An Internal Water-Retention Site in the Rhomboid Intramembrane Protease GlpG Ensures Catalytic Efficiency

Zhou, Yanzi; Moin, Syed M.; Urban, Siniša; Zhang, Yingkai

doi:10.1016/j.str.2012.04.022

Cited by 37 publications

(71 citation statements)

References 31 publications

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“…The exact order of events, and what triggers each step, remain speculative. Membrane thinning surrounding GlpG as observed in molecular dynamics simulations is illustrated (Bondar et al, 2009; Zhou et al, 2012), although its functional consequence remains unclear. Structures 2IC8 (closed GlpG), 2NRF (open GlpG), 1MOX (Spitz-EGF), and 2TGF (TGFα-EGF) were used to diagram the model. DOI: http://dx.doi.org/10.7554/eLife.00173.016…”

Section: Discussionmentioning

confidence: 94%

Membrane immersion allows rhomboid proteases to achieve specificity by reading transmembrane segment dynamics

Moin

Urban

2012

eLife

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119

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Rhomboid proteases reside within cellular membranes, but the advantage of this unusual environment is unclear. We discovered membrane immersion allows substrates to be identified in a fundamentally-different way, based initially upon exposing ‘masked’ conformational dynamics of transmembrane segments rather than sequence-specific binding. EPR and CD spectroscopy revealed that the membrane restrains rhomboid gate and substrate conformation to limit proteolysis. True substrates evolved intrinsically-unstable transmembrane helices that both become unstructured when not supported by the membrane, and facilitate partitioning into the hydrophilic, active-site environment. Accordingly, manipulating substrate and gate dynamics in living cells shifted cleavage sites in a manner incompatible with extended sequence binding, but correlated with a membrane-and-helix-exit propensity scale. Moreover, cleavage of diverse non-substrates was provoked by single-residue changes that destabilize transmembrane helices. Membrane immersion thus bestows rhomboid proteases with the ability to identify substrates primarily based on reading their intrinsic transmembrane dynamics.DOI: http://dx.doi.org/10.7554/eLife.00173.001

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Section: Discussionmentioning

confidence: 94%

Membrane immersion allows rhomboid proteases to achieve specificity by reading transmembrane segment dynamics

Moin

Urban

2012

eLife

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119

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“…The nonnative region contains many residues on the "front face" of GlpG, a region that likely needs to be stable to counterbalance the more flexible active face. Loops 1-3 (the nonnative region) have many packing interactions with GlpG's internal core, whereas loops 4-5 (outside the nonnative region) line the flexible TM5 and are dynamic in molecular dynamics simulations (40). However, there is not a simple stabilization pattern: one of the critical stabilizing residues in GlpG found from Baker and Urban's heat-mapping study (28) is found in the folding nucleus (E166) and another in the nonnative region (R137).…”

Section: An Extensive Nonnative Region Of Folding: Restrictions In Tomentioning

confidence: 99%

Cooperative folding of a polytopic α-helical membrane protein involves a compact N-terminal nucleus and nonnative loops

Paslawski

Lillelund

Kristensen

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Despite the ubiquity of helical membrane proteins in nature and their pharmacological importance, the mechanisms guiding their folding remain unclear. We performed kinetic folding and unfolding experiments on 69 mutants (engineered every 2-3 residues throughout the 178-residue transmembrane domain) of GlpG, a membraneembedded rhomboid protease from Escherichia coli. The only clustering of significantly positive ϕ-values occurs at the cytosolic termini of transmembrane helices 1 and 2, which we identify as a compact nucleus. The three loops flanking these helices show a preponderance of negative ϕ-values, which are sometimes taken to be indicative of nonnative interactions in the transition state. Mutations in transmembrane helices 3-6 yielded predominantly ϕ-values near zero, indicating that this part of the protein has denaturedstate-level structure in the transition state. We propose that loops 1-3 undergo conformational rearrangements to position the folding nucleus correctly, which then drives folding of the rest of the domain. A compact N-terminal nucleus is consistent with the vectorial nature of cotranslational membrane insertion found in vivo. The origin of the interactions in the transition state that lead to a large number of negative ϕ-values remains to be elucidated.GlpG | membrane protein | rhomboid | folding | kinetics T he biologically active structure of a protein is encoded in its sequence, and protein-folding studies aim to elucidate how this native state is reached. Great progress has been made in understanding the mechanisms of folding of water-soluble proteins based on comprehensive protein-engineering studies in combination with computational efforts (1, 2) and application of theoretical models (3-5). Much less is known about the folding mechanisms of membrane proteins that present extra challenges such as low expression levels and the need for a membrane-like environment to fold (6-11). In vivo, α-helical membrane proteins insert into the membrane cotranslationally via the signal recognition particle and Sec-translocon complex (12). Transmembrane helices exit one by one or in pairs into the lipid environment through a lateral gate in the translocon. Folding to the native state occurs spontaneously after helices are inserted into the membrane. To mimic this process, most in vitro membrane protein-folding experiments first denature the protein in SDS; renaturation is then achieved by adding excess nonionic surfactants such as dodecyl maltoside (DDM) (13).A complete protein folding mechanism must include descriptions of the denatured state (D), the native state (N), any metastable intermediates, and the transiently populated transition states (TS) that connect them. TS can only be analyzed indirectly using methods based on kinetic experiments, such as Fersht's ϕ-value approach (14, 15). The ϕ-value is the ratio between the energy perturbation to N (from equilibrium measurements or a combination of folding and unfolding kinetics) and the energy perturbation to TS (from kinetic measurements) ca...

