Natural effectors for conformational control of coronavirus spike proteins

Sialic acid (Sia) at the cell surface is a putative SARS-CoV-2 co-receptor via interactions with Spike N-terminal domain (NTD), and Sia-based inhibitors have shown efficacy against SARS-CoV-2 in culture. However, protein:small-molecule complexes pose difficult cryo-EM targets because small ligands typically act in relatively poorly resolved peripheral regions, and structural details of Spike:Sia interactions via the highly flexible NTD remain elusive. Spike receptor-binding domain (RBD) binds linoleic acid (LA), which interacts with neighboring RBD to lock-in a closed trimer with rigid, well-resolved NTDs. While endogenous LA from certain expression systems serendipitously permits visualization of bound Sia, necessary and sufficient conditions for reliable extraction of this complex remain unclear. Here, we supplement Spike with pure LA in vitro to recover the rigidified state observed with endogenous LA, and leverage enhanced NTD resolvability to visualize binding of the Sia ganglioside GM1. Our structure reveals how Spike might employ plasma membrane gangliosides as co-receptors for viral entry, perhaps driving accelerated search of the 2D cell surface (rather than 3D space) for its high-affinity protein receptor ACE2. While natural effectors exerting control of protein conformation represent powerful biophysical tools, endogenous effectors may conversely trap undesired conformations, precluding observation of alternate states. Stabilization of pre-fusion Spike with exposed receptor-binding surfaces can aid antibody isolation campaigns and vaccine antigen design, but Spikes from some human coronaviruses, such as hCoV-OC43, are observed only with closed RBDs. Noting OC43 Spike features a homologous LA binding site, populated in known structures, we hypothesize that endogenous LA is responsible for strictly closed RBD. In vitro removal of native effectors like LA might enable characterization of cryptic functional states. We apply reverse ligand affinity purification for removal of LA from OC43 Spike, potentially a general strategy for preparation of open-RBD coronavirus Spike conformations.