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Sinisa Urban - One of the best experts on this subject based on the ideXlab platform.
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Decoding the Functional Evolution of an Intramembrane Protease Superfamily by Statistical Coupling Analysis.
Structure (London England : 1993), 2020Co-Authors: Ljubica Mihaljević, Sinisa UrbanAbstract:How evolution endowed membrane enzymes with specific abilities, and then tuned them to the needs of different cells, is poorly understood. We examined whether statistical coupling analysis (SCA) can be applied to rhomboid Proteases, the most widely distributed membrane proteins, to identify amino acid "sectors" that evolved independently to acquire a specific function. SCA revealed three coevolving residue networks that form two sectors. Sector 1 determines substrate specificity, but is paradoxically scattered across the protein, consistent with dynamics driving rhomboid-substrate interactions. Sector 2 is hierarchically composed of a subgroup that maintains the catalytic site, and another that maintains the overall fold, forecasting evolution of rhomboid pseudoProteases. Changing only sector 1 residues of a "recipient" rhomboid converted its substrate specificity and catalytic efficiency to that of the "donor." While used only twice over a decade ago, SCA should be generally applicable to membrane proteins, and our sector grafting approach provides an efficient strategy for designing enzymes.
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cytosolic extensions directly regulate a rhomboid Protease by modulating substrate gating
Nature, 2015Co-Authors: Rosanna P Baker, Sinisa UrbanAbstract:Calcium potently stimulates proteolysis by endogenous rhomboid-4, an Intramembrane Protease that contains a cytoplasmic calcium-binding EF-hand domain. The active sites of Intramembrane Proteases are deep in the middle of the lipid bilayer. This means that the substrate, which is a transmembrane domain of an integral membrane protein, needs to overcome energetic/entropic barriers to leave the hydrophobic environment of the cellular membrane and enter the active site of the Protease. In this manuscript, Rosanna P. Baker and Sinisa Urban show that addition of calcium leads to the activation of rhomboid-4, an Intramembrane Protease that contains an EF-hand domain helix-loop-helix structural domain, a feature found in in a large family of calcium-binding proteins. They determined that the relevant calcium-binding site is formed by a pair of cytoplasmic loops and showed that calcium increases the catalytic rate of this Protease by triggering the opening of the lateral substrate gate within the membrane. Intramembrane Proteases catalyse the signal-generating step of various cell signalling pathways, and continue to be implicated in diseases ranging from malaria infection to Parkinsonian neurodegeneration1,2,3. Despite playing such decisive roles, it remains unclear whether or how these membrane-immersed enzymes might be regulated directly. To address this limitation, here we focus on Intramembrane Proteases containing domains known to exert regulatory functions in other contexts, and characterize a rhomboid Protease that harbours calcium-binding EF-hands. We find calcium potently stimulates proteolysis by endogenous rhomboid-4 in Drosophila cells, and, remarkably, when rhomboid-4 is purified and reconstituted in liposomes. Interestingly, deleting the amino-terminal EF-hands activates proteolysis prematurely, while residues in cytoplasmic loops connecting distal transmembrane segments mediate calcium stimulation. Rhomboid regulation is not orchestrated by either dimerization or substrate interactions. Instead, calcium increases catalytic rate by promoting substrate gating. Substrates with cleavage sites outside the membrane can be cleaved but lose the capacity to be regulated. These observations indicate substrate gating is not an essential step in catalysis, but instead evolved as a mechanism for regulating proteolysis inside the membrane. Moreover, these insights provide new approaches for studying rhomboid functions by investigating upstream inputs that trigger proteolysis.
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Mechanisms and cellular functions of Intramembrane Proteases.
Biochimica et biophysica acta, 2013Co-Authors: Sinisa UrbanAbstract:The turn of the millennium coincided with the branding of a fundamentally different class of enzyme - Proteases that reside immersed inside the membrane. This new field was the convergence of completely separate lines of research focused on cholesterol homeostasis, Alzheimer's disease, and developmental genetics. None intended their ultimate path, but soon became a richly-integrated fabric for an entirely new field: regulated Intramembrane proteolysis. Our aim in this Special Issue is to focus on the ancient and nearly ubiquitous enzymes that catalyze this unexpected yet important reaction. The pace of progress has been dramatic, resulting in a rapidly-expanding universe of known cellular functions, and a paradigm shift in the biochemical understanding of these once heretical enzymes. More recently, the first therapeutic successes have been attained by targeting an Intramembrane Protease. We consider these advances and identify oncoming opportunities in four parts: growing spectra of cellular roles, insights into biochemical mechanisms, therapeutic strategies, and newly-emerging topics. Recent studies also expose challenges for the future, including non-linear relationships between substrate identification and physiological functions, and the need for potent and specific, not broad-class, inhibitors.
