two-component' regulators with phosphorylation-independent signal transduction mechanisms, we propose the term PIARR, standing for phosphorylation-independent activation of response regulator.

The regulatory factor SipA provides a link between NblS and NblR signal transduction pathways in the cyanobacterium Synechococcus sp. PCC 7942.

Cyanobacteria respond to environmental stress conditions by adjusting its photosynthesis machinery. When subjected to nutrient and high light stress, Synechococcus sp. PCC 7942 and other non-diazotrophic cyanobacteria degrade their phycobilisome, the light-harvesting complexes for photosynthesis. Phycobilisome degradation requires convergence of multiple signals onto the nblA gene. Despite considerable efforts to identify regulatory proteins involved in acclimation responses, the signal transduction mechanisms involved remain largely unknown. However, we show here that SipA, a protein that binds to the ATP-binding domain of the histidine kinase NblS, counteracts the function of the response regulator NblR in acclimation to stress, and is also involved in downregulation of the nblA gene. The integrity of the HLR1 element overlapping P(nblA-1) and P(nblA-2) promoters is required for downregulation of the nblA gene. Induction by NblR is strongly dependent on DNA sequences located at least 44 bp upstream transcription initiation from P(nblA-2), and is also hampered by point mutations at HLR1. Genetic evidence of the antagonistic roles of NblR and SipA at regulation of the nblA gene, chlorosis and survival from stress is presented.