<p dir="ltr">Ebola virus is an enveloped filamentous shaped RNA virus which causes severe hemorrhagic fever in humans. Multiple outbreaks of different strains of ebolavirus have been reported in the past years with limited therapeutics available for treatment. Despite some advances in treatment, there remains a lack of knowledge about the mechanisms of ebolavirus replication in host cells.</p><p dir="ltr">Ebolavirus encodes for seven structural proteins with matrix protein (VP40) being the most abundantly expressed viral protein. VP40 is essential for viral assembly and budding as expression of VP40 alone is sufficient for formation of virus-like particles (VLPs). VP40 also disassembles during viral entry to help in viral and host cell membrane fusion. Oligomerization of VP40 has been reported to decrease viral replication and transcription. VP40 can perform these diverse functions by virtue of changes in conformation and oligomerization state. VP40 predominantly exists as a dimer through hydrophobic interactions between the alpha helices of the two protomers. Furthermore, VP40 oligomerizes into a hexamer which serves as the structural unit for cylindrical matrix layer formation. VP40 also forms a ring-shaped octamer for regulation of viral transcription. The different oligomeric forms of VP40 exist in an equilibrium for successful viral infection. However, the exact mechanism of formation, stability, and energetics of conversion between these oligomeric forms is unknown.</p><p dir="ltr">In this study, we performed biophysical analysis on the dimerization interface and identified keystone interactions which when abrogated lead to complete disruption of dimer interface. In addition, peptidomimetics approach was used to design and synthesize a library of compounds to probe the dimerization interface. The compounds were screened using thermal shift assay and then compared using MST and ITC studies. We identified that a peptide mimicking the alpha helical region stabilized by a p-xylene di-cysteine staple was able to bind to VP40 dimer. We also determined that this peptide binds near the dimer interface and was able to slightly shift equilibrium of VP40 dimer towards monomer formation.</p><p dir="ltr">Overall, this report sheds light on critical interactions required for VP40 dimer formation and stability and introduces use of peptidomimetics to probe for VP40 dimerization interface to understand energetics of oligomerization equilibrium, thereby increasing our knowledge about disease mechanism and paving way for development of therapeutics.</p>