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

Takuya Miyake,Yohei Hizukuri,Yoshinori Akiyama

doi:10.3389/fmicb.2020.607381

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 Escherichia coli 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-σE) 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.

Highlights

Intramembrane proteases (IMPs) are a unique class of proteases that exhibit catalytic residues for peptide-bond hydrolysis within the lipid bilayer
We focused on a periplasmic region (named the PDZ carboxyl terminal (PCT) region) located downstream of the PDZ domain of RseP
We showed that H1 forms a peripheral amphiphilic membrane helix that directly interacts with the DegS-cleaved form of RseA

Summary

INTRODUCTION

Intramembrane proteases (IMPs) are a unique class of proteases that exhibit catalytic residues for peptide-bond hydrolysis within the lipid bilayer. TMH1 and TMH3 segments contain a zinc binding motif HExxH and a zinc ligand Asp residue, respectively This family of proteases can be classified into several subgroups: The members of Group I possess one or more PDZ (PSD-95/Dlg/ZO-1) domain(s), which is generally involved in protein-protein interactions in many prokaryotic and eukaryotic proteins, between TMH2 and TMH3, while the homologs from Group II to IV do not (Kinch et al, 2006; Kroos and Akiyama, 2013). A subsequent study showed that the PDZ tandem would act as a size-exclusion filter to prevent the access of intact (full-length) RseA to the recessed active site in the membrane-embedded protease domain (Hizukuri et al, 2014). We propose that the H1 segment acts as an adaptor that mediates both the structural and functional interaction between the PDZ domains and the protease domain of RseP and supports the proper substrate positioning for cleavage

MATERIALS AND METHODS

RESULTS

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DATA AVAILABILITY STATEMENT