Cryo-EM Structures Delineate a pH-Dependent Switch that Mediates Endosomal Positioning of SARS-CoV-2 Spike Receptor-Binding Domains

Abstract

The SARS-CoV-2 spike employs mobile receptor-binding domains (RBDs) to engage the ACE2 receptor and to facilitate virus entry. But what controls RBD positioning? As SARS-CoV-2 utilizes endosomal pathways as a major mode of entry, we investigated the impact of both low pH and ACE2 binding. Cryo-EM structures –at serological and endosomal pH– delineated spike recognition of up to three ACE2 molecules, revealing RBD to freely adopt the ‘up’ conformation required for ACE2 interaction, primarily through RBD movement combined with subtle alterations in position and orientation of neighboring domains. In the absence of ACE2, single-RBD-up conformations dominated at pH 5.5, resolving into a single all-down conformation at lower pH, which biochemical studies suggested might provide immune evasion from RBD-up-recognizing antibody. Notably, a pH-dependent refolding region (residues 824-858) at the spike-interdomain interface displayed dramatic structural rearrangements and appeared to mediate RBD positioning in concert with coordinated movements through the entire trimer apex. Funding: Support for this work was provided by the Intramural Research Program of the Vaccine Research Center, National Institute of Allergy and Infectious Diseases (NIAID), Federal funds from the Frederick National Laboratory for Cancer Research under Contract HHSN261200800001E (A.S., T.S., Y.T.). Cryo-EM data for the spike-ACE2 complexes were collected at Columbia University Cryo-EM Center at the Zuckerman Institute, and at the National Center for CryoEM Access and Training (NCCAT) and the Simons Electron Microscopy Center located at the New York Structural Biology Center, supported by the NIH Common Fund Transformative High Resolution Cryo-Electron Microscopy program (U24 GM129539,) and by grants from the Simons Foundation (SF349247) and NY State Assembly. Cryo-EM datasets for individual spike proteins were collected at the National CryoEM Facility (NCEF) of the National Cancer Institute. This research was, in part, supported by the National Cancer Institute’s National Cryo-EM Facility at the Frederick National Laboratory for Cancer Research under contract HSSN261200800001E. We are especially grateful to U. Baxa, A. Wier, M. Hutchison, and T. Edwards of NCEF for collecting cryo-EM data and for technical assistance with cryo-EM data processing. Frederick Research Computing Environment (FRCE) high-performance computing cluster was used for processing cryo-EM datasets of individual spike proteins. Conflict of Interest: The authors declare no competing interest.