Cryo-EM structures and binding of mouse ACE2 to SARS-CoV-2 variants of concern (preprint)

Investigation of potential hosts of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is crucial to understanding future risks of spillover and spillback. SARS-CoV-2 has been reported to be transmitted from humans to various animals after requiring relatively few mutations. Mice are well adapted to human environments, frequently come in contact with humans, are used widely as infection models, and may act as reservoirs for SARS-CoV-2. Structural and binding data of the mouse ACE2 receptor with the Spike protein of newly identified SARS-CoV-2 variants are needed to better understand the impact of variants of concern (VOC). Previous studies have developed mouse-adapted variants and have identified some determinants of binding. Here we report the cryo-EM structures of mouse ACE2 bound to Spike ectodomains of four different VOC: Beta, Omicron BA.1, Omicron BA.2.12.1 and Omicron BA.4/5. These variants represent the oldest to the newest variants that are able to bind the mouse ACE2 receptor. Our high-resolution structural data complemented with bio-layer interferometry (BLI) binding assays reveal a requirement for a combination of mutations in the Spike protein to enable the binding to mouse ACE2.

De novo design of site-specific protein interactions with learned surface fingerprints (preprint)

Physical interactions between proteins are essential for most biological processes governing life. However, the molecular determinants of such interactions have been challenging to understand, even as genomic, proteomic, and structural data grows. This knowledge gap has been a major obstacle for the comprehensive understanding of cellular protein-protein interaction (PPI) networks and for the de novo design of protein binders that are crucial for synthetic biology and translational applications. We exploit a geometric deep learning framework operating on protein surfaces that generates fingerprints to describe geometric and chemical features critical to drive PPIs. We hypothesized these fingerprints capture the key aspects of molecular recognition that represent a new paradigm in the computational design of novel protein interactions. As a proof-of-principle, we computationally designed four de novo protein binders to engage three protein targets: SARS-CoV-2 spike, PD-1, and PD-L1. The designs bound the target sites with nanomolar affinity upon experimental optimization, structural and mutational characterization showed highly accurate predictions. Overall, our surface-centric approach captures the physical and chemical determinants of molecular recognition, enabling a novel approach for the de novo design of protein interactions and, more broadly, of artificial proteins with function.

SARS-CoV-2 Omicron potently neutralized by a novel antibody with unique Spike binding properties (preprint)

The SARS-CoV-2 Omicron variant exhibits very high levels of transmission, pronounced resistance to authorized therapeutic human monoclonal antibodies and reduced sensitivity to vaccine-induced immunity. Here we describe P2G3, a human monoclonal antibody (mAb) isolated from a previously infected and vaccinated donor, which displays picomolar-range neutralizing activity against Omicron BA.1, BA.1.1, BA.2 and all other current variants, and is thus markedly more potent than all authorized or clinically advanced anti-SARS-CoV-2 mAbs. Structural characterization of P2G3 Fab in complex with the Omicron Spike demonstrates unique binding properties to both down and up spike trimer conformations at an epitope that partially overlaps with the receptor-binding domain (RBD), yet is distinct from those bound by all other characterized mAbs. This distinct epitope and angle of attack allows P2G3 to overcome all the Omicron mutations abolishing or impairing neutralization by other anti-SARS-COV-2 mAbs, and P2G3 accordingly confers complete prophylactic protection in the SARS-CoV-2 Omicron monkey challenge model. Finally, although we could isolate in vitro SARS-CoV2 mutants escaping neutralization by P2G3 or by P5C3, a previously described broadly active Class 1 mAb, we found these viruses to be lowly infectious and their key mutations extremely rare in the wild, and we could demonstrate that P2G3/P5C3 efficiently cross-neutralized one another's escapees. We conclude that this combination of mAbs has great prospects in both the prophylactic and therapeutic settings to protect from Omicron and other VOCs.

Structural analysis of the Spike of the Omicron SARS-COV-2 variant by cryo-EM and implications for immune evasion (preprint)

ABSTRACT The Omicron (B.1.1.529) SARS-COV-2 was reported on November 24, 2021 and declared a variant of concern a couple of days later. 1,2 With its constellation of mutations acquired by this variant on its Spike glycoprotein and the speed at which this new variant has replaced the previously dominant variant Delta in South Africa and the United Kingdom, it is crucial to have atomic structural insights to reveal the mechanism of its rapid proliferation. Here we present a high-resolution cryo-EM structure of the Spike protein of the Omicron variant.

Structural investigation of ACE2 dependent disassembly of the trimeric SARS-CoV-2 Spike glycoprotein (preprint)

The human membrane protein Angiotensin-converting enzyme 2 (hACE2) acts as the main receptor for host cells invasion of the new coronavirus SARS-CoV-2. The viral surface glycoprotein Spike binds to hACE2, which triggers virus entry into cells. As of today, the role of hACE2 for virus fusion is not well understood. Blocking the transition of Spike from its prefusion to post-fusion state might be a strategy to prevent or treat COVID-19. Here we report a single particle cryo-electron microscopy analysis of SARS-CoV-2 trimeric Spike in presence of the human ACE2 ectodomain. The binding of purified hACE2 ectodomain to Spike induces the disassembly of the trimeric form of Spike and a structural rearrangement of its S1 domain to form a stable, monomeric complex with hACE2. This observed hACE2 dependent dissociation of the Spike trimer suggests a mechanism for the therapeutic role of recombinant soluble hACE2 for treatment of COVID-19.