Abstract
Intramembrane proteases function in numerous signaling pathways that impact health, but how their membrane-embedded active sites interact with modulators is poorly understood. We examined inhibition of intramembrane metalloprotease SpoIVFB by proteins BofA and SpoIVFA. We found that BofA residues in and near a predicted transmembrane segment are required for SpoIVFB inhibition, and cross-linking experiments indicated that this transmembrane segment occupies the SpoIVFB active site region. BofA and SpoIVFA neither prevented SpoIVFB from interacting with substrate in co-purification assays nor interfered with cross-linking between the C-terminal regions of substrate and SpoIVFB. However, the inhibitory proteins did interfere with cross-linking between the SpoIVFB active site region and the substrate N-terminal Proregion, which is normally cleaved. We conclude that BofA and SpoIVFA block substrate access to the membrane-embedded active site of SpoIVFB. A structural model was built of SpoIVFB in complex with BofA and parts of SpoIVFA and substrate, using partial homology and constraints from cross-linking and co-evolutionary analyses. The model predicts that conserved BofA residues interact to stabilize a transmembrane segment and a membrane-embedded C-terminal region. SpoIVFA is predicted to bridge the BofA C-terminal region and SpoIVFB, forming a membrane-embedded inhibitory complex. Implications for design of intramembrane metalloprotease inhibitors are discussed.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
The title and abstract have been revised.