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• W2967792612 abstract "•Mechanical, kinetic, and energetic properties of PAR1•Properties changing upon binding of an agonist or antagonist•Secondary structures change conformational variability and lifetime•Secondary structures change free energy and mechanical rigidity G protein-coupled receptors (GPCRs) show complex relationships between functional states and conformational plasticity that can be qualitatively and quantitatively described by contouring their free energy landscape. However, how ligands modulate the free energy landscape to direct conformation and function of GPCRs is not entirely understood. Here, we employ single-molecule force spectroscopy to parametrize the free energy landscape of the human protease-activated receptor 1 (PAR1), and delineate the mechanical, kinetic, and energetic properties of PAR1 being set into different functional states. Whereas in the inactive unliganded state PAR1 adopts mechanically rigid and stiff conformations, upon agonist or antagonist binding the receptor mechanically softens, while increasing its conformational flexibility, and kinetic and energetic stability. By mapping the free energy landscape to the PAR1 structure, we observe key structural regions putting this conformational plasticity into effect. Our insight, complemented with previously acquired knowledge on other GPCRs, outlines a more general framework to understand how GPCRs stabilize certain functional states. G protein-coupled receptors (GPCRs) show complex relationships between functional states and conformational plasticity that can be qualitatively and quantitatively described by contouring their free energy landscape. However, how ligands modulate the free energy landscape to direct conformation and function of GPCRs is not entirely understood. Here, we employ single-molecule force spectroscopy to parametrize the free energy landscape of the human protease-activated receptor 1 (PAR1), and delineate the mechanical, kinetic, and energetic properties of PAR1 being set into different functional states. Whereas in the inactive unliganded state PAR1 adopts mechanically rigid and stiff conformations, upon agonist or antagonist binding the receptor mechanically softens, while increasing its conformational flexibility, and kinetic and energetic stability. By mapping the free energy landscape to the PAR1 structure, we observe key structural regions putting this conformational plasticity into effect. Our insight, complemented with previously acquired knowledge on other GPCRs, outlines a more general framework to understand how GPCRs stabilize certain functional states. G protein-coupled receptors (GPCRs) encompass a large family of transmembrane proteins that sense a wide range of extracellular cues including neurotransmitters, peptides, hormones, light, or mechanical forces. Upon sensing the cues, GPCRs trigger specific cellular responses through intracellular signaling pathways (Calebiro and Jobin, 2019Calebiro D. Jobin M.L. Hot spots for GPCR signaling: lessons from single-molecule microscopy.Curr. Opin. Cell Biol. 2019; 57: 57-63Crossref PubMed Scopus (14) Google Scholar, Hilger et al., 2018Hilger D. Masureel M. Kobilka B.K. Structure and dynamics of GPCR signaling complexes.Nat. Struct. Mol. Biol. 2018; 25: 4-12Crossref PubMed Scopus (429) Google Scholar, Palczewski, 2006Palczewski K. G protein-coupled receptor rhodopsin.Annu. Rev. Biochem. 