D Xu, W Jiang, . Wu, RG Gaudet, E-S Park, M Su, SK Cheppali, NR Cheemarla, P Kumar, PD Uchil, JR Grover, EF Foxman, CM Brown, PJ Stansfeld, J Bewersdorf, W Mothes, E Karatekin, CB Wilen, and JD MacMicking

Understanding protective immunity to COVID-19 facilitates preparedness for future pandemics and combats new SARS-CoV-2 variants emerging in the human population. Neutralizing antibodies have been widely studied; however, on the basis of large-scale exome sequencing of protected versus severely ill patients with COVID-19, local cell-autonomous defence is also crucial. Here we identify phospholipid scramblase 1 (PLSCR1) as a potent cell-autonomous restriction factor against live SARS-CoV-2 infection in parallel genome-wide CRISPR–Cas9 screens of human lung epithelia and hepatocytes before and after stimulation with interferon-γ (IFNγ). IFNγ-induced PLSCR1 not only restricted SARS-CoV-2 USA-WA1/2020, but was also effective against the Delta B.1.617.2 and Omicron BA.1 lineages. . Our mechanistic studies, together with reports that COVID-associated PLSCR1 mutations are found in some susceptible people, identify an anti-coronavirus protein that interferes at a late entry step before viral RNA is released into the host-cell cytosol.

Nature. July 2023

Fuss MF, Wieferig JP, Corey RA, Hellmich Y, Tascón I, Sousa JS, Stansfeld PJ, Vonck J, Hänelt I

Cyclic di-AMP is the only known essential second messenger in bacteria and archaea, regulating different proteins indispensable for numerous physiological processes. In particular, it controls various potassium and osmolyte transporters involved in osmoregulation. In this study, we reveal the molecular basis of how c-di-AMP binding inhibits KimA. We report cryo-EM structures of KimA with bound c-di-AMP in detergent solution and reconstituted in amphipols. By combining structural data with functional assays and molecular dynamics simulations we reveal how c-di-AMP modulates transport. We show that an intracellular loop in the transmembrane domain interacts with c-di-AMP bound to the adjacent cytosolic domain. This reduces the mobility of transmembrane helices at the cytosolic side of the K⁺ binding site and therefore traps KimA in an inward-occluded conformation.

Nature Communications. June 2023

Jonathan Cook, Tyler C Bavistock, Martin BL McAndrew, David I Roper, Phillip J Stansfeld and Allister Crow

Here, we present a high-resolution structure of the isolated AmiA protein, confirming that it is autoinhibited in the same manner as AmiB and AmiC, and a complex of the AmiB enzymatic domain bound to the activating EnvC LytM domain. In isolation, the active site of AmiA is blocked by an autoinhibitory helix that binds directly to the catalytic zinc and fills the volume expected to accommodate peptidoglycan binding. In the complex, binding of the EnvC LytM domain induces a conformational change that displaces the amidase autoinhibitory helix and reorganizes the active site for activity. Our structures, together with complementary mutagenesis work, defines the conformational changes required to activate AmiA and/or AmiB through their interaction with other cognate activator EnvC.

PNAS.; June 2023