Hydrogen-Deuterium Exchange Mass Spectrometry Reveals an Antibody-Induced Allosteric Pathway Driving SARS-CoV-2 Spike Trimer Disassembly.

The rapid evolution of SARS-CoV-2 necessitates a deeper understanding of antibody neutralization mechanisms to guide the development of broad-spectrum therapies. Here, we integrate pulsed hydrogen-deuterium exchange mass spectrometry (HDX-MS), biolayer interferometry (BLI), and negative-stain electron microscopy (nsEM) to dissect the dynamic regulation of two engineered spike (S) protein constructs, the prefusion-stabilized S-6P and the wild-type-like S-R, by two antibody classes: the noncompetitive RBD class V antibody R1-32 and the competitive class I antibody B38. Our results show that both spike's native receptor, human angiotensin-converting enzyme 2 (ACE2), and B38 primarily protect epitopes within the receptor-binding domain (RBD), inducing a similar level of fusion-priming structural transitions without causing trimer disassembly. In contrast, R1-32 binds to a semicryptic epitope and uniquely triggers long-range allosteric destabilization across the "tripartite interface", a conserved structural hub centered around Domain C. This local perturbation propagates through region 962-977 to the trimer interface, driving time-dependent S-trimer disassembly, as further supported by nsEM. Combining these insights, eight fully conserved residues within the tripartite interface were identified as critical hotspots, representing potential targets for broad-spectrum antibodies capable of inducing trimer disassembly. Together, these findings establish a structure-dynamics framework for rational antibody design, emphasizing the targeting of conserved, conformation-sensitive epitopes to outpace viral immune evasion. Moreover, this work highlights pulsed HDX-MS as a powerful approach for resolving antibody-mediated dynamic regulation of the SARS-CoV-2 spike protein, facilitating the development of next-generation broad-spectrum therapeutics against emerging variants.