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Inhibitory proteins block substrate access by occupying the active site cleft of Bacillus subtilis intramembrane protease SpoIVFB.

ABSTRACT: Intramembrane proteases (IPs) function in numerous signaling pathways that impact health, but elucidating the regulation of membrane-embedded proteases is challenging. We examined inhibition of intramembrane metalloprotease SpoIVFB by proteins BofA and SpoIVFA. We found that SpoIVFB inhibition requires BofA residues in and near a predicted transmembrane segment (TMS). This segment of BofA occupies the SpoIVFB active site cleft based on cross-linking experiments. SpoIVFB inhibition also requires SpoIVFA. The inhibitory proteins block access of the substrate N-terminal region to the membrane-embedded SpoIVFB active site, based on additional cross-linking experiments; however, the inhibitory proteins did not prevent interaction between the substrate C-terminal region and the SpoIVFB soluble domain. We built a structural model of SpoIVFB in complex with BofA and parts of SpoIVFA and substrate, using partial homology and constraints from cross-linking and co-evolutionary analyses. The model predicts that conserved BofA residues interact to stabilize a TMS and a membrane-embedded C-terminal region. The model also predicts that SpoIVFA bridges the BofA C-terminal region and SpoIVFB, forming a membrane-embedded inhibition complex. Our results reveal a novel mechanism of IP inhibition with clear implications for relief from inhibition in vivo and design of inhibitors as potential therapeutics.

SUBMITTER: Olenic S

PROVIDER: S-EPMC9042235 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature

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Inhibitory proteins block substrate access by occupying the active site cleft of <i>Bacillus subtilis</i> intramembrane protease SpoIVFB.

Olenic Sandra S Heo Lim L Feig Michael M Kroos Lee L

eLife 20220426

Intramembrane proteases (IPs) function in numerous signaling pathways that impact health, but elucidating the regulation of membrane-embedded proteases is challenging. We examined inhibition of intramembrane metalloprotease SpoIVFB by proteins BofA and SpoIVFA. We found that SpoIVFB inhibition requires BofA residues in and near a predicted transmembrane segment (TMS). This segment of BofA occupies the SpoIVFB active site cleft based on cross-linking experiments. SpoIVFB inhibition also requires ...[more]

PMID: 35471152

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Project description:RasP is a predicted intramembrane metalloprotease of Bacillus subtilis that has been proposed to cleave the stress response anti-sigma factors RsiW and RsiV, the cell division protein FtsL, and remnant signal peptides within their transmembrane segments. To provide evidence for direct effects of RasP on putative substrates, we developed a heterologous coexpression system. Since expression of catalytically inactive RasP E21A inhibited expression of other membrane proteins in Escherichia coli, we added extra transmembrane segments to RasP E21A, which allowed accumulation of most other membrane proteins. A corresponding active version of RasP appeared to promiscuously cleave coexpressed membrane proteins, except those with a large periplasmic domain. However, stable cleavage products were not observed, even in clpP mutant E. coli Fusions of transmembrane segment-containing parts of FtsL and RsiW to E. coli maltose-binding protein (MBP) also resulted in proteins that appeared to be RasP substrates upon coexpression in E. coli, including FtsL with a full-length C-terminal domain (suggesting that prior cleavage by a site 1 protease is unnecessary) and RsiW designed to mimic the PrsW site 1 cleavage product (suggesting that further trimming by extracytoplasmic protease is unnecessary). Purified RasP cleaved His6-MBP-RsiW(73-118) in vitro within the RsiW transmembrane segment based on mass spectrometry analysis, demonstrating that RasP is an intramembrane protease. Surprisingly, purified RasP failed to cleave His6-MBP-FtsL(23-117). We propose that the lack of α-helix-breaking residues in the FtsL transmembrane segment creates a requirement for the membrane environment and/or an additional protein(s) in order for RasP to cleave FtsL.IMPORTANCE Intramembrane proteases govern important signaling pathways in nearly all organisms. In bacteria, they function in stress responses, cell division, pathogenesis, and other processes. Their membrane-associated substrates are typically inferred from genetic studies in the native bacterium. Evidence for direct effects has come sometimes from coexpression of the enzyme and potential substrate in a heterologous host and rarely from biochemical reconstitution of cleavage in vitro We applied these two approaches to the B. subtilis enzyme RasP and its proposed substrates RsiW and FtsL. We discovered potential pitfalls and solutions in heterologous coexpression experiments in E. coli, providing evidence that both substrates are cleaved by RasP in vivo but, surprisingly, that only RsiW was cleaved in vitro, suggesting that FtsL has an additional requirement.

Dataset Information

Inhibitory proteins block substrate access by occupying the active site cleft of Bacillus subtilis intramembrane protease SpoIVFB.

Publications

Inhibitory proteins block substrate access by occupying the active site cleft of <i>Bacillus subtilis</i> intramembrane protease SpoIVFB.

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