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“…Thus, understanding the molecular mechanism by which ␥-secretase produces A␤ 42 is important for the development of therapeutic agents against AD. ␥-Secretase comprises four transmembrane proteins: presenilin (PS), anterior pharynx-defective 1, Nicastrin, and presenilin enhancer 2 (Pen-2; Takasugi et al, 2003). PS is an aspartic protease subunit that shows proteolytic activity and autoproteolysis to generate Nand C-terminal fragments (NTF and CTF, respectively) on formation of the full complex.…”

Section: Introductionmentioning

confidence: 99%

“…However, unlike PS, mmPSH can catalyze the proteolysis of substrates without additional cofactors, and, furthermore, it is difficult to resolve the crystal structure of multimeric membrane proteins. We previ-ously analyzed the structure of membrane-embedded, proteolytically active PS1 using the substituted cysteine accessibility method (SCAM) and cross-linking experiments (Sato et al, , 2008Takagi et al, 2010). SCAM enables identification of the hydrophilic environment by the accessibility of sulfhydryl reagents to cysteine residues (Cys) introduced at a desired position.…”

Section: Introductionmentioning

confidence: 99%

“…Using a combination of these approaches, we found that PS1 harbors a hydrophilic "catalytic pore" structure formed by TMD1, TMD6, TMD7, and TMD9 in the membrane. In this study, we analyzed the structure of PS1 TMD4 and TMD5, which are thought to play important roles in the binding of PS1 with Pen-2 and stabilization of the complex, respectively (Kim and Sisodia, 2005;Watanabe et al, 2005Watanabe et al, , 2010. We found that both TMD4 and TMD5 face the hydrophilic pore in the membrane and are involved in forming the catalytic site structure of ␥-secretase.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Conformational Changes in Transmembrane Domain 4 of Presenilin 1 Are Associated with Altered Amyloid-β 42 Production

Tominaga¹,

Cai²,

Takagi-Niidome³

et al. 2016

J. Neurosci.

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␥-Secretase is an intramembrane-cleaving protease that produces amyloid-␤ peptide 42 (A␤ 42 ), which is the toxic and aggregation-prone species of A␤ that causes Alzheimer's disease. Here, we used the substituted cysteine accessibility method to analyze the structure of transmembrane domains (TMDs) 4 and 5 of human presenilin 1 (PS1), a catalytic subunit of ␥-secretase. We revealed that TMD4 and TMD5 face the intramembranous hydrophilic milieu together with TMD1, TMD6, TMD7, and TMD9 of PS1 to form the catalytic pore structure. Notably, we found a correlation in the distance between the cytosolic sides of TMD4/TMD7 and A␤ 42 production levels, suggesting that allosteric conformational changes of the cytosolic side of TMD4 affect A␤ 42 -generating ␥-secretase activity. Our results provide new insights into the relationship between the structure and activity of human PS1.

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An Internal Water-Retention Site in the Rhomboid Intramembrane Protease GlpG Ensures Catalytic Efficiency

Cited by 37 publications

References 31 publications

Membrane immersion allows rhomboid proteases to achieve specificity by reading transmembrane segment dynamics

Membrane immersion allows rhomboid proteases to achieve specificity by reading transmembrane segment dynamics

Cooperative folding of a polytopic α-helical membrane protein involves a compact N-terminal nucleus and nonnative loops

Conformational Changes in Transmembrane Domain 4 of Presenilin 1 Are Associated with Altered Amyloid-β 42 Production

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