2006; 75: 743-767Crossref PubMed Scopus (559) Google Scholar, Wootten et al., 2018Wootten D. Christopoulos A. Marti-Solano M. Babu M.M. Sexton P.M. Mechanisms of signalling and biased agonism in G protein-coupled receptors.Nat. Rev. Mol. Cell Biol. 2018; 19: 638-653Crossref PubMed Scopus (319) Google Scholar). Being virtually involved in almost every cellular process, the seven transmembrane α-helical receptors regulate numerous physiological processes, making them favored drug targets (Campbell and Smrcka, 2018Campbell A.P. Smrcka A.V. Targeting G protein-coupled receptor signalling by blocking G proteins.Nat. Rev. Drug Discov. 2018; 17: 789-803Crossref PubMed Scopus (77) Google Scholar, Hauser et al., 2017Hauser A.S. Attwood M.M. Rask-Anderson M. Schiöth H.B. Gloriam D.E. Trends in GPCR drug discovery: new agents, targets and indications.Nat. Rev. Drug Discov. 2017; 16: 829-842Crossref PubMed Scopus (1255) Google Scholar). GPCRs are dynamic structures that show a high conformational variability, of which certain conformations induce specific intracellular signaling pathways (Deupi and Kobilka, 2010Deupi X. Kobilka B.K. Energy landscapes as a tool to integrate GPCR structure, dynamics, and function.Physiology (Bethesda). 2010; 25: 293-303Crossref PubMed Scopus (220) Google Scholar, Venkatakrishnan et al., 2013Venkatakrishnan A.J. Deupi X. Lebon G. Tate C.G. Schertler G.F. Babu M.M. Molecular signatures of G-protein-coupled receptors.Nature. 2013; 494: 185-194Crossref PubMed Scopus (1093) Google Scholar, Wingler et al., 2019Wingler L.M. Elgeti M. Hilger D. Latorraca N.R. Lerch M.T. Staus D.P. Dror R.O. Kobilka B.K. Hubbell W.L. Lefkowitz R.J. Angiotensin analogs with divergent bias stabilize distinct receptor conformations.Cell. 2019; 176: 468-478Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). The probability to possess one or a set of specific conformations depends on which ligand binds the GPCR and on cofactors including receptor assembly, lipids, or chemical compounds (Katritch et al., 2013Katritch V. Cherezov V. Stevens R.C. Structure-function of the G protein-coupled receptor superfamily.Annu. Rev. Pharmacol. Toxicol. 2013; 53: 531-556Crossref PubMed Scopus (788) Google Scholar, Smith et al., 2018Smith J.S. Lefkowitz R.J. Rajagopal S. Biased signalling: from simple switches to allosteric microprocessors.Nat. Rev. Drug Discov. 2018; 17: 243-260Crossref PubMed Scopus (377) Google Scholar, Weis and Kobilka, 2018Weis W.I. Kobilka B.K. The molecular basis of G protein-coupled receptor activation.Annu. Rev. Biochem. 2018; 87: 897-919Crossref PubMed Scopus (455) Google Scholar, Zocher et al., 2012bZocher M. Zhang C. Rasmussen S.G. Kobilka B.K. Muller D.J. Cholesterol increases kinetic, energetic, and mechanical stability of the human beta2-adrenergic receptor.Proc. Natl. Acad. Sci. U S A. 2012; 109: E3463-E3472Crossref PubMed Scopus (125) Google Scholar). Although high-resolution structures of GPCRs are the pinnacle of corroborating our present understanding of most functional studies (Hilger et al., 2018Hilger D. Masureel M. Kobilka B.K. Structure and dynamics of GPCR signaling complexes.Nat. Struct. Mol. Biol. 2018; 25: 4-12Crossref PubMed Scopus (429) Google Scholar), static structures only provide insights into a specific stabilized state frozen in time and space (Steyaert and Kobilka, 2011Steyaert J. Kobilka B.K. Nanobody stabilization of G protein-coupled receptor conformational states.Curr. Opin. Struct. Biol. 2011; 21: 567-572Crossref PubMed Scopus (184) Google Scholar) out of the many different dynamic conformations (Deupi and Kobilka, 2010Deupi X. Kobilka B.K. Energy landscapes as a tool to integrate GPCR structure, dynamics, and function.Physiology (Bethesda). 2010; 25: 293-303Crossref PubMed Scopus (220) Google Scholar). Therefore, to understand how the binding of ligands or cofactors can guide GPCRs to adopt certain conformations requires complementary methods (García-Nafría et al., 2018García-Nafría J. Nehmé R. Edwards P.C. Tate C.G. Cryo-EM structure of the serotonin 5-HT1B receptor coupled to heterotrimeric Go.Nature. 