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Architectural and thermodynamic principles underlying Intramembrane Protease function
Nature Chemical Biology, 2012Co-Authors: Rosanna P Baker, Sinisa UrbanAbstract:Intramembrane Proteases hydrolyze peptide bonds within the membrane as a signaling paradigm universal to all life forms and with implications in disease. Deciphering the architectural strategies supporting Intramembrane proteolysis is an essential but unattained goal. We integrated new, quantitative and high-throughput thermal light-scattering technology, reversible equilibrium unfolding and refolding and quantitative Protease assays to interrogate rhomboid architecture with 151 purified variants. Rhomboid Proteases maintain low intrinsic thermodynamic stability (Δ G = 2.1–4.5 kcal mol^−1) resulting from a multitude of generally weak transmembrane packing interactions, making them highly responsive to their environment. Stability is consolidated by two buried glycines and several packing leucines, with a few multifaceted hydrogen bonds strategically deployed to two peripheral regions. Opposite these regions lie transmembrane segment 5 and connected loops that are notably exempt of structural responsibility, suggesting Intramembrane proteolysis involves considerable but localized protein dynamics. Our analyses provide a comprehensive 'heat map' of the physiochemical anatomy underlying membrane-immersed enzyme function at, what is to our knowledge, unprecedented resolution. Application of a new thermal light scattering technique to quantitatively analyze nearly 150 mutants of the rhomboid Intramembrane Protease GlpG, coupled with thermodynamic measurements and Protease assays, reveals how interactions throughout the molecule collaborate to support enzyme structure and function.
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Architectural and thermodynamic principles underlying Intramembrane Protease function
Nature chemical biology, 2012Co-Authors: Rosanna P Baker, Sinisa UrbanAbstract:Application of a new thermal light scattering technique to quantitatively analyze nearly 150 mutants of the rhomboid Intramembrane Protease GlpG, coupled with thermodynamic measurements and Protease assays, reveals how interactions throughout the molecule collaborate to support enzyme structure and function.
Rosanna P Baker - One of the best experts on this subject based on the ideXlab platform.
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cytosolic extensions directly regulate a rhomboid Protease by modulating substrate gating
Nature, 2015Co-Authors: Rosanna P Baker, Sinisa UrbanAbstract:Calcium potently stimulates proteolysis by endogenous rhomboid-4, an Intramembrane Protease that contains a cytoplasmic calcium-binding EF-hand domain. The active sites of Intramembrane Proteases are deep in the middle of the lipid bilayer. This means that the substrate, which is a transmembrane domain of an integral membrane protein, needs to overcome energetic/entropic barriers to leave the hydrophobic environment of the cellular membrane and enter the active site of the Protease. In this manuscript, Rosanna P. Baker and Sinisa Urban show that addition of calcium leads to the activation of rhomboid-4, an Intramembrane Protease that contains an EF-hand domain helix-loop-helix structural domain, a feature found in in a large family of calcium-binding proteins. They determined that the relevant calcium-binding site is formed by a pair of cytoplasmic loops and showed that calcium increases the catalytic rate of this Protease by triggering the opening of the lateral substrate gate within the membrane. Intramembrane Proteases catalyse the signal-generating step of various cell signalling pathways, and continue to be implicated in diseases ranging from malaria infection to Parkinsonian neurodegeneration1,2,3. Despite playing such decisive roles, it remains unclear whether or how these membrane-immersed enzymes might be regulated directly. To address this limitation, here we focus on Intramembrane Proteases containing domains known to exert regulatory functions in other contexts, and characterize a rhomboid Protease that harbours calcium-binding EF-hands. We find calcium potently stimulates proteolysis by endogenous rhomboid-4 in Drosophila cells, and, remarkably, when rhomboid-4 is purified and reconstituted in liposomes. Interestingly, deleting the amino-terminal EF-hands activates proteolysis prematurely, while residues in cytoplasmic loops connecting distal transmembrane segments mediate calcium stimulation. Rhomboid regulation is not orchestrated by either dimerization or substrate interactions. Instead, calcium increases catalytic rate by promoting substrate gating. Substrates with cleavage sites outside the membrane can be cleaved but lose the capacity to be regulated. These observations indicate substrate gating is not an essential step in catalysis, but instead evolved as a mechanism for regulating proteolysis inside the membrane. Moreover, these insights provide new approaches for studying rhomboid functions by investigating upstream inputs that trigger proteolysis.
