Involvement of a Membrane-Bound Amphiphilic Helix in Substrate Discrimination and Binding by an S2P Peptidase RseP

Overview

Journal Front Microbiol

Specialty Microbiology

Date 2020 Dec 17

PMID 33329500

Citations 6

Authors

Takuya Miyake

Yohei Hizukuri

Yoshinori Akiyama

Affiliations

Soon will be listed here.

Abstract

Intramembrane proteases (IMPs) are a unique class of proteases that catalyze the proteolysis within the membrane and regulate diverse cellular processes in various organisms. RseP, an site-2 protease (S2P) family IMP, is involved in the regulation of an extracytoplasmic stress response through the cleavage of membrane-spanning anti-stress-response transcription factor (anti-σ) protein RseA. Extracytoplasmic stresses trigger a sequential cleavage of RseA, in which first DegS cleaves off its periplasmic domain, and RseP catalyzes the second cleavage of RseA. The two tandem-arranged periplasmic PDZ (PDZ tandem) domains of RseP serve as a size-exclusion filter which prevents the access of an intact RseA into the active site of RseP IMP domain. However, RseP's substrate recognition mechanism is not fully understood. Here, we found that a periplasmic region of RseP, located downstream of the PDZ tandem, contains a segment (named H1) predicted to form an amphiphilic helix. Bacterial S2P homologs with various numbers of PDZ domains have a similar amphiphilic helix in the corresponding region. We demonstrated that the H1 segment forms a partially membrane-embedded amphiphilic helix on the periplasmic surface of the membrane. Systematic and random mutagenesis analyses revealed that the H1 helix is important for the stability and proteolytic function of RseP and that mutations in the H1 segment can affect the PDZ-mediated substrate discrimination. Cross-linking experiments suggested that H1 directly interacts with the DegS-cleaved form of RseA. We propose that H1 acts as an adaptor required for proper arrangement of the PDZ tandem domain to perform its filter function and for substrate positioning for its efficient cleavage.

Citing Articles

Structure of mitochondrial pyruvate carrier and its inhibition mechanism.

He Z, Zhang J, Xu Y, Fine E, Suomivuori C, Dror R Nature. 2025; .

PMID: 40044865 DOI: 10.1038/s41586-025-08667-y.

Cryo-EM structure of the bacterial intramembrane metalloprotease RseP in the substrate-bound state.

Asahi K, Hirose M, Aruga R, Shimizu Y, Tajiri M, Tanaka T Sci Adv. 2025; 11(9):eadu0925.

PMID: 40009668 PMC: 11864173. DOI: 10.1126/sciadv.adu0925.

S2P intramembrane protease RseP degrades small membrane proteins and suppresses the cytotoxicity of intrinsic toxin HokB.

Yokoyama T, Yamagata Y, Honna S, Mizuno S, Katagiri S, Oi R mBio. 2023; 14(4):e0108623.

PMID: 37409810 PMC: 10470546. DOI: 10.1128/mbio.01086-23.

In vivo characterization of the bacterial intramembrane-cleaving protease RseP using the heme binding tag-based assay iCliPSpy.

Kupke T, Gotz R, Richter F, Beck R, Lolicato F, Nickel W Commun Biol. 2023; 6(1):287.

PMID: 36934128 PMC: 10024687. DOI: 10.1038/s42003-023-04654-z.

Mechanistic insights into intramembrane proteolysis by site-2 protease homolog RseP.

Imaizumi Y, Takanuki K, Miyake T, Takemoto M, Hirata K, Hirose M Sci Adv. 2022; 8(34):eabp9011.

PMID: 36001659 PMC: 9401612. DOI: 10.1126/sciadv.abp9011.

References

Jones D . Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol. 1999; 292(2):195-202. DOI: 10.1006/jmbi.1999.3091. View

Douchin V, Bohn C, Bouloc P . Down-regulation of porins by a small RNA bypasses the essentiality of the regulated intramembrane proteolysis protease RseP in Escherichia coli. J Biol Chem. 2006; 281(18):12253-9. DOI: 10.1074/jbc.M600819200. View

Maegawa S, Koide K, Ito K, Akiyama Y . The intramembrane active site of GlpG, an E. coli rhomboid protease, is accessible to water and hydrolyses an extramembrane peptide bond of substrates. Mol Microbiol. 2007; 64(2):435-47. DOI: 10.1111/j.1365-2958.2007.05679.x. View

Kreutzberger A, Ji M, Aaron J, Mihaljevic L, Urban S . Rhomboid distorts lipids to break the viscosity-imposed speed limit of membrane diffusion. Science. 2019; 363(6426). PMC: 6368390. DOI: 10.1126/science.aao0076. View