2018; 558: 620-623Crossref PubMed Scopus (146) Google Scholar, Maeda and Schertler, 2013Maeda S. Schertler G.F.X. Production of GPCR and GPCR complexes for structure determination.Curr. Opin. Struct. Biol. 2013; 23: 381-392Crossref PubMed Scopus (25) Google Scholar, Manglik et al., 2017Manglik A. Kobilka B.K. Steyaert J. Nanobodies to study G protein-coupled receptor structure and function.Annu. Rev. Pharmacol. Toxicol. 2017; 57: 19-37Crossref PubMed Scopus (147) Google Scholar, Rosenbaum et al., 2007Rosenbaum D.M. Cherezov V. Hanson M.A. Rasmussen S.G. Thian F.S. Kobilka T.S. Choi H.J. Yao X.J. Weis W.I. Stevens R.C. et al.GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function.Science. 2007; 318: 1266-1273Crossref PubMed Scopus (1189) Google Scholar, Serrano-Vega et al., 2008Serrano-Vega M.J. Magnani F. Shibata Y. Tate C.G. Conformational thermostabilization of the β1-adrenergic receptor in a detergent-resistant form.Proc. Natl. Acad. Sci. U S A. 2008; 105: 877-882Crossref PubMed Scopus (364) Google Scholar). Protease-activated receptors (PARs) form a functionally unique and medically important sub-family among GPCRs. PARs are unique because their N-terminal end, after enzymatic cleavage, exposes a tethered peptide sequence that auto-activates the receptor to induce signal transduction pathways. The PAR family consists of four known members PAR1-4, each of which is activated by a specific set of proteases (Adams et al., 2011Adams M.N. Ramachandran R. Yau M.K. Suen J.Y. Fairlie D.P. Hollenberg M.D. Hooper J.D. Structure, function and pathophysiology of protease activated receptors.Pharmacol. Ther. 2011; 130: 248-282Crossref PubMed Scopus (284) Google Scholar, Coughlin, 2000Coughlin S.R. Thrombin signalling and protease-activated receptors.Nature. 2000; 407: 258-264Crossref PubMed Scopus (2137) Google Scholar). Of the PAR members, PAR1 is responsible for hemostasis, thrombosis, and inflammation. Its key role in the cardiovascular, musculoskeletal, gastrointestinal, respiratory, and central nervous systems makes PAR1 a sought-after drug target (Hamilton and Trejo, 2017Hamilton J.R. Trejo J. Challenges and opportunities in protease-activated receptor drug development.Annu. Rev. Pharmacol. Toxicol. 2017; 57: 349-373Crossref PubMed Scopus (42) Google Scholar, Wojtukiewicz et al., 2015Wojtukiewicz M.Z. Hempel D. Sierko E. Tucker S.C. Honn K.V. Protease-activated receptors (PARs)–biology and role in cancer invasion and metastasis.Cancer Metastasis Rev. 2015; 34: 775-796Crossref PubMed Scopus (94) Google Scholar). PAR1 activation is initiated by serine-proteases including thrombin that cleave a part of the receptor’s extracellular N-terminal end. The truncated N-terminal end exposes an SFLLRN peptide sequence, which binds and activates PAR1 (Coughlin, 2000Coughlin S.R. Thrombin signalling and protease-activated receptors.Nature. 2000; 407: 258-264Crossref PubMed Scopus (2137) Google Scholar). This binding of the tethered SFLLRN ligand can be inhibited by an irreversible antagonist, vorapaxar, a US Food and Drug Administration-approved drug for anti-platelet therapy against thrombosis (Hamilton and Trejo, 2017Hamilton J.R. Trejo J. Challenges and opportunities in protease-activated receptor drug development.Annu. Rev. Pharmacol. Toxicol. 2017; 57: 349-373Crossref PubMed Scopus (42) Google Scholar). The vorapaxar-inhibited conformation of PAR1 is the only structure solved by X-ray crystallography (Zhang et al., 2012Zhang C. Srinivasan Y. Arlow D.H. Fung J.J. Palmer D. Zheng Y. Green H.F. Pandey A. Dror R.O. Shaw D.E. et al.High-resolution crystal structure of human protease-activated receptor 1.Nature. 