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-K during Bacillus subtilis endospore formation. When catalytically inactive SpoIVFB was coexpressed with C-terminally truncated Pro-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 K (21-126) parts contributed to improving SpoIVFB solubilization from membranes, but only the 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-K (1-126) and for normal interaction with the substrate. The inactive SpoIVFB⅐Pro-K (1-126)-His 6 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 SpoIVFB⅐Pro-K (1-126)-His 6 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 enzyme⅐substrate complex and future structural studies.Many critical cellular processes are regulated by intramembrane proteolysis (1). Intramembrane proteases (IPs) 3 cleave their substrates within a transmembrane segment (TMS) or near the membrane surface. There are three classes of IPs: rhomboids, aspartyl IPs, and IMMPs (often called site-2 proteases or S2Ps). Rhomboids are serine IPs that promote animal cellular signaling, coordinate bacterial quorum sensing, regulate mitochondrial homeostasis, and control protozoan infection (2-5). Presenilin, an aspartyl IP, is the catalytic component of ␥-secretase, which is involved in the processing of the amyloid precursor protein, Notch, and many other subtrates (6, 7). Dysfunction of ␥-secretase contributes to the pathogenesis of Alzheimer disease (8) and many other diseases (7). Aspartyl IPs also include preflagellin and prepilin peptidases involved in bacterial pathogenesis (9), and signal peptide peptidases, which facilitate the clearance of signal peptides from membranes, participate in viral infection, and generate small peptides as signal molecules for immune systems (10, 11). IMMPs also play critical roles in a wide variety of biological functions. In eukaryotes, cholesterol metabolism, the unfolded protein response, and the acute-phase response are regulated by IMMPs (1, 12, 13). In bacteria, IMMPs control sporulation, envelope stress responses, mating signal production, polar morphogenesis, virulence, ...

Interaction of intramembrane metalloprotease SpoIVFB with substrate Pro-σ K

“…The cross-linking results supported the model of SpoIVFB and provided initial insight into its interaction with Pro-σ K (1-127) (25). Biochemical approaches showed that the SpoIVFB-Pro-σ K (1-127) complex can be solubilized from E. coli membranes with mild detergents and purified (26). Disulfide cross-linking of purified complex containing single-Cys versions of the two proteins suggested that it resembles the complex formed in vivo.…”

“…Side chains of residues in the two regions may directly interact, although the Ala substitutions could subtly change SpoIVFB structure and indirectly perturb other interactions with Pro-σ K . Disulfide cross-linking of single-Cys versions of SpoIVFB and Pro-σ K , both in E. coli (25) and in vitro (26) in the presence or absence of ATP, should further elucidate how the SpoIVFB linker mediates ATP-dependent coordination between the CBS and membrane domains.…”

“…CBS domains adopt a variety of arrangements in different proteins (19,36). SpoIVFB is tetrameric with one CBS domain per monomer (18,26). In our model, the four CBS domains are arranged as an "antiparallel CBS module" (36) in which the A and D chain CBS domain pair is head-to-tail relative to the C and B chain CBS domain pair (Fig.…”

“…A tetrameric model of SpoIVFB with two bound Pro-σ K (i.e., residues 1-127) was built in stages using a combination of homology modeling and simulationbased assembly contingent on constraints derived from crosslinking experiments (see SI Appendix for details). Briefly, the N-terminal membrane domain of SpoIVFB was modeled using the structure of mjS2P (27), with two subunits of the SpoIVFB tetramer in the open conformation (chains A and C) presumed to bind Pro-σ K (chains X and Y) and two subunits in the closed conformation (chains B and D) presumed unable to bind Pro-σ K , consistent with the observed 4:2 SpoIVFB-Pro-σ K complex (26). The C-terminal CBS domain of SpoIVFB was modeled using the Thermotoga maritima TM0935 CBS tetramer structure (30).…”

“…This approach facilitated mutational studies that showed the C-terminal half of Pro-σ K is dispensable for cleavage (22) and revealed the preferences of SpoIVFB for residues in Pro-σ K (1-127) (i.e., the N-terminal half) near the cleavage site (23). [Note: Pro-σ K (1-127) has been called Pro-σ K (1-126) previously (18,(21)(22)(23)(24)(25)(26), but mass spectrometry data suggest that the first of two adjacent potential start codons is used (18,26), adding one residue at the N-terminal end. We adopt the revised numbering of Pro-σ K herein.]…”