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Architectural and thermodynamic principles underlying Intramembrane Protease function
Nature Chemical Biology, 2012Co-Authors: Rosanna P Baker, Sinisa UrbanAbstract:Intramembrane Proteases hydrolyze peptide bonds within the membrane as a signaling paradigm universal to all life forms and with implications in disease. Deciphering the architectural strategies supporting Intramembrane proteolysis is an essential but unattained goal. We integrated new, quantitative and high-throughput thermal light-scattering technology, reversible equilibrium unfolding and refolding and quantitative Protease assays to interrogate rhomboid architecture with 151 purified variants. Rhomboid Proteases maintain low intrinsic thermodynamic stability (Δ G = 2.1–4.5 kcal mol^−1) resulting from a multitude of generally weak transmembrane packing interactions, making them highly responsive to their environment. Stability is consolidated by two buried glycines and several packing leucines, with a few multifaceted hydrogen bonds strategically deployed to two peripheral regions. Opposite these regions lie transmembrane segment 5 and connected loops that are notably exempt of structural responsibility, suggesting Intramembrane proteolysis involves considerable but localized protein dynamics. Our analyses provide a comprehensive 'heat map' of the physiochemical anatomy underlying membrane-immersed enzyme function at, what is to our knowledge, unprecedented resolution. Application of a new thermal light scattering technique to quantitatively analyze nearly 150 mutants of the rhomboid Intramembrane Protease GlpG, coupled with thermodynamic measurements and Protease assays, reveals how interactions throughout the molecule collaborate to support enzyme structure and function.
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Architectural and thermodynamic principles underlying Intramembrane Protease function
Nature chemical biology, 2012Co-Authors: Rosanna P Baker, Sinisa UrbanAbstract:Application of a new thermal light scattering technique to quantitatively analyze nearly 150 mutants of the rhomboid Intramembrane Protease GlpG, coupled with thermodynamic measurements and Protease assays, reveals how interactions throughout the molecule collaborate to support enzyme structure and function.
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In vivo analysis reveals substrate-gating mutants of a rhomboid Intramembrane Protease display increased activity in living cells.
Biological chemistry, 2008Co-Authors: Sinisa Urban, Rosanna P BakerAbstract:Intramembrane Proteases hydrolyze peptide bonds within cell membranes. Recent crystal structures revealed that rhomboid Intramembrane Proteases contain a hydrated active site that opens to the outside of the cell, but is protected laterally from membrane lipids by protein segments. Using Escherichia coli rhomboid (GlpG) structures as a guide, we previously took a mutational approach to identify the GlpG gating mechanism that allows substrates to enter the active site laterally from the membrane. Mutations that weaken contacts keeping the gate closed increase enzyme activity and implicate transmembrane segment 5 as the substrate gate. Since these analyses were performed in vitro with pure proteins in detergent micelles, we have now examined GlpG in its natural environment, within the membrane of live E. coli cells. In striking congruity with in vitro analysis, gate-opening mutants in transmembrane segment 5 display up to a 10-fold increase in Protease activity in living cells. Conversely, mutations in other parts of the Protease, including the membrane-inserted L1 loop previously thought to be the gate, decrease enzyme activity. These observations provide evidence for the existence of both closed and open forms of GlpG in cells, and show that inter-conversion between them via substrate gating is rate limiting physiologically.
