Residues in Conserved Loops of Intramembrane Metalloprotease SpoIVFB Interact with Residues near the Cleavage Site in Pro-σ ^K

Abstract

bIntramembrane metalloproteases (IMMPs) control critical biological processes by cleaving membrane-associated proteins within a transmembrane segment or at a site near the membrane surface. Phylogenetic analysis divides IMMPs into four groups. SpoIVFB is a group III IMMP that regulates Bacillus subtilis endospore formation by cleaving Pro-K and releasing the active sigma factor from a membrane. To elucidate the enzyme-substrate interaction, single-cysteine versions of catalytically inactive SpoIVFB and C-terminally truncated Pro-K (1-126) (which can be cleaved by active SpoIVFB) were coexpressed in Escherichia coli, and proximity was tested by disulfide cross-linking in vivo. As expected, the results provided evidence that catalytic residue Glu-44 of SpoIVFB is near the cleavage site in the substrate. Also near the cleavage site were two residues of SpoIVFB in predicted conserved loops; Pro-135 in a short loop and Val-70 in a longer loop. Pro-135 corresponds to Pro-399 of RseP, a group I IMMP, and Pro-399 was reported previously to interact with substrate near the cleavage site, suggesting a conserved interaction across IMMP subfamilies. Val-70 follows a newly recognized conserved motif, PXGG (X is a large hydrophobic residue), which is in a hydrophobic region predicted to be a membrane reentrant loop. Following the hydrophobic region is a negatively charged region that is conserved in IMMPs of groups I and III. At least two residues with a negatively charged side chain are required in this region for activity of SpoIVFB. The region exhibits other features in IMMPs of groups II and IV. Its possible roles, as well as that of the short loop, are discussed. New insights into IMMP-substrate interaction build toward understanding how IMMPs function and may facilitate manipulation of their activity.

Involvement of a conserved GFG motif region in substrate binding by RseP, an Escherichia coliS2P protease

“…The observation that Y69C in the MRE β‐loop can be crosslinked to at least two different positions in the TM segment of RseA suggests a flexible nature for the RseP–substrate interaction, as suggested previously (Koide et al ., ; Zhang et al ., ). Obviously structural analysis of the RseP–substrate complex will be needed to understand the molecular details of the interaction between RseP and a substrate.…”

“…Structural analysis of mjS2P, an archaeal S2P homolog, showed that it has a b-hairpin-like-loop inserted from the cytoplasmic side into the membrane domain in the vicinity of the proteolytic active site (Feng et al, 2007). This bhairpin-like-loop is conserved among Group I and III S2P homologs, including E. coli RseP and B. subtilis SpoIVFB (Zhang et al, 2013;Akiyama et al, 2015). A study on SpoIVFB demonstrated that its b-hairpin-like-loop could be crosslinked with a substrate (Zhang et al, 2013).…”

“…This bhairpin-like-loop is conserved among Group I and III S2P homologs, including E. coli RseP and B. subtilis SpoIVFB (Zhang et al, 2013;Akiyama et al, 2015). A study on SpoIVFB demonstrated that its b-hairpin-like-loop could be crosslinked with a substrate (Zhang et al, 2013). We named this conserved region MRE b-loop (membranereentrant b-loop) and analyzed its function (Akiyama et al, 2015).…”

Bacillus subtilis Intramembrane Protease RasP Activity in Escherichia coli and In Vitro

“…S1C) is nonphysiological. SpoIVFB appears to have three short (3-to 8-residue) periplasmic loops (21) and BofA appears to have a short (31-residue) C-terminal periplasmic domain (27), based on analysis of PhoA fusion proteins expressed in E. coli and based on a homology model of SpoIVFB (28). In contrast, SpoIVFA appears to have a long (173-residue) C-terminal extracytoplasmic domain (21,29) and resisted cleavage by 2TM-RasP (Fig.…”

“…1. Here, some additional features of RasP are depicted, including the HELGH motif with its catalytic E21 residue (9), a putative membrane-reentrant ␤-loop between TMS 1 and TMS 2 (28,50), a PSD-95/Dgl/ZO-1 (PDZ) domain between TMS 2 and TMS 3 (30), and a short loop interrupting TMS 3 that is predicted on the basis of the structure of a Methanocaldococcus jannaschii IMMP (51) and is followed by a conserved DG sequence whose D residue together with the two H residues of the HELGH motif are predicted to coordinate a zinc ion (30). The topology of RsiW was determined in B. subtilis (9) and is predicted to be the same in E. coli.…”