title: Features of Pro-σK important for cleavage by SpoIVFB, an intramembrane metalloprotease.
abstract: Intramembrane proteases regulate diverse processes by cleaving substrates within a transmembrane segment or near the membrane surface. Bacillus subtilis SpoIVFB is an intramembrane metalloprotease that cleaves Pro-σ(K) during sporulation. To elucidate features of Pro-σ(K) important for cleavage by SpoIVFB, coexpression of the two proteins in Escherichia coli was used along with cell fractionation. In the absence of SpoIVFB, a portion of the Pro-σ(K) was peripherally membrane associated. This portion was not observed in the presence of SpoIVFB, suggesting that it serves as the substrate. Deletion of Pro-σ(K) residues 2 to 8, addition of residues at its N terminus, or certain single-residue substitutions near the cleavage site impaired cleavage. Certain multiresidue substitutions near the cleavage site changed the position of cleavage, revealing preferences for a small residue preceding the cleavage site N-terminally (i.e., at the P1 position) and a hydrophobic residue at the second position following the cleavage site C-terminally (i.e., P2'). These features appear to be conserved among Pro-σ(K) orthologs. SpoIVFB did not tolerate an aromatic residue at P1 or P2' of Pro-σ(K). A Lys residue at P3' of Pro-σ(K) could not be replaced with Ala unless a Lys was provided farther C-terminally (e.g., at P9'). α-Helix-destabilizing residues near the cleavage site were not crucial for SpoIVFB to cleave Pro-σ(K). The preferences and tolerances of SpoIVFB are somewhat different from those of other intramembrane metalloproteases, perhaps reflecting differences in the interaction of the substrate with the membrane and the enzyme.
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan, USA. Ruanbao.Zhou@sdstate.edu
authors.forename: Ruanbao
authors.initials: R
authors.lastname: Zhou
authors.forename: Kangming
authors.initials: K
authors.lastname: Chen
authors.forename: Xianling
authors.initials: X
authors.lastname: Xiang
authors.forename: Liping
authors.initials: L
authors.lastname: Gu
authors.forename: Lee
authors.initials: L
authors.lastname: Kroos
mesh_terms: D001412:Bacillus subtilis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry; D004252:DNA Mutational Analysis; D010450:Endopeptidases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D055786:Gene Knockout Techniques; D006868:Hydrolysis; D061268:Insulin Lispro; D008565:Membrane Proteins / Q000378:metabolism*; D008958:Models, Molecular; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D061268:Insulin Lispro; D008565:Membrane Proteins; D011498:Protein Precursors; D011994:Recombinant Proteins; D014157:Transcription Factors; C084190:sigma K; D010450:Endopeptidases; C071442:spoIVFB protein, Bacillus subtilis