2012; 492: 387-392Crossref PubMed Scopus (356) Google Scholar). Besides the lack of additional structural models providing insight into other PAR1 conformations, their chemical and physical properties also remain to be characterized. Atomic force microscopy (AFM) is well-established for the high-resolution imaging (≤1 nm) of single membrane proteins at physiologically relevant conditions (Dufrene et al., 2017Dufrene Y.F. Ando T. Garcia R. Alsteens D. Martinez-Martin D. Engel A. Gerber C. Muller D.J. Imaging modes of atomic force microscopy for application in molecular and cell biology.Nat. Nanotechnol. 2017; 12: 295-307Crossref PubMed Scopus (555) Google Scholar) and for quantifying the structural stability of membrane proteins using AFM-based single-molecule force spectroscopy (SMFS) (Bippes and Muller, 2011Bippes C.A. Muller D.J. High-resolution atomic force microscopy and spectroscopy of native membrane proteins.Rep. Prog. Phys. 2011; 74: 086601Crossref Scopus (111) Google Scholar, Thoma et al., 2018Thoma J. Sapra K.T. Müller D.J. Single-molecule force spectroscopy of transmembrane β-barrel proteins.Annu. Rev. Anal. Chem. 2018; 11: 375-395Crossref PubMed Scopus (14) Google Scholar). The exceptional sensitivity of SMFS enables to map the stability of the structural segments (e.g., single α helices and polypeptide loops) of a membrane protein and to monitor how they change stability in response to ligand or inhibitor binding (Bippes et al., 2009Bippes C. Zeltina A. Casagrande F. Ratera M. Palacin M. Muller D.J. Fotiadis D. Substrate binding tunes conformational flexibility and kinetic stability of an amino acid antiporter.J. Biol. Chem. 2009; 28: 18651-18663Crossref Scopus (32) Google Scholar, Kedrov et al., 2006Kedrov A. Ziegler C. Muller D.J. Differentiating ligand and inhibitor interactions of a single antiporter.J. Mol. Biol. 2006; 362: 925-932Crossref PubMed Scopus (34) Google Scholar, Kedrov et al., 2008Kedrov A. Appel M. Baumann H. Ziegler C. Muller D.J. Examining the dynamic energy landscape of an antiporter upon inhibitor binding.J. Mol. Biol. 2008; 375: 1258-1266Crossref PubMed Scopus (29) Google Scholar, Serdiuk et al., 2014Serdiuk T. Madej M.G. Sugihara J. Kawamura S. Mari S.A. Kaback H.R. Muller D.J. Substrate-induced changes in the structural properties of LacY.Proc. Natl. Acad. Sci. U S A. 2014; 111: E1571-E1580Crossref PubMed Scopus (31) Google Scholar, Zocher et al., 2012aZocher M. Fung J.J. Kobilka B.K. Muller D.J. Ligand-specific interactions modulate kinetic, energetic, and mechanical properties of the human beta2 adrenergic receptor.Structure. 2012; 20: 1391-1402Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), temperature (Janovjak et al., 2007Janovjak H. Knaus H. Muller D.J. Transmembrane helices have rough energy surfaces.J. Am. Chem. Soc. 2007; 129: 246-247Crossref PubMed Scopus (43) Google Scholar), pH (Damaghi et al., 2010Damaghi M. Bippes C. Koster S. Yildiz O. Mari S.A. Kuhlbrandt W. Muller D.J. pH-dependent interactions guide the folding and gate the transmembrane pore of the beta-barrel membrane protein OmpG.J. Mol. Biol. 2010; 397: 878-882Crossref PubMed Scopus (33) Google Scholar), ions (Park et al., 2007Park P.S. Sapra K.T. Kolinski M. Filipek S. Palczewski K. Muller D.J. Stabilizing effect of Zn2+ in native bovine rhodopsin.J. Biol. Chem. 2007; 282: 11377-11385Crossref PubMed Scopus (55) Google Scholar), mutations (Kawamura et al., 2012Kawamura S. Colozo A.T. Ge L. Muller D.J. Park P.S. Structural, energetic, and mechanical perturbations in rhodopsin mutant that causes congenital stationary night blindness.J. Biol. Chem. 2012; 287: 21826-21835Crossref PubMed Scopus (25) Google Scholar, Sapra et al., 2008Sapra K.T. Balasubramanian G.P. Labudde D. Bowie J.U. Muller D.J. Point mutations in membrane proteins reshape energy landscape and populate different unfolding pathways.J. Mol. Biol. 2008; 376: 1076-1090Crossref PubMed Scopus (46) Google Scholar), or membrane lipid composition (Serdiuk et al., 2015Serdiuk T. Sugihara J. Mari S.A. Kaback H.R. Muller D.J. Observing a lipid-dependent alteration in single lactose permeases.Structure. 