title: Holoenzyme structures of endothelial nitric oxide synthase - an allosteric role for calmodulin in pivoting the FMN domain for electron transfer.
abstract: While the three-dimensional structures of heme- and flavin-binding domains of the NOS isoforms have been determined, the structures of the holoenzymes remained elusive. Application of electron cryo-microscopy and structural modeling of the bovine endothelial nitric oxide synthase (eNOS) holoenzyme produced detailed models of the intact holoenzyme in the presence and absence of Ca(2+)/calmodulin (CaM). These models accommodate the cross-electron transfer from the reductase in one monomer to the heme in the opposite monomer. The heme domain acts as the anchoring dimeric structure for the entire enzyme molecule, while the FMN domain is activated by CaM to move flexibly to bridge the distance between the reductase and oxygenase domains. Our results indicate that the key regulatory role of CaM involves the stabilization of structural intermediates and precise positioning of the pivot for the FMN domain tethered shuttling motion to accommodate efficient and rapid electron transfer in the homodimer of eNOS.
authors.affiliation: Bioinformatics and Systems Biology Program, Sanford Burnham Medical Research Institute, La Jolla, CA 92075, USA. Electronic address: niels@burnham.org.
authors.forename: Niels
authors.initials: N
authors.lastname: Volkmann
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA; Department of Pediatrics, First School of Medicine, Charles University, 12109 Prague, Czech Republic.
authors.forename: Pavel
authors.initials: P
authors.lastname: Martásek
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA.
authors.forename: Linda J
authors.initials: LJ
authors.lastname: Roman
authors.affiliation: Bioinformatics and Systems Biology Program, Sanford Burnham Medical Research Institute, La Jolla, CA 92075, USA.
authors.forename: Xiao-Ping
authors.initials: XP
authors.lastname: Xu
authors.affiliation: Bioinformatics and Systems Biology Program, Sanford Burnham Medical Research Institute, La Jolla, CA 92075, USA.
authors.forename: Christopher
authors.initials: C
authors.lastname: Page
authors.affiliation: Bioinformatics and Systems Biology Program, Sanford Burnham Medical Research Institute, La Jolla, CA 92075, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Swift
authors.affiliation: Bioinformatics and Systems Biology Program, Sanford Burnham Medical Research Institute, La Jolla, CA 92075, USA. Electronic address: dorit@burnham.org.
authors.forename: Dorit
authors.initials: D
authors.lastname: Hanein
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA.
authors.forename: Bettie Sue
authors.initials: BS
authors.lastname: Masters
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D004579:Electron Transport; D005486:Flavin Mononucleotide / Q000737:chemistry*; D006418:Heme / Q000737:chemistry; D020035:Holoenzymes / Q000737:chemistry*; D007700:Kinetics; D052250:Nitric Oxide Synthase Type III / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary
chemical_list: D002147:Calmodulin; D020035:Holoenzymes; D006418:Heme; D005486:Flavin Mononucleotide; D052250:Nitric Oxide Synthase Type III; D002118:Calcium
keywords: Calmodulin; Electron cryomicroscopy; Electron transfer; Image processing; Nitric oxide synthase; Three-dimensional reconstruction