title: Critical Role of Interdomain Interactions in the Conformational Change and Catalytic Mechanism of Endoplasmic Reticulum Aminopeptidase 1.
abstract: Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an intracellular enzyme that is important for the generation of antigenic epitopes and major histocompatibility class I-restricted adaptive immune responses. ERAP1 processes a vast variety of different peptides but still shows length and sequence selectivity, although the mechanism behind these properties is poorly understood. X-ray crystallographic analysis has revealed that ERAP1 can assume at least two distinct conformations in which C-terminal domain IV is either proximal or distal to active site domain II. To improve our understanding of the role of this conformational change in the catalytic mechanism of ERAP1, we used site-directed mutagenesis to perturb key salt bridges between domains II and IV. Enzymatic analysis revealed that these mutations, although located away from the catalytic site, greatly reduce the catalytic efficiency and change the allosteric kinetic behavior. The variants were more efficiently activated by small peptides and bound a competitive inhibitor with weaker affinity and faster dissociation kinetics. Molecular dynamics analysis suggested that the mutations affect the conformational distribution of ERAP1, reducing the population of closed states. Small-angle X-ray scattering indicated that both the wild type and the ERAP1 variants are predominantly in an open conformational state in solution. Overall, our findings suggest that electrostatic interactions between domains II and IV in ERAP1 are crucial for driving a conformational change that regulates the structural integrity of the catalytic site. The extent of domain opening in ERAP1 probably underlies its specialization for antigenic peptide precursors and should be taken into account in inhibitor development efforts.
authors.affiliation: National Centre for Scientific Research Demokritos , Agia Paraskevi, 15341 Athens, Greece.
authors.forename: Athanasios
authors.initials: A
authors.lastname: Stamogiannos
authors.affiliation: Department of Pathology, University of Massachusetts Medical School , Worcester, Massachusetts 01655, United States.
authors.forename: Zachary
authors.initials: Z
authors.lastname: Maben
authors.affiliation: National Centre for Scientific Research Demokritos , Agia Paraskevi, 15341 Athens, Greece.
authors.forename: Athanasios
authors.identifier: 0000-0003-3931-6232
authors.initials: A
authors.lastname: Papakyriakou
authors.affiliation: National Centre for Scientific Research Demokritos , Agia Paraskevi, 15341 Athens, Greece.
authors.forename: Anastasia
authors.initials: A
authors.lastname: Mpakali
authors.affiliation: Laboratory of Organic Chemistry, Department of Chemistry, University of Athens , Panepistimiopolis, Zografou, 15771 Athens, Greece.
authors.forename: Paraskevi
authors.initials: P
authors.lastname: Kokkala
authors.affiliation: Laboratory of Organic Chemistry, Department of Chemistry, University of Athens , Panepistimiopolis, Zografou, 15771 Athens, Greece.
authors.forename: Dimitris
authors.initials: D
authors.lastname: Georgiadis
authors.affiliation: Department of Pathology, University of Massachusetts Medical School , Worcester, Massachusetts 01655, United States.
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Stern
authors.affiliation: National Centre for Scientific Research Demokritos , Agia Paraskevi, 15341 Athens, Greece.
authors.forename: Efstratios
authors.identifier: 0000-0002-3566-2309
authors.initials: E
authors.lastname: Stratikos
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain; D002460:Cell Line; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D007915:Lepidoptera / Q000166:cytology / Q000378:metabolism; D015778:Minor Histocompatibility Antigens / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D009154:Mutation; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012492:Salts / Q000737:chemistry; D055672:Static Electricity; D013816:Thermodynamics
chemical_list: D015778:Minor Histocompatibility Antigens; D011994:Recombinant Proteins; D012492:Salts; D000626:Aminopeptidases; C464841:ERAP1 protein, human