title: Complex Formed between Intramembrane Metalloprotease SpoIVFB and Its Substrate, Pro-σK.
abstract: Intramembrane metalloproteases (IMMPs) are conserved from bacteria to humans and control many important signaling pathways, but little is known about how IMMPs interact with their substrates. SpoIVFB is an IMMP that cleaves Pro-σ(K) during Bacillus subtilis endospore formation. When catalytically inactive SpoIVFB was coexpressed with C-terminally truncated Pro-σ(K)(1-126) (which can be cleaved by active SpoIVFB) in Escherichia coli, the substrate dramatically improved solubilization of the enzyme from membranes with mild detergents. Both the Pro(1-20) and σ(K)(21-126) parts contributed to improving SpoIVFB solubilization from membranes, but only the σ(K) part was needed to form a stable complex with SpoIVFB in a pulldown assay. The last 10 residues of SpoIVFB were required for improved solubilization from membranes by Pro-σ(K)(1-126) and for normal interaction with the substrate. The inactive SpoIVFB·Pro-σ(K)(1-126)-His6 complex was stable during affinity purification and gel filtration chromatography. Disulfide cross-linking of the purified complex indicated that it resembled the complex formed in vivo Ion mobility-mass spectrometry analysis resulted in an observed mass consistent with a 4:2 SpoIVFB·Pro-σ(K)(1-126)-His6 complex. Stepwise photobleaching of SpoIVFB fused to a fluorescent protein supported the notion that the enzyme is tetrameric during B. subtilis sporulation. The results provide the first evidence that an IMMP acts as a tetramer, give new insights into how SpoIVFB interacts with its substrate, and lay the foundation for further biochemical analysis of the enzyme·substrate complex and future structural studies.
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824 and.
authors.forename: Yang
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824 and.
authors.forename: Sabyasachi
authors.initials: S
authors.lastname: Halder
authors.affiliation: the Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Kerr
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824 and.
authors.forename: Daniel
authors.initials: D
authors.lastname: Parrell
authors.affiliation: the Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109.
authors.forename: Brandon
authors.initials: B
authors.lastname: Ruotolo
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824 and kroos@msu.edu.
authors.forename: Lee
authors.initials: L
authors.lastname: Kroos
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology* / Q000254:growth & development; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D015151:Immunoblotting; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D011498:Protein Precursors; D010450:Endopeptidases; C071442:spoIVFB protein, Bacillus subtilis
keywords: Bacillus; bacterial signal transduction; enzyme purification; enzyme-substrate interaction; intramembrane proteolysis; mass spectrometry (MS); metalloenzyme; protein complex; sigma factor; sporulation