Structural studies of a signal peptide in complex with signal peptidase I cytoplasmic domain: The stabilizing effect of membrane‐mimetics on the acquired fold

Bona, Paolo De; Deshmukh, Lalit; Gorbatyuk, Vitaliy; Vinogradova, Olga; Kendall, Debra A.

doi:10.1002/prot.23238

Cited by 9 publications

(4 citation statements)

References 35 publications

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“…The helix-breaking M26P mutation supports our conclusion that lack of co-translational Env signal-peptide cleavage is primarily the result of secondary structure around the cleavage site. NMR studies of E.coli signal peptidase in complex with alkaline phosphatase signal peptide revealed that the cleavage region adopted a poorly structured ‘U-turn’ shape ( De Bona et al, 2012 ). The loop originated from proline in position −5 to the cleavage site confirming the role of proline in separating hydrophobic and C-terminal region of signal peptides by inducing formation of unstructured turns or loops predicted in the literature earlier ( von Heijne, 1983 ; Jain et al, 1994 ).…”

Section: Discussionmentioning

confidence: 99%

Structure and topology around the cleavage site regulate post-translational cleavage of the HIV-1 gp160 signal peptide

Snapp

McCaul

Quandte

et al. 2017

eLife

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Like all other secretory proteins, the HIV-1 envelope glycoprotein gp160 is targeted to the endoplasmic reticulum (ER) by its signal peptide during synthesis. Proper gp160 folding in the ER requires core glycosylation, disulfide-bond formation and proline isomerization. Signal-peptide cleavage occurs only late after gp160 chain termination and is dependent on folding of the soluble subunit gp120 to a near-native conformation. We here detail the mechanism by which co-translational signal-peptide cleavage is prevented. Conserved residues from the signal peptide and residues downstream of the canonical cleavage site form an extended alpha-helix in the ER membrane, which covers the cleavage site, thus preventing cleavage. A point mutation in the signal peptide breaks the alpha helix allowing co-translational cleavage. We demonstrate that postponed cleavage of gp160 enhances functional folding of the molecule. The change to early cleavage results in decreased viral fitness compared to wild-type HIV.

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Section: Discussionmentioning

confidence: 99%

Structure and topology around the cleavage site regulate post-translational cleavage of the HIV-1 gp160 signal peptide

Snapp

McCaul

Quandte

et al. 2017

eLife

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“…Positioned at the N‐terminus of secreted proteins, signal peptides in E. coli must be recognised by either SRP or SecA if they are to be targeted to the translocon. The flexibility of the C‐terminal end of the signal‐peptide, often rich in structure‐disrupting residues such as proline and glycine, was thought to facilitate access by signal peptidases I and II (De Bona et al , 2012). However, Smets et al (2022) demonstrate a novel function for this region in disrupting the folding of downstream regions (Fig 1E).…”

Section: Figure Folding‐dependent Protein Translocation Across the Pl...mentioning

confidence: 99%

Hold the fold: how delayed folding aids protein secretion

McCaul

Braakman

2022

The EMBO Journal

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“…Alternatively, mutating a proline (P71) on the C-terminal side of the cleavage site does not have a significant effect on σ V activity suggesting the proline at position 61 has a role in site-1 processing. It is thought this residue is important for creating a U turn in the structure that creates disorder at positions -5 to -1, thus arranging them to interact with the signal peptidase active site (Auclair et al, 2012;; Bona et al, 2012). It is also thought this residue stops the formation of the helix from the transmembrane domain, which contributes to positioning the signal peptidase cleavage site close to the membrane and signal peptidase (Auclair et al, 2011;; Bona et al, 2012).…”

Section: Analyzing Constitutive Rsiv Mutantsmentioning

confidence: 99%

The activation and response of Bacillus subtilis ECF sigma factor sigma V to lysozyme

Hastie¹

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Extra-cytoplasmic function (ECF) σ factors are a subset of σ factors bacteria use to transcribe specific genes in response to environmental cues. In the absence of an inducing signal, ECF σ factors are inhibited by an anti-σ factor that prevents the ECF σ factor from interacting with RNA polymerase. The ECF σ factor σ V from Bacillus subtilis is activated in response to lysozyme stress. Lysozyme damages bacterial peptidoglycan by cleaving at the 1,4-ß-linkage between N-acetylmuramic acid and Nacetylglucosamine. In the absence of lysozyme, the activity of σ V is inhibited by the antiσ factor RsiV, a single pass transmembrane protein. The two main components of this project have been to elucidate the mechanism of σ V activation and examine how this system senses lysozyme stress. In Chapter 2 we show the activation of σ V is specific to lysozyme and the anti-σ factor RsiV is degraded by a step-wise proteolytic cascade known as Regulated Intramembrane Proteolysis (RIP). In the presence of lysozyme, the extracellular domain of RsiV is removed by cleavage at site-1. Upon removal of the extracellular domain, the site-2 protease, RasP, cleaves RsiV within the membrane. The remainder of RsiV is degraded by cytosolic proteases allowing σ V to interact with RNA polymerase. In response to lysozyme stress σ V activates an O-actyltransferase, OatA, which modifies the peptidoglycan to prevent further lysozyme damage. Our studies in Chapter 3 identifed the protease(s) responsible for site-1 cleavage of RsiV and revealed RsiV directly binds to lysozyme. We determined the cleavage site of the site-1 protease using N-terminal sequencing and demonstrate that disruption of site-1 cleavage blocks σ V activation. Site-1 cleavage occurs at what appears to be a signal peptide cleavage site. We demonstrate that four out of the five signal peptidases from B. subtilis are able to cleave RsiV at site-1 in vitro only in the presence of lysozyme. Additionally, we show that the extracellular domain of RsiV directly binds the inducing substrate lysozyme. v In Chapter 4 we focus on determining if the interaction between RsiV and lysozyme is necessary for σ V activation. Based on the co-crystal structure of RsiV and lysozyme we mutated sveral residues predicted to be involved in binding. One combination of RsiV mutations (S169W, P259A, Y261A) was unable to activate σ V and subsequently was unable to bind lysozyme. We propose a RIP dependent mechanism of σ V activation that is contingent upon the anti-σ factor (RsiV) directly binding the inducing signal (lysozyme) to present the site-1 cleavage site to signal peptidase. The co-crystal structure of RsiV and lysozyme revealed that RsiV interacts with the active site of lysozyme. We demonstrate that purified RsiV inhibits lysozyme activity suggesting RsiV provides an additional lysozyme response mechanism. Thus, the anti-σ factor RsiV senses the presence of lysozyme, activates σ V , and protects against further lysozyme damage. vi

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Structural studies of a signal peptide in complex with signal peptidase I cytoplasmic domain: The stabilizing effect of membrane‐mimetics on the acquired fold

Cited by 9 publications

References 35 publications

Structure and topology around the cleavage site regulate post-translational cleavage of the HIV-1 gp160 signal peptide

Structure and topology around the cleavage site regulate post-translational cleavage of the HIV-1 gp160 signal peptide

Hold the fold: how delayed folding aids protein secretion

The activation and response of Bacillus subtilis ECF sigma factor sigma V to lysozyme

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