2015; 23: 754-761Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, Zocher et al., 2012bZocher M. Zhang C. Rasmussen S.G. Kobilka B.K. Muller D.J. Cholesterol increases kinetic, energetic, and mechanical stability of the human beta2-adrenergic receptor.Proc. Natl. Acad. Sci. U S A. 2012; 109: E3463-E3472Crossref PubMed Scopus (125) Google Scholar). Furthermore, applying SMFS in the dynamic mode can quantify the mechanical, kinetic and energetic properties and contour the free energy landscape of functionally activated or inhibited GPCR structures (Kawamura et al., 2013Kawamura S. Gerstung M. Colozo A.T. Helenius J. Maeda A. Beerenwinkel N. Park P.S. Muller D.J. Kinetic, energetic, and mechanical differences between dark-state rhodopsin and opsin.Structure. 2013; 21: 426-437Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, Spoerri et al., 2018Spoerri P.M. Kato H.E. Pfreundschuh M. Mari S.A. Serdiuk T. Thoma J. Sapra K.T. Zhang C. Kobilka B.K. Müller D.J. Structural properties of the human protease-activated receptor 1 changing by a strong antagonist.Structure. 2018; 26: 829-838Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, Zocher et al., 2012aZocher M. Fung J.J. Kobilka B.K. Muller D.J. Ligand-specific interactions modulate kinetic, energetic, and mechanical properties of the human beta2 adrenergic receptor.Structure. 2012; 20: 1391-1402Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). So far, however, a common trend describing the intrinsic chemical and physical properties of unliganded, activated, and inhibited GPCR conformations could not be highlighted. Here, we apply SMFS to quantify how the mechanical, kinetic, and energetic properties of unliganded inactive human PAR1 change upon activating the receptor via agonist binding (SFLLRN peptide) or inhibiting the receptor via antagonist binding (peptide-mimic BMS 200261 [BMS]). Using dynamic SMFS we map the parameters characterizing the free energy landscape of PAR1 and structurally delineate the mechanical, kinetic and energetic properties of all three functional states. In the inactive unliganded state PAR1 adopts mechanically rigid and stiff conformations. However, upon agonist or antagonist binding the receptor mechanically softens, while increasing its conformational flexibility, and kinetic and energetic stability. Thereby, we observe key structural regions of PAR1 putting this conformational plasticity into effect. The results highlight how ligand binding tunes the conformational plasticity of human PAR1 in specific ways and show a common trend of how agonist and antagonist binding change conformational properties of GPCRs. For SMFS, purified human PAR1 was reconstituted into liposomes made of 1,2-dioleoyl-sn-glycero-3-phosphocholine and the cholesterol analog cholesteryl hemisuccinate at a 10:1 ratio (w/w) (STAR Methods; Figure S1). The proteoliposomes were adsorbed onto mica (Muller and Engel, 2007Muller D.J. Engel A. Atomic force microscopy and spectroscopy of native membrane proteins.Nat. Protoc. 2007; 2: 2191-2197Crossref PubMed Scopus (180) Google Scholar, Spoerri et al., 2018Spoerri P.M. Kato H.E. Pfreundschuh M. Mari S.A. Serdiuk T. Thoma J. Sapra K.T. Zhang C. Kobilka B.K. Müller D.J. Structural properties of the human protease-activated receptor 1 changing by a strong antagonist.Structure. 