Identification of key functional sites for ATPase activity in the OBC protein complex using artificial intelligence approaches

Abstract Microorganisms evolve diverse immune defense systems to protect against phage and viral invasion. The BREX system represents a common bacterial genomic defense mechanism, where preliminary studies indicate that the interaction between BrxC and OrbA proteins (forming the OBC complex) can alter the aggregation pattern of defense system components and modulate immune function. Notably, the ATPase domain of this system plays a pivotal role. This study employs ONS, an artificial intelligence-based integrated analytical approach combining machine learning and deep learning, to identify critical enzymatic active sites in target macromolecular complexes and provide crucial insights for potential drug target discovery. Through comprehensive ONS analysis of multiple sites, we identified lysine (K73) as a key residue governing the ATPase activity of this protein complex. Following plasmid construction and expression, we successfully purified the OBC complex, along with newly engineered OBCK (K73 mutant) and negative control OBCE complexes. ATPase activity assays demonstrated complete loss of enzymatic function in the OBCK mutant, confirming K73 as the essential catalytic site for BREX system protease activity. Comparative analysis revealed minimal differences between OBC and OBCE complexes, but significant divergence from OBCK. The single K73 mutation alone was sufficient to abolish critical enzymatic activity and disrupt BREX system function, achieving efficient complex engineering without requiring multiple mutations. Building on this artificial intelligence methodology, we further analyzed potential functional sites and antigenic epitopes in OBC and OBCK complexes, establishing a research foundation for developing anti-drug-resistant pathogen therapeutics.