Switching Spike Plasticity Shapes ACE2 Engagement Across SARS‐CoV‐2 Variants

Abstract

ABSTRACT Conformational plasticity allows class I viral fusion proteins, including the SARS‐CoV‐2 spike, to undergo major structural rearrangements that support receptor binding and membrane fusion, making them key vaccine and antiviral targets. Using high‐speed atomic force microscopy (AFM) and single‐molecule force spectroscopy, we analyzed the ancestral and nine spike variants and found an evolutionary trend from increased flexibility in early strains to greater rigidification beginning with Delta, fluctuating plasticity in Omicron sublineages, and pronounced compaction in JN.1. Delta favored multivalent ACE2 engagement through its three receptor‐binding domains, whereas later Omicron variants shifted toward predominantly single‐site interactions. This change was compensated by stronger RBD–ACE2 bond stability. Overall, spike evolution appears to balance openness and compaction to regulate receptor accessibility, mobility, and immune escape while preserving or enhancing entry efficiency. These single‐molecule measurements define functional constraints on viral adaptation and may inform prediction of future variants and design of improved countermeasures.