2018; 26: 829-838Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar), where they collapsed and formed membrane patches exposing the terminal ends of PAR1 in buffer solution (Figures S1C–S1E). To non-specifically attach the stylus of the AFM cantilever to the N terminus of single receptors, the stylus was pushed to a membrane applying a force of 700 pN for 0.5 s. Then, the cantilever was retracted from the membrane at constant speed. This retraction stretched the N terminus and the pulling force built up during stretching unfolded and extracted a single PAR1 from the membrane (Figure S2A–B). Simultaneously, a force-distance (FD) curve recorded a sequence of force peaks corresponding to the stepwise unfolding and extraction of the receptor (Muller and Engel, 2007Muller D.J. Engel A. Atomic force microscopy and spectroscopy of native membrane proteins.Nat. Protoc. 2007; 2: 2191-2197Crossref PubMed Scopus (180) Google Scholar, Spoerri et al., 2018Spoerri P.M. Kato H.E. Pfreundschuh M. Mari S.A. Serdiuk T. Thoma J. Sapra K.T. Zhang C. Kobilka B.K. Müller D.J. Structural properties of the human protease-activated receptor 1 changing by a strong antagonist.Structure. 2018; 26: 829-838Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). Next, we repeated the SMFS experiments several thousand times to record the common unfolding pattern of human PAR1 in the unliganded inactive state (STAR Methods), the inhibited antagonist-bound state, and the activated agonist-bound state (Figures 1 and S2C). Whereas the antagonist-bound state was measured in the presence of the antagonist peptide-mimic BMS-200261 (BMS) known to inhibit PAR1-mediated platelet aggregation (Bernatowicz et al., 1996Bernatowicz M.S. Klimas C.E. Hartl K.S. Peluso M. Allegretto N.J. Seiler S.M. Development of potent thrombin receptor antagonist peptides.J. Med. Chem. 1996; 39: 4879-4887Crossref PubMed Scopus (138) Google Scholar), the agonist-bound state was measured in the presence of the synthetic agonist peptide (SFLLRN peptide) corresponding to the native N-terminal SFLLRN sequence that activates PAR1 (Coughlin, 2000Coughlin S.R. Thrombin signalling and protease-activated receptors.Nature. 2000; 407: 258-264Crossref PubMed Scopus (2137) Google Scholar). Although the exact structural locations of the agonist and antagonist binding to PAR1 are not known, both binding sites are known to localize on the extracellular side of PAR1 (Zhang et al., 2012Zhang C. Srinivasan Y. Arlow D.H. Fung J.J. Palmer D. Zheng Y. Green H.F. Pandey A. Dror R.O. Shaw D.E. et al.High-resolution crystal structure of human protease-activated receptor 1.Nature. 2012; 492: 387-392Crossref PubMed Scopus (356) Google Scholar). Previously, we have shown that mechanical unfolding of PAR1 by SMFS predominantly occurred by non-specifically attaching the extracellular N terminus to the AFM stylus and retracting the stylus (Spoerri et al., 2018Spoerri P.M. Kato H.E. Pfreundschuh M. Mari S.A. Serdiuk T. Thoma J. Sapra K.T. Zhang C. Kobilka B.K. Müller D.J. Structural properties of the human protease-activated receptor 1 changing by a strong antagonist.Structure. 2018; 26: 829-838Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). In this study, we used PAR1 carrying a shorter N-terminal (−35 amino acids [aa]) and a longer C-terminal (+76 aa) end. Nevertheless, unfolding PAR1 with the altered terminal ends, and in the absence and presence of ligands, produced FD curves of similar force peak patterns as obtained previously. This suggests that a shorter N terminus and a longer C terminus and the presence of ligands did not change the preference of the AFM stylus to non-specifically attach to the PAR1 N terminus. To confirm if the preferential attachment to the N terminus was indeed the case, we reduced the highly conserved disulfide bond formed between Cys175 (α helix H3) and Cys254 (extracellular loop E2) of PAR1 with DTT and conducted SMFS (Figure S3). Consequently, the force peak pattern of the FD curves increased distance by about 25 nm (≈79 aa), which corresponds to the contour length of the fully unfolded and stretched polypeptide comprising α helices H3 and H4 (Sapra et al., 2006Sapra K.T. Park P.S. Filipek S. Engel A. Palczewski K. Muller D.J. Detecting molecular interactions that stabilize bovine rhodopsin.J. Mol. Biol. 2006; 358: 255-269Crossref PubMed Scopus (60) Google Scholar, Spoerri et al., 2018Spoerri P.M. Kato H.E. Pfreundschuh M. Mari S.A. Serdiuk T. Thoma J. Sapra K.T. Zhang C. Kobilka B.K. Müller D.J. Structural properties of the human protease-activated receptor 1 changing by a strong antagonist.Structure. 2018; 26: 829-838Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). Thus, the PAR1 construct preferentially attached with the N terminus to the AFM stylus from which it was mechanically extended, unfolded, and extracted. Superimposition of the FD curves recorded for unliganded, antagonist-, and agonist-bound PAR1 showed recurring patterns of force peaks (Figure 1). Fitting each force peak with the worm-like chain model followed by statistical analysis unveiled eight main classes of force peaks corresponding to the contour lengths of the polypeptide stretched in each of the eight unfolding steps (Figure 1) (Bippes and Muller, 2011Bippes C.A. Muller D.J. High-resolution atomic force microscopy and spectroscopy of native membrane proteins.Rep. Prog. Phys. 2011; 74: 086601Crossref Scopus (111) Google Scholar, Muller and Engel, 2007Muller D.J. Engel A. Atomic force microscopy and spectroscopy of native membrane proteins.Nat. Protoc. 2007; 2: 2191-2197Crossref PubMed Scopus (180) Google Scholar). The contour lengths of the eight force peak classes were then used to locate the eight structural segments via which PAR1 unfolded (Figure 2): structural segment N1 describes the distal free end of the N terminus; segment N2 the proximal end of N terminus and top of α helix H1; segment H1-C1-H2 describes α helix H1, cytoplasmic loop C1, and α helix H2; segment E1 the extracellular loop E1; segment H3-C2-H4-E2 describes α helix H3, cytoplasmic loop C2, α helix H4, and extracellular loop E2; segment H5-C3-H6-E3 describes α helix H5, cytoplasmic loop C3, α helix H6, and extracellular loop E3; segment H7 describes α helix H7; and segment CT the C terminus. The eight structural segments were the same as detected previously for the mechanical unfolding of PAR1 in the inactive and vorapaxar-bound states (Spoerri et al., 2018Spoerri P.M. Kato H.E. Pfreundschuh M. Mari S.A. Serdiuk T. Thoma J. Sapra K.T. Zhang C. Kobilka B.K. Müller D.J. Structural properties of the human protease-activated receptor 1 changing by a strong antagonist.Structure. 2018; 26: 829-838Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). We thus conclude that human PAR1, independently of whether it was unliganded or liganded, stabilizes its structure against unfolding using the same structural segments. To detect the structural segments stabilizing PAR1 in the inactive, active, and inhibited states, we mechanically pulled and unfolded the receptor (Figure 1; STAR Methods). However, the force required to unfold and extract structural segments of proteins from membranes depends on the loading rate (e.g., pulling speed) applied by the AFM cantilever and is thus less suitable to interpret the stability of the membrane protein (Bippes and Muller, 2011Bippes C.A. Muller D.J. High-resolution atomic force microscopy and spectroscopy of native membrane proteins.Rep. Prog. Phys. 2011; 74: 086601Crossref Scopus (111) Google Scholar). Yet, measuring the unfolding force at a wide range of loading rates allows to approximate the parameters describing the kinetic stability of the