Zoll S, Stanchev S, Began J, Skerle J, Lepsik M, Peclinovska L . Substrate binding and specificity of rhomboid intramembrane protease revealed by substrate-peptide complex structures. EMBO J. 2014; 33(20):2408-21. PMC: 4253528. DOI: 10.15252/embj.201489367. View

Hizukuri Y, Akiyama K, Akiyama Y . Biochemical Characterization of Function and Structure of RseP, an Escherichia coli S2P Protease. Methods Enzymol. 2017; 584:1-33. DOI: 10.1016/bs.mie.2016.09.044. View

Halder S, Parrell D, Whitten D, Feig M, Kroos L . Interaction of intramembrane metalloprotease SpoIVFB with substrate Pro-σ. Proc Natl Acad Sci U S A. 2017; 114(50):E10677-E10686. PMC: 5740621. DOI: 10.1073/pnas.1711467114. View

Kornilova A, Bihel F, Das C, Wolfe M . The initial substrate-binding site of gamma-secretase is located on presenilin near the active site. Proc Natl Acad Sci U S A. 2005; 102(9):3230-5. PMC: 552920. DOI: 10.1073/pnas.0407640102. View

Fukumori A, Steiner H . Substrate recruitment of γ-secretase and mechanism of clinical presenilin mutations revealed by photoaffinity mapping. EMBO J. 2016; 35(15):1628-43. PMC: 4883025. DOI: 10.15252/embj.201694151. View

10.

Beard H, Barniol-Xicota M, Yang J, Verhelst S . Discovery of Cellular Roles of Intramembrane Proteases. ACS Chem Biol. 2019; 14(11):2372-2388. DOI: 10.1021/acschembio.9b00404. View

11.

Liu L, Deber C . Uncoupling hydrophobicity and helicity in transmembrane segments. Alpha-helical propensities of the amino acids in non-polar environments. J Biol Chem. 1998; 273(37):23645-8. DOI: 10.1074/jbc.273.37.23645. View

12.

Shokhen M, Albeck A . How does the exosite of rhomboid protease affect substrate processing and inhibition?. Protein Sci. 2017; 26(12):2355-2366. PMC: 5699488. DOI: 10.1002/pro.3294. View

13.

Koide K, Ito K, Akiyama Y . Substrate recognition and binding by RseP, an Escherichia coli intramembrane protease. J Biol Chem. 2008; 283(15):9562-70. DOI: 10.1074/jbc.M709984200. View

14.

Alba B, Zhong H, Pelayo J, Gross C . degS (hhoB) is an essential Escherichia coli gene whose indispensable function is to provide sigma (E) activity. Mol Microbiol. 2001; 40(6):1323-33. DOI: 10.1046/j.1365-2958.2001.02475.x. View

15.

Hizukuri Y, Akiyama Y . PDZ domains of RseP are not essential for sequential cleavage of RseA or stress-induced σ(E) activation in vivo. Mol Microbiol. 2012; 86(5):1232-45. DOI: 10.1111/mmi.12053. View

16.

Strisovsky K, Sharpe H, Freeman M . Sequence-specific intramembrane proteolysis: identification of a recognition motif in rhomboid substrates. Mol Cell. 2010; 36(6):1048-59. PMC: 2941825. DOI: 10.1016/j.molcel.2009.11.006. View

17.

Koide K, Maegawa S, Ito K, Akiyama Y . Environment of the active site region of RseP, an Escherichia coli regulated intramembrane proteolysis protease, assessed by site-directed cysteine alkylation. J Biol Chem. 2006; 282(7):4553-4560. DOI: 10.1074/jbc.M607339200. View

18.

Gautier R, Douguet D, Antonny B, Drin G . HELIQUEST: a web server to screen sequences with specific alpha-helical properties. Bioinformatics. 2008; 24(18):2101-2. DOI: 10.1093/bioinformatics/btn392. View

19.

Schneider J, Glickman M . Function of site-2 proteases in bacteria and bacterial pathogens. Biochim Biophys Acta. 2013; 1828(12):2808-14. PMC: 4097180. DOI: 10.1016/j.bbamem.2013.04.019. View

20.

Grigorova I, Chaba R, Zhong H, Alba B, Rhodius V, Herman C . Fine-tuning of the Escherichia coli sigmaE envelope stress response relies on multiple mechanisms to inhibit signal-independent proteolysis of the transmembrane anti-sigma factor, RseA. Genes Dev. 2004; 18(21):2686-97. PMC: 525548. DOI: 10.1101/gad.1238604. View