The Ebola virus (EBOV) matrix protein VP40 is the main driver of viral assembly and budding, processes which depend on its ability to oligomerize at the inner leaflet of the plasma membrane (PM). VP40 forms dimers in the cytoplasm that traffic to the membrane and assemble into higher-order oligomers, but the specific molecular determinants governing the stability of these oligomeric interfaces are not fully understood. Here, we investigate the role of the C-terminal lysine residue, Lys326, in mediating VP40-lipid and VP40-VP40 interactions. Using a combination of molecular dynamics simulations and computational site-saturation mutagenesis, we predicted that while Lys326 has a minimal role in the stability of the isolated VP40 dimer, it is a critical determinant in stabilizing the interfaces of higher-order structures like tetramers and hexamers. Experimentally, mutation of this residue to alanine or aspartic acid (K326A or K326D) impaired VP40 PM localization, oligomerization, and in a charge-dependent manner, virus-like particle production. Our results indicate that a cationic residue is crucial not only for interactions with anionic phospholipids in the PM but, more importantly, for forming stabilizing networks across VP40 dimer-dimer interfaces. Together, these findings identify Lys326 as a keystone residue that couples membrane binding to the stabilization of essential higher-order oligomers, revealing a critical interface for EBOV assembly that could be exploited for future antiviral strategies.
