Inhibitory proteins block substrate access by occupying the active site cleft of Bacillus subtilis intramembrane protease SpoIVFB
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Curated by eLife
Evaluation Summary:
A member of a large class of metalloproteases with representatives in mammals as well as bacteria, its status as a multipass membrane protein has made illuminating the molecular basis of SpoIVFB inhibition challenging. In this study, Olenic and colleagues combine genetics, cross-linking, and co-evolutionary analysis to develop a structural model of interaction between SpoIVFB and its inhibitors SpoIVFA and BofA. Given the conservation and importance of this family of metalloproteases, this work should have broad impact, influencing our understanding of the regulation of this class of proteins across the tree of life.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)
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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.
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Evaluation Summary:
A member of a large class of metalloproteases with representatives in mammals as well as bacteria, its status as a multipass membrane protein has made illuminating the molecular basis of SpoIVFB inhibition challenging. In this study, Olenic and colleagues combine genetics, cross-linking, and co-evolutionary analysis to develop a structural model of interaction between SpoIVFB and its inhibitors SpoIVFA and BofA. Given the conservation and importance of this family of metalloproteases, this work should have broad impact, influencing our understanding of the regulation of this class of proteins across the tree of life.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and …
Evaluation Summary:
A member of a large class of metalloproteases with representatives in mammals as well as bacteria, its status as a multipass membrane protein has made illuminating the molecular basis of SpoIVFB inhibition challenging. In this study, Olenic and colleagues combine genetics, cross-linking, and co-evolutionary analysis to develop a structural model of interaction between SpoIVFB and its inhibitors SpoIVFA and BofA. Given the conservation and importance of this family of metalloproteases, this work should have broad impact, influencing our understanding of the regulation of this class of proteins across the tree of life.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
This paper by Olenic et al focuses on interaction between the intramembrane metalloprotease SpoIVFB, and its inhibitors, SpoIVFA and BofA. A member of a large class of proteases with representatives in mammals as well as bacteria, its status as a multipass membrane protein has made illuminating the molecular basis of SpoIVFB inhibition challenging. Here, Olenic and colleagues combine genetics, cross-linking, and co-evolutionary bioinformatic analysis to develop a structural model of interaction between SpoIVFB and SpoIVFA and BofA. Overall, I found the experiments appropriate and the results compelling. Given the conservation and importance of this family of metalloproteases, I anticipate this work will be of interest to investigators working outside B. subtilis and should be appealing to a broad audience …
Reviewer #1 (Public Review):
This paper by Olenic et al focuses on interaction between the intramembrane metalloprotease SpoIVFB, and its inhibitors, SpoIVFA and BofA. A member of a large class of proteases with representatives in mammals as well as bacteria, its status as a multipass membrane protein has made illuminating the molecular basis of SpoIVFB inhibition challenging. Here, Olenic and colleagues combine genetics, cross-linking, and co-evolutionary bioinformatic analysis to develop a structural model of interaction between SpoIVFB and SpoIVFA and BofA. Overall, I found the experiments appropriate and the results compelling. Given the conservation and importance of this family of metalloproteases, I anticipate this work will be of interest to investigators working outside B. subtilis and should be appealing to a broad audience (subject to making the manuscript more accessible).
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Reviewer #2 (Public Review):
Interestingly Co-IP experiments suggest that SpoIVFP, SpoIVFA, BofA and Pro-sigmaK are all simultaneously bound in complex.
They use crosslinking studies to show that BofA binds to the active site of SpoIVFB.
The authors provide strong evidence using cross-linking studies that the inhibitory proteins prevent Pro-sigmaK from interacting with the SpoIVFB active site.
The authors also identify mutations in BofA that lose the inhibitory effect of Pro-sigmaK processing. One limitation of this study is the instability of the proteins in the absence of the complex. This makes it difficult to interpret how mutations in one protein affect the stability of that protein.
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Reviewer #3 (Public Review):
Olenic et al describe a very comprehensive investigation of the molecular mechanism of the regulation of the intramembrane metallo protease SpoIVFB from Bacillus subtilis. This protease plays essential roles in regulating the transcription of genes involved in spore coat formation by cleaving and thus releasing the transcriptional co-activator Pro-σK. The authors show that the activity of the enzyme is regulated by complex formation with two other proteins, BofA and SpoIVFA. They use a sophisticated plasmid design to make sure that all three components as well as the substrate are expressed at equivalent levels. Through a combination of functional assays and alanine-scanning they identify three residues in BofA that are important for inhibition, they further show that only the full complex is stable and that …
Reviewer #3 (Public Review):
Olenic et al describe a very comprehensive investigation of the molecular mechanism of the regulation of the intramembrane metallo protease SpoIVFB from Bacillus subtilis. This protease plays essential roles in regulating the transcription of genes involved in spore coat formation by cleaving and thus releasing the transcriptional co-activator Pro-σK. The authors show that the activity of the enzyme is regulated by complex formation with two other proteins, BofA and SpoIVFA. They use a sophisticated plasmid design to make sure that all three components as well as the substrate are expressed at equivalent levels. Through a combination of functional assays and alanine-scanning they identify three residues in BofA that are important for inhibition, they further show that only the full complex is stable and that removal or mutations of one component leads to degradation. By introducing cysteine residues at certain locations and investigating the cross linking between the cysteines they identify an interface between SpoIVFB and BofA and extend the analysis to obtain a model of the inhibitory complex. In this model BofA blocks the access to the active site of the enzyme but without inhibiting the binding of the substrate to a specialized soluble domain of the enzyme.
Overall, the mechanism is an interesting new addition to the field of activity regulation of intramembrane proteolysis and is based on an extensive characterization of the entire system.
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