Search

RNA can directly control protein activity in a process called riboregulation; only a few mechanisms of riboregulation have been described in detail, none of which have been characterized on structural grounds. Here, we present a comprehensive structural, functional, and phylogenetic analysis of riboregulation of cytosolic serine hydroxymethyltransferase (SHMT1), the enzyme interconverting serine and glycine in one-carbon metabolism. We have determined the cryoelectron microscopy (cryo-EM) structure of human SHMT1 in its free- and RNA-bound states, and we show that the RNA modulator competes with polyglutamylated folates and acts as an allosteric switch, selectively altering the enzyme's reactivity vs. serine. In addition, we identify the tetrameric assembly and a flap structural motif as key structural elements necessary for binding of RNA to eukaryotic SHMT1. The results presented here suggest that riboregulation may have played a role in evolution of eukaryotic SHMT1 and in compartmentalization of one-carbon metabolism. Our findings provide insights for RNA-based therapeutic strategies targeting this cancer-linked metabolic pathway. [Display omitted] • Cryo-EM structure of human SHMT1 at 3.29 Å reveals different active site conformations • Cryo-EM structure of the SHMT1:RNA complex at 3.52 Å reveals a 4:1 binding stoichiometry • The tetrameric assembly and a flap structural motif are necessary for RNA binding • RNA riboregulates SHMT1 by competing with folate and acting as an allosteric switch Using cryo-EM, Spizzichino et al. have solved the structure of the metabolic enzyme SHMT1 in the unbound form and in complex with RNA. Functional results showed that RNA riboregulates SHMT1 activity by acting as an allosteric switch, paving the way for the design of innovative RNA-based inhibitors. [ABSTRACT FROM AUTHOR]

The HCN1–4 channel family is responsible for the hyperpolarization-activated cation current I f /I h 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. [Display omitted] • 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. [ABSTRACT FROM AUTHOR]

In the arms race against bacteria, bacteriophages have evolved diverse anti-CRISPR proteins (Acrs) that block CRISPR-Cas immunity. Acrs play key roles in the molecular coevolution of bacteria with their predators, use a variety of mechanisms of action, and provide tools to regulate Cas-based genome manipulation. Here, we present structural and functional analyses of AcrIIA6, an Acr from virulent phages, exploring its unique anti-CRISPR action. Our cryo-EM structures and functional data of AcrIIA6 binding to Streptococcus thermophilus Cas9 (St1Cas9) show that AcrIIA6 acts as an allosteric inhibitor and induces St1Cas9 dimerization. AcrIIA6 reduces St1Cas9 binding affinity for DNA and prevents DNA binding within cells. The PAM and AcrIIA6 recognition sites are structurally close and allosterically linked. Mechanistically, AcrIIA6 affects the St1Cas9 conformational dynamics associated with PAM binding. Finally, we identify a natural St1Cas9 variant resistant to AcrIIA6 illustrating Acr-driven mutational escape and molecular diversification of Cas9 proteins. • AcrIIA6 allosterically inhibits St1Cas9 and can induce its dimerization • AcrIIA6 alters St1Cas9 dynamics associated with PAM binding • AcrIIA6 reduces St1Cas9 DNA binding affinity, thereby blocking DNA binding in cells • A natural St1Cas9 variant illustrates an anti-CRISPR driven mutational escape Fuchsbauer et al. present structural and functional data highlighting the allosteric inhibition mechanism used by AcrIIA6 to inactivate St1Cas9. AcrIIA6 modifies St1Cas9 dynamics and structure, which ultimately prevents its binding to DNA within cells. A naturally resistant St1Cas9 variant illustrates how bacterial cells can escape phages anti-CRISPR systems. [ABSTRACT FROM AUTHOR]

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., Competing Interests: Declaration of interests O.M., E.M.P., and R.D.S. are employees of Alfasigma SpA and are named as inventors in a patent application on the name of the same company., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)