structural segments. To access the parameters describing the free energy landscape of each structural segment of PAR1 (Figure S4), we performed dynamic SMFS (DFS) and mechanically unfolded PAR1 over a wide range of pulling speeds of 300, 600, 900, 1,200, 2,500, and 5,000 nm s−1 (Figure S5; STAR Methods). For every structural segment of PAR1, a DFS plot showed that the mean force required to unfold the segment increased linearly with the logarithm of the mean loading rate (Figures 3 and S6). This behavior was independent of whether PAR1 was liganded or not. However, the unfolding forces characterizing the stability of the structural segments of unliganded and SFLLRN-bound PAR1 were considerably below those of the segments of BMS-bound PAR1. Although such changes in forces may suggest higher or lower mechanical stability of a structural segment at certain applied loading rates, which are far from equilibrium, they do not necessarily reflect the mechanical, kinetic, or energetic stability of the segment at equilibrium. To approach the parameters describing these properties of PAR1 at equilibrium we fitted the Bell-Evans model (Figure S4; STAR Methods) to the slope of each DFS plot (Figure 3). The fits estimated the distance xu a folded structural segment had to be stretched along the pulling direction to reach its transition state toward unfolding (Table 1). The xu values approximate the width of the free energy valley stabilizing a structural segment and thus the conformational flexibility of the segment. The DFS plot also estimated the unfolding rate k0 of a structural segment under zero force, which is reciprocal of the lifetime. Furthermore, the unfolding free energy ΔGu‡, which describes the height of the free energy barrier stabilizing a structural segment, and κ, which defines the mechanical stiffness (i.e., spring constant), were estimated from k0 and xu, respectively (Figure S4).Table 1Parameters Characterizing the Properties of Unliganded, BMS-, and SFLLRN-Bound PAR1Structural SegmentUnliganded PAR1BMS-Bound PAR1SFLLRN-Bound PAR1xu ± SD (nm)N10.21 ± 0.020.23 ± 0.04 (+10%)0.23 ± 0.04 (+10%)N20.19 ± 0.020.20 ± 0.04 (+5%)0.23 ± 0.03 (+21%)H1-C1-H20.17 ± 0.030.22 ± 0.05 (+29%)0.17 ± 0.01 (0%)E10.31 ± 0.070.92 ± 0.23 (+200%)0.38 ± 0.06 (+23%)H3-C2-H4-E20.24 ± 0.060.32 ± 0.07 (+33%)0.28 ± 0.03 (+17%)H5-C3-H6-E30.17 ± 0.030.15 ± 0.01 (−12%)0.24 ± 0.04 (+41%)H70.56 ± 0.230.95 ± 0.25 (+70%)0.76 ± 0.21 (+36%)CT0.18 ± 0.040.32 ± 0.07 (+78%)0.16 ± 0.01 (−11%)k0 ± SD (s−1)N12.25 ± 1.000.44 ± 0.53 (−80%)1.31 ± 1.35 (−42%)N21.32 ± 0.780.35 ± 0.45 (−73%)0.64 ± 0.48 (−52%)H1-C1-H21.71 ± 1.630.17 ± 0.34 (−90%)3.10 ± 1.17 (+81%)E10.60 ± 0.951.9 × 10−7 ± 1.6 × 10−6 (−100%)0.24 ± 0.27 (−60%)H3-C2-H4-E20.26 ± 0.450.04 ± 0.01 (−85%)0.09 ± 0.08 (−35%)H5-C3-H6-E30.75 ± 0.810.62 ± 0.20 (−17%)0.15 ± 0.18 (−80%)H70.01 ± 0.059.3 × 10−8 ± 5.6 ×10−7 (−100%)4.1 × 10−4 ± 1.6 × 10−3 (−96%)CT0.39 ± 0.570.004 ± 0.010 (−99%)0.89 ± 0.45 (+128%)ΔGu‡ ± SD (kBT)N117.6 ± 0.419.2 ± 1.2 (+9%)18.2 ± 1.0 (+3%)N218.1 ± 0.619.5 ± 1.3 (+8%)18.9 ± 0.8 (+4%)H1-C1-H217.9 ± 1.020.2 ± 2.0 (+13%)17.3 ± 0.4 (−3%)E118.9 ± 1.633.9 ± 5.7 (+79%)19.8 ± 1.1 (+5%)H3-C2-H4-E219.8 ± 1.721.6 ± 0.2 (+9%)20.8 ± 0.9 (+5%)H5-C3-H6-E318.7 ± 1.118.9 ± 0.3 (+1%)20.3 ± 1.3 (+9%)H722.9 ± 4.434.6 ± 6.0 (+51%)26.2 ± 3.9 (+14%)CT19.4 ± 1.523.9 ± 2.4 (+23%)18.5 ± 0.5 (−5%)κ ± SD (N m−1)N13.45 ± 0.612.91 ± 1.02 (−16%)2.74 ± 1.00 (−21%)N24.08 ±" @default.
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• W2967792612 date "2019-10-01" @default.
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• W2967792612 title "Conformational Plasticity of Human Protease-Activated Receptor 1 upon Antagonist- and Agonist-Binding" @default.
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