title: Architecture of the nitric-oxide synthase holoenzyme reveals large conformational changes and a calmodulin-driven release of the FMN domain.
abstract: Nitric-oxide synthase (NOS) is required in mammals to generate NO for regulating blood pressure, synaptic response, and immune defense. NOS is a large homodimer with well characterized reductase and oxygenase domains that coordinate a multistep, interdomain electron transfer mechanism to oxidize l-arginine and generate NO. Ca(2+)-calmodulin (CaM) binds between the reductase and oxygenase domains to activate NO synthesis. Although NOS has long been proposed to adopt distinct conformations that alternate between interflavin and FMN-heme electron transfer steps, structures of the holoenzyme have remained elusive and the CaM-bound arrangement is unknown. Here we have applied single particle electron microscopy (EM) methods to characterize the full-length of the neuronal isoform (nNOS) complex and determine the structural mechanism of CaM activation. We have identified that nNOS adopts an ensemble of open and closed conformational states and that CaM binding induces a dramatic rearrangement of the reductase domain. Our three-dimensional reconstruction of the intact nNOS-CaM complex reveals a closed conformation and a cross-monomer arrangement with the FMN domain rotated away from the NADPH-FAD center, toward the oxygenase dimer. This work captures, for the first time, the reductase-oxygenase structural arrangement and the CaM-dependent release of the FMN domain that coordinates to drive electron transfer across the domains during catalysis.
authors.affiliation: From the Department of Biological Chemistry, the Program in Chemical Biology, and.
authors.forename: Adam L
authors.initials: AL
authors.lastname: Yokom
authors.affiliation: the Department of Pharmacology.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Morishima
authors.affiliation: the Department of Pharmacology.
authors.forename: Miranda
authors.initials: M
authors.lastname: Lau
authors.affiliation: the Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109.
authors.forename: Min
authors.initials: M
authors.lastname: Su
authors.affiliation: the Program in Chemical Biology, and.
authors.forename: Alisa
authors.initials: A
authors.lastname: Glukhova
authors.affiliation: the Department of Pharmacology.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Osawa
authors.affiliation: From the Department of Biological Chemistry, the Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109 dsouth@umich.edu.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Southworth
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D052248:Nitric Oxide Synthase Type I / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D051381:Rats
chemical_list: D002147:Calmodulin; D020035:Holoenzymes; D052248:Nitric Oxide Synthase Type I
keywords: Calmodulin; Electron Microscopy (EM); Flavoproteins; Nitric-oxide Synthase; Oxidation-Reduction
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