Structural insights into the catalytic mechanism and substrate recognition of a type V CifB deubiquitinase

Cytoplasmic incompatibility (CI) is induced by the toxin protein CifB from Wolbachia and related bacteria through its deubiquitinase (DUB) domain; however, the substrate recognition and catalytic mechanisms of this domain remain unclear. Here, we report the high-resolution crystal structure of the type V CifB DUB domain from Rickettsia felis (a close relative of Wolbachia) in complex with ubiquitin. We found that DUB^R.felis adopts a canonical CE clan/Ulp1-like fold. Mutational analysis revealed that the catalytic residue Cys1152 is essential for enzymatic activity, whereas mutations of His1095 or Asp1114 only partially reduce activity. Structural analysis further suggests the presence of a water-mediated compensatory hydrogen-bonding network within the active site. Further analysis of the complex structure shows that the interaction between DUB^R.felis and ubiquitin can be divided into three distinct interfaces. DUB^R.felis undergoes conformational rearrangements via an induced-fit mechanism: the VR3 region moves outward to form a channel for the ubiquitin C-terminal Gly-Gly motif, while the VR2 loop undergoes an approximately 60° flip to remodel the substrate-binding pocket. These changes, together with a hydrogen-bonding network between catalytic His1095 and ubiquitin Gly76, enable precise substrate positioning and efficient cleavage. This study provides a theoretical framework for understanding the catalytic mechanism of type V CI factor DUBs and their role in CI.