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enzymatic analysis of a rhomboid Intramembrane Protease implicates transmembrane helix 5 as the lateral substrate gate
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Rosanna P Baker, Keith E. Young, Liang Feng, Sinisa UrbanAbstract:Intramembrane proteolysis is a core regulatory mechanism of cells that raises a biochemical paradox of how hydrolysis of peptide bonds is accomplished within the normally hydrophobic environment of the membrane. Recent high-resolution crystal structures have revealed that rhomboid Proteases contain a catalytic serine recessed into the plane of the membrane, within a hydrophilic cavity that opens to the extracellular face, but protected laterally from membrane lipids by a ring of transmembrane segments. This architecture poses questions about how substrates enter the internal active site laterally from membrane lipid. Because structures are static glimpses of a dynamic enzyme, we have taken a structure–function approach analyzing >40 engineered variants to identify the gating mechanism used by rhomboid Proteases. Importantly, our analyses were conducted with a substrate that we show is cleaved at two Intramembrane sites within the previously defined Spitz substrate motif. Engineered mutants in the L1 loop and active-site region of the GlpG rhomboid Protease suggest an important structural, rather than dynamic, gating function for the L1 loop that was first proposed to be the substrate gate. Conversely, three classes of mutations that promote transmembrane helix 5 displacement away from the Protease core dramatically enhanced enzyme activity 4- to 10-fold. Our functional analyses have identified transmembrane helix 5 movement to gate lateral substrate entry as a rate-limiting step in Intramembrane proteolysis. Moreover, our mutagenesis also underscores the importance of other residue interactions within the enzyme that warrant further scrutiny.
Michael S. Glickman - One of the best experts on this subject based on the ideXlab platform.
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m tuberculosis Intramembrane Protease rip1 controls transcription through three anti sigma factor substrates
Molecular Microbiology, 2010Co-Authors: Joseph G. Sklar, Hideki Makinoshima, Jessica S. Schneider, Michael S. GlickmanAbstract:Regulated Intramembrane proteolysis (RIP) is a mechanism of transmembrane signal transduction that functions through Intramembrane proteolysis of substrates. We previously reported that the RIP metalloProtease Rv2869c (Rip1) is a determinant of Mycobacterium tuberculosis (Mtb) cell envelope composition and virulence, but the substrates of Rip1 were undefined. Here we show that Rip1 cleaves three transmembrane anti-sigma factors: anti-SigK, anti-SigL and anti-SigM, negative regulators of Sigma K, L and M. We show that transcriptional activation of katG in response to phenanthroline requires activation of SigK and SigL by Rip1 cleavage of anti-SigK and anti-SigL. We also demonstrate a Rip1-dependent pathway that activates the genes for the mycolic acid biosynthetic enzyme KasA and the resuscitation promoting factor RpfC, but represses the bacterioferritin encoding gene bfrB. Regulation of these three genes by Rip1 is not reproduced by deletion of Sigma K, L or M, either indicating a requirement for multiple Rip1 substrates or additional arms of the Rip1 pathway. These results identify a branched proteolytic signal transduction system in which a single Intramembrane Protease cleaves three anti-sigma factor substrates to control multiple downstream pathways involved in lipid biosynthesis and defence against oxidative stress.
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M. tuberculosis Intramembrane Protease Rip1 controls transcription through three anti‐sigma factor substrates
Molecular microbiology, 2010Co-Authors: Joseph G. Sklar, Hideki Makinoshima, Jessica S. Schneider, Michael S. GlickmanAbstract:Regulated Intramembrane proteolysis (RIP) is a mechanism of transmembrane signal transduction that functions through Intramembrane proteolysis of substrates. We previously reported that the RIP metalloProtease Rv2869c (Rip1) is a determinant of Mycobacterium tuberculosis (Mtb) cell envelope composition and virulence, but the substrates of Rip1 were undefined. Here we show that Rip1 cleaves three transmembrane anti-sigma factors: anti-SigK, anti-SigL and anti-SigM, negative regulators of Sigma K, L and M. We show that transcriptional activation of katG in response to phenanthroline requires activation of SigK and SigL by Rip1 cleavage of anti-SigK and anti-SigL. We also demonstrate a Rip1-dependent pathway that activates the genes for the mycolic acid biosynthetic enzyme KasA and the resuscitation promoting factor RpfC, but represses the bacterioferritin encoding gene bfrB. Regulation of these three genes by Rip1 is not reproduced by deletion of Sigma K, L or M, either indicating a requirement for multiple Rip1 substrates or additional arms of the Rip1 pathway. These results identify a branched proteolytic signal transduction system in which a single Intramembrane Protease cleaves three anti-sigma factor substrates to control multiple downstream pathways involved in lipid biosynthesis and defence against oxidative stress.
Kvido Strisovsky - One of the best experts on this subject based on the ideXlab platform.
