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Interaction of intramembrane metalloprotease SpoIVFB with substrate Pro-σK

  1. Lee Kroosa,2
  1. aDepartment of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824
  1. Edited by Richard Losick, Harvard University, Cambridge, MA, and approved November 3, 2017 (received for review June 26, 2017)

Significance

Most proteases catalyze peptide bond hydrolysis of substrate proteins in aqueous environments. Intramembrane proteases (IPs) are unusual, cleaving substrates in hydrophobic cellular membranes. IPs regulate many processes that impact health, but potential benefits of manipulating IP activities remain elusive due to insufficient knowledge about how IPs interact with substrates. We report experimental and modeling results that illuminate how intramembrane metalloprotease SpoIVFB interacts with its substrate Pro-σK. A 26-residue linker between two domains of SpoIVFB is crucial, perhaps allowing an ATP-induced conformational change to position Pro-σK for cleavage. SpoIVFB and Pro-σK are broadly conserved in endospore-forming bacteria. Endospores are highly resistant cells that promote persistence of some important human pathogens. The work may lead to new strategies to control endospore formation.

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 Bacillus subtilis IP SpoIVFB with its substrate Pro-σK 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-σK. Chemical cross-linking and mass spectrometry of purified, inactive SpoIVFB–Pro-σK 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-σK loops into the membrane domain of SpoIVFB, and the rest of Pro-σK 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-σK cleavage by the membrane domain.

Footnotes

  • ?1Present address: Analytical Development Department, Stelis Biopharma Pvt. Ltd., Bangalore, Karnataka, 560 105, India.

  • ?2To whom correspondence should be addressed. Email: kroos{at}msu.edu.
  • Author contributions: S.H., D.P., M.F., and L.K. designed research; S.H., D.P., D.W., and M.F. performed research; D.W. and M.F. contributed new reagents/analytic tools; S.H., D.P., D.W., M.F., and L.K. analyzed data; and S.H., D.P., D.W., M.F., and L.K. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • This article contains supporting information online at www.danielhellerman.com/lookup/suppl/doi:10.1073/pnas.1711467114/-/DCSupplemental.

Published under the PNAS license.

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