Interaction of intramembrane metalloprotease SpoIVFB with substrate Pro-σ^K.

Abstract

Intramembrane proteases (IPs) cleave membrane-associated substrates in nearly all organisms and regulate diverse processes. A better understanding of how these enzymes interact with their substrates is necessary for rational design of IP modulators. We show that interaction of <i>Bacillus subtilis</i> IP SpoIVFB with its substrate Pro-σ^K depends on particular residues in the interdomain linker of SpoIVFB. The linker plus either the N-terminal membrane domain or the C-terminal cystathione-β-synthase (CBS) domain of SpoIVFB was sufficient for the interaction but not for cleavage of Pro-σ^K Chemical cross-linking and mass spectrometry of purified, inactive SpoIVFB-Pro-σ^K complex indicated residues of the two proteins in proximity. A structural model of the complex was built via partial homology and by using constraints based on cross-linking data. In the model, the Proregion of Pro-σ^K loops into the membrane domain of SpoIVFB, and the rest of Pro-σ^K interacts extensively with the linker and the CBS domain of SpoIVFB. The extensive interaction is proposed to allow coordination between ATP binding by the CBS domain and Pro-σ^K cleavage by the membrane domain.