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Conformational surveillance of Orai1 by a rhomboid Intramembrane Protease prevents inappropriate CRAC channel activation
2020Co-Authors: Adam Graham Grieve, Kvido Strisovsky, Yi Chun Yeh, Lucrezia Zarcone, Johannes Breuning, Nicholas Johnson, Marion H. Brown, Anant B. Parekh, Matthew FreemanAbstract:SummaryCalcium influx through plasma membrane calcium release-activated calcium (CRAC) channels, which are formed of hexamers of Orai1, is a potent trigger for many important biological processes, most notably in T cell mediated immunity. Through a bioinformatics-led cell biological screen, we have identified Orai1 as a substrate for the rhomboid Intramembrane Protease, RHBDL2. We show that RHBDL2 prevents stochastic signalling in unstimulated cells through conformational surveillance and cleavage of inappropriately activated Orai1. A conserved, disease-linked proline residue is responsible for RHBDL2 recognising only the active conformation of Orai1, and cleavage by RHBDL2 is required to sharpen switch-like signalling triggered by store-operated calcium entry. Loss of RHBDL2 control of Orai1 causes severe dysregulation of CRAC channel effectors including transcription factor activation, inflammatory cytokine expression and T cell activation. We propose that this seek-and-destroy function may represent an ancient activity of rhomboid Proteases in degrading unwanted signalling proteins.
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rhomboid Intramembrane Protease rhbdl4 triggers er export and non canonical secretion of membrane anchored tgfα
Scientific Reports, 2016Co-Authors: Lina Wunderle, Kvido Strisovsky, Colin Adrain, Julia D Knopf, Nathalie Kuhnle, Aymeric Morle, Beate Hehn, Matthew Freeman, Marius K LembergAbstract:Rhomboid Intramembrane Proteases are the enzymes that release active epidermal growth factor receptor (EGFR) ligands in Drosophila and C. elegans, but little is known about their functions in mammals. Here we show that the mammalian rhomboid Protease RHBDL4 (also known as Rhbdd1) promotes trafficking of several membrane proteins, including the EGFR ligand TGFα, from the endoplasmic reticulum (ER) to the Golgi apparatus, thereby triggering their secretion by extracellular microvesicles. Our data also demonstrate that RHBDL4-dependent trafficking control is regulated by G-protein coupled receptors, suggesting a role for this rhomboid Protease in pathological conditions, including EGFR signaling. We propose that RHBDL4 reorganizes trafficking events within the early secretory pathway in response to GPCR signaling. Our work identifies RHBDL4 as a rheostat that tunes secretion dynamics and abundance of specific membrane protein cargoes.
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substrate binding and specificity of rhomboid Intramembrane Protease revealed by substrate peptide complex structures
The EMBO Journal, 2014Co-Authors: Sebastian Zoll, Jakub Began, Stancho Stanchev, Jan Škerle, Martin Lepšík, Lucie Peclinovská, Pavel Majer, Kvido StrisovskyAbstract:The mechanisms of Intramembrane Proteases are incompletely understood due to the lack of structural data on substrate complexes. To gain insight into substrate binding by rhomboid Proteases, we have synthesised a series of novel peptidyl-chloromethylketone (CMK) inhibitors and analysed their interactions with Escherichia coli rhomboid GlpG enzymologically and structurally. We show that peptidyl-CMKs derived from the natural rhomboid substrate TatA from bacterium Providencia stuartii bind GlpG in a substrate-like manner, and their co-crystal structures with GlpG reveal the S1 to S4 subsites of the Protease. The S1 subsite is prominent and merges into the ‘water retention site’, suggesting intimate interplay between substrate binding, specificity and catalysis. Unexpectedly, the S4 subsite is plastically formed by residues of the L1 loop, an important but hitherto enigmatic feature of the rhomboid fold. We propose that the homologous region of members of the wider rhomboid-like protein superfamily may have similar substrate or client-protein binding function. Finally, using molecular dynamics, we generate a model of the Michaelis complex of the substrate bound in the active site of GlpG.
