Architecture of the Nitric-oxide Synthase Holoenzyme Reveals Large Conformational Changes and a Calmodulin-driven Release of the FMN Domain

Yokom, Adam L.; Morishima, Yosuke; Lau, Miranda; Su, Min; Glukhova, Alisa; Osawa, Yoichi; Southworth, Daniel R.

doi:10.1074/jbc.m114.564005

Cited by 40 publications

(70 citation statements)

References 48 publications

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“…Furthermore, the enforcement of C2 symmetry during the reconstruction steps may bias the analysis. It is notable that the 2D class averages obtained by Yokom et al (13) for the nNOS-CaM complex in the absence of chemical cross-linking are in complete agreement with those reported here.…”

Section: Discussionsupporting

confidence: 81%

“…Each of these linkers could adopt a variety of orientations, providing significant conformational flexibility between the reductase and oxidase domains. The observed conformational flexibility was consistent with the 2D class averages of a recent nNOS EM study (13). This conformational flexibility allowed us to examine snapshots of the NOS holoenzyme in the input and the output state as well as many intermediate states.…”

Section: Discussionsupporting

confidence: 55%

“…Taken together, these observations suggest that only one reductase domain at a time contributes to NOS catalysis, while the other reductase domain remains in the input state. In contrast, a recent negatively stained EM structure of a chemically cross-linked nNOS-CaM complex showed both reductase domains in the output state (13). Because of the high flexibility observed here for NOS isoforms, it is possible that crosslinking the particles may have trapped a minor NOS conformation that is not populated under normal conditions.…”

Section: Discussionmentioning

confidence: 52%

“…To transition into the output state, a large conformational change occurs that allows the FMN subdomain to donate an electron to the oxidase domain. Although progress has been made elucidating the interaction surfaces present in the input and output states (7)(8)(9)(10)(11)(12), the trajectory of this conformational change between the input and output is only beginning to be explored (13). Further understanding of this conformational change is particularly important as electron transfer is rate-limiting during NOS catalysis (6).…”

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confidence: 99%

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Molecular architecture of mammalian nitric oxide synthases

Campbell

Smith

Potter

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

122

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NOSs are homodimeric multidomain enzymes responsible for producing NO. In mammals, NO acts as an intercellular messenger in a variety of signaling reactions, as well as a cytotoxin in the innate immune response. Mammals possess three NOS isoformsinducible, endothelial, and neuronal NOS-that are composed of an N-terminal oxidase domain and a C-terminal reductase domain. Calmodulin (CaM) activates NO synthesis by binding to the helical region connecting these two domains. Although crystal structures of isolated domains have been reported, no structure is available for full-length NOS. We used high-throughput single-particle EM to obtain the structures and higher-order domain organization of all three NOS holoenzymes. The structures of inducible, endothelial, and neuronal NOS with and without CaM bound are similar, consisting of a dimerized oxidase domain flanked by two separated reductase domains. NOS isoforms adopt many conformations enabled by three flexible linkers. These conformations represent snapshots of the continuous electron transfer pathway from the reductase domain to the oxidase domain, which reveal that only a single reductase domain participates in electron transfer at a time, and that CaM activates NOS by constraining rotational motions and by directly binding to the oxidase domain. Direct visualization of these large conformational changes induced during electron transfer provides significant insight into the molecular underpinnings governing NO formation.heme | flavin | electron microscopy | conformational heterogeneity N O has emerged as an integral signaling molecule in biology.NOSs are the only enzymes responsible for NO production in mammals. Disruptions in NO signaling are linked to hypertension, erectile dysfunction, neurodegeneration, stroke, and heart disease (1-3). Three NOS isoforms, which vary slightly in size and composition from 260 to 321 kDa for the NOS homodimer, are present in mammals; each is a distinct gene product differing in subcellular localization, tissue distribution, and mode of regulation. The constitutively expressed NOS isoforms are found primarily in endothelial cells [endothelial NOS (eNOS)] and neuronal cells [neuronal NOS (nNOS)], and NO produced by these isoforms initiates diverse signaling processes, including vasodilation, platelet aggregation, myocardial functions, and neurotransmission (4). The activity of the constitutive NOS isoforms is dependent on intracellular Ca 2+ concentrations. The third isoform, inducible NOS (iNOS), is transcriptionally controlled and produces cytotoxic concentrations of NO at sites of infection or inflammation. Unlike eNOS and nNOS, the activity of iNOS is independent of the intracellular Ca 2+ concentration.Mammalian NOS isoforms use a complex assembly of domains and cofactors to convert L-arginine to L-citrulline and NO, via the intermediate N-hydroxy-L-arginine. Mammalian NOS isoforms are active as homodimers and composed of two primary domains: the N-terminal oxidase domain and the C-terminal reductase domain (Fig. 1A). The re...

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Section: Discussionsupporting

confidence: 81%

Section: Discussionsupporting

confidence: 55%

Section: Discussionmentioning

confidence: 52%

mentioning

confidence: 99%

See 2 more Smart Citations

Molecular architecture of mammalian nitric oxide synthases

Campbell

Smith

Potter

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

122

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“…Although several structures of isolated domains of the NOS isoforms have been determined (Li et al, 2001; Raman et al, 1998; Zhang et al, 2001; Crane et al, 1997; Crane et al, 1998; Garcin et al, 2004), currently there is no full-length atomic structure available for any of the NOS isoforms, as multiple attempts to determine the X-ray structures have met with failure. A recent electron microscopy study employing negative staining of chemically cross-linked nNOS holoenzymes (Yokom et al, 2014) is not only of a different isoform, it also suffers from potential sample preparation artifacts and an electron cryo-microscopy study of eNOS holoenzymes only shows incomplete density that only accommodates the oxygenase domains (Persechini et al, 2013), leaving the conformations of the eNOS holoenzyme an open question.…”

Section: Introductionmentioning

confidence: 99%

Holoenzyme structures of endothelial nitric oxide synthase – An allosteric role for calmodulin in pivoting the FMN domain for electron transfer

Volkmann

Martásek

Roman

et al. 2014

Journal of Structural Biology

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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 Ca2+/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.

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Role of an isoform‐specific residue at the calmodulin–heme (NO synthase) interface in the FMN – heme electron transfer

Zheng

Wang

et al. 2018

FEBS Letters

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The interface between calmodulin (CaM) and the NO synthase (NOS) heme domain is the least characterized interprotein interface that the NOS isoforms must traverse through during catalysis. Our previous molecular dynamics simulations predicted a salt bridge between K497 in human inducible NOS (iNOS) heme domain and D118(CaM). Herein, the FMN - heme interdomain electron transfer (IET) rate was found to be notably decreased by charge-reversal mutation, while the IET in the iNOS K497D mutant is significantly restored by the CaM D118K mutation. The results of wild-type protein with added synthetic peptides further demonstrate the critical nature of K497 relative to the rest of the peptide sequence in modulating the IET. These data provide definitive evidence supporting the regulatory role of the isoform-specific K497 residue.

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Architecture of the Nitric-oxide Synthase Holoenzyme Reveals Large Conformational Changes and a Calmodulin-driven Release of the FMN Domain

Cited by 40 publications

References 48 publications

Molecular architecture of mammalian nitric oxide synthases

Molecular architecture of mammalian nitric oxide synthases

Holoenzyme structures of endothelial nitric oxide synthase – An allosteric role for calmodulin in pivoting the FMN domain for electron transfer

Role of an isoform‐specific residue at the calmodulin–heme (NO synthase) interface in the FMN – heme electron transfer

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