Biophysical Characterization of Ebola VP35 Protein Binding to Host Mindbomb Protein 2

Mindbomb protein 2 (MIB2) is an E3 ubiquitin ligase that plays critical roles in immune regulation, inflammation, apoptosis, and cancer progression. Recent studies identified MIB2 as a host factor that binds Ebola virus (EBOV) protein VP35 (eVP35). Disruption of MIB2-VP35 binding diminishes EBOV-induced cytotoxicity, but the molecular basis of this interaction remains incompletely defined. How MIB2 function is regulated by eVP35 is not well understood. Here, we use biophysical and mass spectrometry (MS)-based approaches, including crosslinking-MS (XL-MS) and hydrogen-deuterium exchange-MS (HDX-MS), to characterize the molecular mechanism. Results from these studies reveal conformational rearrangements among MIB2 domains upon eVP35 binding. Intramolecular XL-MS coupled with molecular docking indicates that MIB2 alone adopts a compact conformation where the MZM and MREP domains are closely oriented to the ankyrin repeats. Differential HDX-MS analysis of MIB2-eVP35 binding revealed that the interaction interface involves the ²⁰¹NNLNS²⁰⁵ motif in eVP35 and the conserved MIB2 residues ¹²¹NYR¹²³ within the MZM, which are critical for substrate binding. Our results also suggest that the MZM and MREP domains reorient relative to the ankyrin repeats upon eVP35 binding. These structural rearrangements of MIB2 domains likely contribute to viral immune evasion.