Substrate binding and specificity of rhomboid intramembrane protease revealed by substrate–peptide complex structures

Zoll, Sebastian; Stanchev, Stancho; Began, Jakub; Škerle, Jan; Lepšı́k, Martin; Peclinovská, Lucie; Majer, Pavel; Střı́šovský, Kvido

doi:10.15252/embj.201489367

Cited by 86 publications

(112 citation statements)

References 55 publications

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“…6). Whether or not TM5 must undergo significant conformational rearrangements in order for full-sized substrates to access the proteolytic site is a matter of some controversy (5,25,34). Our model suggests that the conformation of TM5 is highly dynamic even under folding conditions, which is consistent with the experimental observation that tethering TM5 to TM2 eliminates enzymatic (Fig.…”

Section: Folding Can Be Initiated In Either the N-or C-terminal Domaisupporting

confidence: 85%

“…GlpG's homologs are found across all kingdoms of life. GlpG has been the subject of several biophysical experimental studies aimed toward understanding membrane protein folding and the relationships among protein structure, dynamics, and function (1)(2)(3)(4)(5). An extensive experimental φ-value analysis found φ-values significantly different from zero, indicative of structural changes during the rate-limiting step of folding, in transmembrane helices 1 through 5 (TM1-5) and the intervening loops (4).…”

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confidence: 99%

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Topological constraints and modular structure in the folding and functional motions of GlpG, an intramembrane protease

Schafer

Truong

Otzen

et al. 2016

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

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We investigate the folding of GlpG, an intramembrane protease, using perfectly funneled structure-based models that implicitly account for the absence or presence of the membrane. These two models are used to describe, respectively, folding in detergent micelles and folding within a bilayer, which effectively constrains GlpG's topology in unfolded and partially folded states. Structural free-energy landscape analysis shows that although the presence of multiple folding pathways is an intrinsic property of GlpG's modular functional architecture, the large entropic cost of organizing helical bundles in the absence of the constraining bilayer leads to pathways that backtrack (i.e., local unfolding of previously folded substructures is required when moving from the unfolded to the folded state along the minimum free-energy pathway). This backtracking explains the experimental observation of thermodynamically destabilizing mutations that accelerate GlpG's folding in detergent micelles. In contrast, backtracking is absent from the model when folding is constrained within a bilayer, the environment in which GlpG has evolved to fold. We also characterize a near-native state with a highly mobile transmembrane helix 5 (TM5) that is significantly populated under folding conditions when GlpG is embedded in a bilayer. Unbinding of TM5 from the rest of the structure exposes GlpG's active site, consistent with studies of the catalytic mechanism of GlpG that suggest that TM5 serves as a substrate gate to the active site.membrane proteins | micelle folding | bilayer folding | folding mechanism | intramembrane proteolysis G lpG is a rhomboid protease that sits and functions in the cell membrane. GlpG's homologs are found across all kingdoms of life. GlpG has been the subject of several biophysical experimental studies aimed toward understanding membrane protein folding and the relationships among protein structure, dynamics, and function (1-5). An extensive experimental φ-value analysis found φ-values significantly different from zero, indicative of structural changes during the rate-limiting step of folding, in transmembrane helices 1 through 5 (TM1-5) and the intervening loops (4). Most of the nonzero φ-values, particularly in TM3-5 and in the large loop L1, were negative, meaning that although the corresponding mutation destabilizes the native state, the mutation nonetheless accelerates folding. The preponderance of negative φ-values was puzzling and unprecedented, and at the time, these effects were tentatively ascribed to nonnative interactions in the transition state ensemble. In this work, we show that, in fact, simple models with perfectly funneled energy landscapes that lack nonnative interactions are able to explain the origin of these negative φ-values and how the values arise when folding in detergent micelles rather than bilayers.α-Helical membrane protein folding is thought to occur in two stages in vivo (6). The first stage, setting up the proper topology of transmembrane helices, is handled by the translocon (7,8)...

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Section: Folding Can Be Initiated In Either the N-or C-terminal Domaisupporting

confidence: 85%

mentioning

confidence: 99%

Topological constraints and modular structure in the folding and functional motions of GlpG, an intramembrane protease

Schafer

Truong

Otzen

et al. 2016

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

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“…Crystallography and cryo-EM have given us a look at the structural properties of several IMPs [46,62,64,80,81] and also of multiple inhibitor-rhomboid protease complexes [9,78,[82][83][84][85][86]. However, this has not yet resulted in the design of more potent rhomboid inhibitors.…”

Section: Potency Of Inhibitorsmentioning

confidence: 99%

Intramembrane proteases as drug targets

Verhelst

2017

The FEBS Journal

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Proteases are considered attractive drug targets. Various drugs targeting classical, soluble proteases have been approved for treatment of human disease. Intramembrane proteases (IMPs) are a more recently discovered group of proteolytic enzymes. They are embedded in lipid bilayers and their active sites are located in the plane of a membrane. All four mechanistic families of IMPs have been linked to disease, but currently, no drugs against IMPs have entered the market. In this review, I will outline the function of IMPs with a focus on the ones involved in human disease, which includes Alzheimer's disease, cancer, and infectious diseases by microorganisms. Inhibitors of IMPs are known for all mechanistic classes, but are not yet very potent or selective – aside from those targeting γ‐secretase. I will here describe the different features of IMP inhibitors and discuss a list of issues that need attention in the near future in order to improve the drug development for IMPs.

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“…Structures with several other inhibitors have also been elucidated: with DFP (7) [41], the phosphonate CAPF (8) [65], the submicromolar isocoumarin S016 (3) [37], several N-carbamoylated b-lactams (5) [53] and a tetrapeptide chloromethyl ketone (10) [34]. The similarities between the structures are striking and several interesting observations can be made.…”

Section: Inhibitor-rhomboid Structuresmentioning

confidence: 98%

“…The early observation that TPCK (9) is a weak inhibitor of DmRho-1, urged Zoll et al to synthesize a number of peptidyl chloromethyl ketone analogs that have a longer peptide chain with residues that are based on the substrate specificity of GlpG in the P4eP1 position (10) [34]. These compounds react with GlpG by forming a hemi-ketal with the active site serine followed by alkylation of the active site histidine (Fig.…”

Section: Othersmentioning

confidence: 99%

Inhibitors of rhomboid proteases

Wolf

Verhelst

2016

Biochimie

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Rhomboid proteases form one of the most widespread families of intramembrane proteases. They utilize a catalytic serine-histidine dyad located several Å below the surface of the membrane for substrate hydrolysis. Multiple studies have implicated rhomboid proteases in biologically and medically relevant processes. Several assays have been developed that are able to monitor rhomboid activity. With the aid of these assays, different types of inhibitors have been found, all based on electrophiles that covalently react with the active site machinery. Although the currently available inhibitors have limited selectivity and moderate potency, they can function as research tools and as starting point for the development of activity-based probes, which are reagents that can specifically detect active rhomboid species. Structural studies on complexes of inhibitors with the Escherichia coli rhomboid GlpG have provided insight into how substrate recognition may occur. Future synthetic efforts, aided by high-throughput screening or structure-based design, may lead to more potent and selective inhibitors for this interesting family of proteases.

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Substrate binding and specificity of rhomboid intramembrane protease revealed by substrate–peptide complex structures

Cited by 86 publications

References 55 publications

Topological constraints and modular structure in the folding and functional motions of GlpG, an intramembrane protease

Topological constraints and modular structure in the folding and functional motions of GlpG, an intramembrane protease

Intramembrane proteases as drug targets

Inhibitors of rhomboid proteases

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