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Substrate binding and specificity of rhomboid Intramembrane Protease revealed by substrate–peptide complex structures
The EMBO journal, 2014Co-Authors: Sebastian Zoll, Jakub Began, Stancho Stanchev, Jan Škerle, Martin Lepšík, Lucie Peclinovská, Pavel Majer, Kvido StrisovskyAbstract:The mechanisms of Intramembrane Proteases are incompletely understood due to the lack of structural data on substrate complexes. To gain insight into substrate binding by rhomboid Proteases, we have synthesised a series of novel peptidyl-chloromethylketone (CMK) inhibitors and analysed their interactions with Escherichia coli rhomboid GlpG enzymologically and structurally. We show that peptidyl-CMKs derived from the natural rhomboid substrate TatA from bacterium Providencia stuartii bind GlpG in a substrate-like manner, and their co-crystal structures with GlpG reveal the S1 to S4 subsites of the Protease. The S1 subsite is prominent and merges into the ‘water retention site’, suggesting intimate interplay between substrate binding, specificity and catalysis. Unexpectedly, the S4 subsite is plastically formed by residues of the L1 loop, an important but hitherto enigmatic feature of the rhomboid fold. We propose that the homologous region of members of the wider rhomboid-like protein superfamily may have similar substrate or client-protein binding function. Finally, using molecular dynamics, we generate a model of the Michaelis complex of the substrate bound in the active site of GlpG.
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Structural and mechanistic principles of Intramembrane proteolysis – lessons from rhomboids
The FEBS journal, 2013Co-Authors: Kvido StrisovskyAbstract:Intramembrane Proteases cleave membrane proteins in their transmembrane helices to regulate a wide range of biological processes. They catalyse hydrolytic reactions within the hydrophobic environment of lipid membranes where water is normally excluded. How? Do the different classes of Intramembrane Proteases share any mechanistic principles? In this review these questions will be discussed in view of the crystal structures of prokaryotic members of the three known catalytic types of Intramembrane Proteases published over the past 7 years. Rhomboids, the Intramembrane serine Proteases that are the best understood family, will be the initial area of focus, and the principles that have arisen from a number of structural and biochemical studies will be considered. The site-2 metalloProtease and GXGD-type aspartyl Protease structures will then be discussed, with parallels drawn and differences highlighted between these enzymes and the rhomboids. Despite the significant advances achieved so far, to obtain a detailed understanding of the mechanism of any Intramembrane Protease, high-resolution structural information on the substrate–enzyme complex is required. This remains a major challenge for the field.
Adam Lange - One of the best experts on this subject based on the ideXlab platform.
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Structure and Dynamics of the Rhomboid Protease GlpG in Liposomes Studied by Solid-State NMR.
Journal of the American Chemical Society, 2019Co-Authors: Chaowei Shi, Carl Oster, Claudia Bohg, Sascha Lange, Veniamin Chevelkov, Adam LangeAbstract:Rhomboid Proteases are Intramembrane Proteases that hydrolyze substrate peptide bonds within the lipid bilayer and are important for a wide range of biological processes. The bacterial Intramembrane Protease GlpG is one of the model systems for structural investigations of the rhomboid family. Two different models of substrate gating have been proposed, based on crystal structures of GlpG in detergent micelles. Here, we present a detailed investigation of enzymatically active GlpG in a native-like lipid environment using solid-state NMR spectroscopy. Proton-detected experiments confirm the presence of water molecules in the catalytic cavity. A secondary chemical shift analysis indicates a previously unobserved kink in the central part of the gating helix TM5. Dynamics measurements revealed a dynamic hotspot of GlpG at the N-terminal part of TM5 and the adjacent loop L4, indicating that this region is important for gating. In addition, relaxation dispersion experiments suggest that TM5 is in conformational exchange between an open and a closed conformation.
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structure and dynamics of the rhomboid Protease glpg in liposomes studied by solid state nmr
Journal of the American Chemical Society, 2019Co-Authors: Carl Oster, Claudia Bohg, Sascha Lange, Veniamin Chevelkov, Longmei Li, Adam LangeAbstract:Rhomboid Proteases are Intramembrane Proteases that hydrolyze substrate peptide bonds within the lipid bilayer and are important for a wide range of biological processes. The bacterial Intramembrane Protease GlpG is one of the model systems for structural investigations of the rhomboid family. Two different models of substrate gating have been proposed, based on crystal structures of GlpG in detergent micelles. Here, we present a detailed investigation of enzymatically active GlpG in a native-like lipid environment using solid-state NMR spectroscopy. Proton-detected experiments confirm the presence of water molecules in the catalytic cavity. A secondary chemical shift analysis indicates a previously unobserved kink in the central part of the gating helix TM5. Dynamics measurements revealed a dynamic hotspot of GlpG at the N-terminal part of TM5 and the adjacent loop L4, indicating that this region is important for gating. In addition, relaxation dispersion experiments suggest that TM5 is in conformational...