title: Reactive Center Loop Insertion in α-1-Antitrypsin Captured by Accelerated Molecular Dynamics Simulation.
abstract: Protease inhibition by metastable serine protease inhibitors (serpins) is mediated by one of the largest functional intradomain conformational changes known in biology. In this extensive structural rearrangement, protease-serpin complex formation triggers cleavage of the serpin reactive center loop (RCL), its subsequent insertion into central β-sheet A, and covalent trapping of the target protease. In this study, we present the first detailed accelerated molecular dynamics simulation of the insertion of the fully cleaved RCL in α-1-antitrypsin (α1AT), the archetypal member of the family of human serpins. Our results reveal internal water pathways that allow the initial incorporation of side chains of RCL residues into the protein interior. We observed structural plasticity of the helix F (hF) element that blocks the RCL path in the native state, which is in excellent agreement with previous experimental reports. Furthermore, the simulation suggested a novel role of hF and the connected turn (thFs3A) as chaperones that support the insertion process by reducing the conformational space available to the RCL. Transient electrostatic interactions of RCL residues potentially fine-tune the serpin inhibitory activity. On the basis of our simulation, we generated the α1AT mutants K168E, E346K, and K168E/E346K and analyzed their inhibitory activity along with their intrinsic stability and heat-induced polymerization. Remarkably, the E346K mutation exhibited enhanced inhibitory activity along with an increased rate of premature structural collapse (polymerization), suggesting a significant role of E346 in the gatekeeping of the strain in the metastable native state.
authors.affiliation: Center for Insoluble Protein Structures (inSPIN) and Interdisciplinary Nanoscience Center (iNANO), Aarhus University , Aarhus, Denmark.|Department of Chemistry, Aarhus University , Aarhus, Denmark.
authors.forename: Ole Juul
authors.initials: OJ
authors.lastname: Andersen
authors.affiliation: Center for Insoluble Protein Structures (inSPIN) and Interdisciplinary Nanoscience Center (iNANO), Aarhus University , Aarhus, Denmark.|Department of Molecular Biology and Genetics, Aarhus University , Aarhus, Denmark.
authors.forename: Michael Wulff
authors.initials: MW
authors.lastname: Risør
authors.affiliation: Center for Insoluble Protein Structures (inSPIN) and Interdisciplinary Nanoscience Center (iNANO), Aarhus University , Aarhus, Denmark.|Department of Molecular Biology and Genetics, Aarhus University , Aarhus, Denmark.
authors.forename: Emil Christian
authors.initials: EC
authors.lastname: Poulsen
authors.affiliation: Center for Insoluble Protein Structures (inSPIN) and Interdisciplinary Nanoscience Center (iNANO), Aarhus University , Aarhus, Denmark.|Department of Chemistry, Aarhus University , Aarhus, Denmark.
authors.forename: Niels Chr
authors.initials: NC
authors.lastname: Nielsen
authors.affiliation: Howard Hughes Medical Institute and Department of Pharmacology, University of California at San Diego , La Jolla, California 92093, United States.
authors.forename: Yinglong
authors.identifier: 0000-0003-3714-1395
authors.initials: Y
authors.lastname: Miao
authors.affiliation: Center for Insoluble Protein Structures (inSPIN) and Interdisciplinary Nanoscience Center (iNANO), Aarhus University , Aarhus, Denmark.|Department of Molecular Biology and Genetics, Aarhus University , Aarhus, Denmark.
authors.forename: Jan J
authors.initials: JJ
authors.lastname: Enghild
authors.affiliation: Center for Insoluble Protein Structures (inSPIN) and Interdisciplinary Nanoscience Center (iNANO), Aarhus University , Aarhus, Denmark.|Department of Chemistry, Aarhus University , Aarhus, Denmark.
authors.forename: Birgit
authors.identifier: 0000-0001-9937-1562
authors.initials: B
authors.lastname: Schiøtt
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry*; D002417:Cattle; D003001:Cloning, Molecular; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D009154:Mutation; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D055672:Static Electricity; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014357:Trypsin / Q000737:chemistry*; D000515:alpha 1-Antitrypsin / Q000737:chemistry* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D000515:alpha 1-Antitrypsin; D001224:Aspartic Acid; D014357:Trypsin