Elucidating nitric oxide synthase domain interactions by molecular dynamics

Hollingsworth, Scott A.; Holden, Jeffrey K.; Li, Huiying; Poulos, T.L.

doi:10.1002/pro.2824

Cited by 17 publications

(22 citation statements)

References 53 publications

(123 reference statements)

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“…As previously noted, our nNOS FMNNOSoxy domain docking model differs somewhat from the computer-generated domain docking models of human or mouse iNOS (31,33,34). The iNOS models shift the docking site of the FMN domain relative to our nNOS model, and consequently predict that it involves different cross-domain charge pairing interactions, a greater FMNto-heme distance, and invoke participation of a conserved aromatic residue (Trp-366 in iNOS) in mediating the FMN-to-heme ET.…”

Section: Discussionmentioning

confidence: 90%

“…Similarly, individual neutralization or reversal of positively-charged surface residues on the NOSoxy domain identified three (Lys-423, Lys-620, and Lys-660) as being important for enabling nNOS heme reduction (30). Subsequently, several groups have generated computer-based domain docking models that suggest complementary charge interactions may form between specific residues on the NOSoxy and FMN domains (30,31,33,34). The various model structures differ in several ways including which residue pairs form cross-domain charge pairing interactions, the distance between the FMN and heme, and whether NOS protein residues help bridge the ET reaction.…”

Section: _______________________________________mentioning

confidence: 99%

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A cross-domain charge interaction governs the activity of NO synthase

Haque

Tejero

Bayachou

et al. 2018

Journal of Biological Chemistry

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NO synthase (NOS) enzymes perform inter-domain electron transfer reactions during catalysis that may rely on complementary charge interactions at domain-domain interfaces. Guided by our previous results and a computer-generated domain docking model, we assessed the importance of cross-domain charge interactions in the FMN to heme electron transfer in neuronal NOS (nNOS). We reversed the charge of three residues that form an electronegative triad on the FMN domain, and then individually reversed the charges of three electropositive residues (Lys-423, Lys-620, Lys-660) on the oxygenase domain (NOSoxy), to potentially restore a crossdomain charge interaction with the triad, but in reversed polarity. Charge reversal of the triad completely eliminated heme reduction and NO synthesis in nNOS. These functions were partly restored by the charge reversal at oxygenase residue Lys-423, but not at Lys-620 or Lys-660. Full recovery of heme reduction was likely muted by an accompanying change in FMN midpoint potential that made electron transfer to the heme thermodynamically unfavorable. Our results provide direct evidence that crossdomain charge pairing is required for the FMN to heme electron transfer in nNOS. The unique ability of charge reversal at position 423 to rescue function indicates that it participates in an essential cross-domain charge interaction with the FMN domain triad. This supports our domain docking model and suggests that it may depict a productive electron transfer complex formed during nNOS catalysis. _______________________________________Nitric oxide (NO) 1 is synthesized in mammals in a two-step oxidation of LArginine (Arg) that is catalyzed by three similar NO synthase isozymes (NOS, EC 1.14.13.39): inducible NOS (iNOS), neuronal NOS (nNOS), and endothelial NOS (eNOS) (1,2). Each NOS is comprised of an N-terminal oxygenase domain (NOSoxy) that is connected to a C-terminal flavoprotein domain by a central calmodulin http://www.jbc.org/cgi

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Section: Discussionmentioning

confidence: 90%

Section: _______________________________________mentioning

confidence: 99%

A cross-domain charge interaction governs the activity of NO synthase

Haque

Tejero

Bayachou

et al. 2018

Journal of Biological Chemistry

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“…1). Knowing the crystal structures of the various NOS modules together with cryoEM [12–14], hydrogen-deuterium exchange [15], molecular dynamics [16,17], and a wealth of mutagenesis data has provided a working model of holo-NOS and the role calmodulin plays in NOS activation [18]. The crystal structure of the nNOS reductase domain is very similar to P450 reductase and in both structures the FMN and FAD are in direct contact.…”

Section: Structural Biologymentioning

confidence: 99%

“…The magenta sphere model is the heme. B) Docking model of the complex formed between the iNOS heme and FMN domains [17]. The FMN domain of the molecule A (cyan) docks to the heme domain of molecule B (green) and vise versa.…”

Section: Figurementioning

confidence: 99%

Nitric oxide synthase and structure-based inhibitor design

Poulos

2017

Nitric Oxide

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Once it was discovered that the enzyme nitric oxide synthase (NOS) is responsible for the biosynthesis of NO, NOS became a drug target. Particularly important is the over production of NO by neuronal NOS (nNOS) in various neurodegenerative disorders. After the various NOS isoforms were identified, inhibitor development proceeded rapidly. It soon became evident, however, that isoform selectivity presents a major challenge. All 3 human NOS isoforms, nNOS, eNOS (endothelial NOS), and iNOS (inducible NOS) have nearly identical active site structures thus making selective inhibitor design especially difficult. Of particular importance is the avoidance of inhibiting eNOS owing to its vital role in the cardiovascular system. This review summarizes some of the history of NOS inhibitor development and more recent advances in developing isoform selective inhibitors using primarily structure-based approaches.

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“…A formal tethered shuttle mechanism was proposed in which the FMN binding domain dissociates from a reductase complex 'input state' and reorients to transfer electrons to the oxygenase domain [7,23,24]. Work from other groups also supports shuttle mechanisms in which reductase function requires major conformational changes, e.g., [12,[25][26][27][28][29][30][31]. In addition to switching on the production of nitric oxide from a negligible rate, CaM binding to nNOS holoenzyme [27,[32][33][34][35] increases NADPH cytochrome c reductase activity, typically by a factor of four, and significantly increases steady state flavin fluorescence [7,12,[32][33][34][35].…”

Section: Introductionmentioning

confidence: 99%

Control of eNOS and nNOS through regulation of obligatory conformational changes in the reductase catalytic cycle

Salerno

Hopper

Ghosh

et al. 2018

Preprint

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Endothelial and neuronal nitric oxide synthases (eNOS, nNOS) are important signal generators in a number of processes including angiogenesis and neurotransmission. The homologous inducible isoform (iNOS) occupies a multitude of conformational states in a catalytic cycle, including subnanosecond input and output states and a distribution of 'open' conformations with average lifetimes of ~4.3 ns. In this study, fluorescence lifetime spectroscopy was used to probe conformational states of purified eNOS and nNOS in the presence of chaotropes, calmodulin, NADP + and NADPH. Two-domain FMN/oxygenase constructs of nNOS were also examined with respect to calmodulin effects. Optical biosensing was used to analyze calmodulin binding in the presence of NADP + and NADPH. Calmodulin binding induced a shift of the population away from the input and to the open and output states of NOS. NADP + shifted the population towards the input state. The oxygenase domain, lacking the input state, provided a measure of calmodulin-induced open-output transitions. A mechanism for regulation by calmodulin and an elucidation of the catalytic mechanism are suggested by a 'conformational lockdown' model. Calmodulin speeds transitions between input and open and between open and output states, effectively reducing the conformational manifold, speeding catalysis. Conformational control of catalysis involves reorientation of the FMN binding domain, of which fluorescence lifetime is an indicator. The approach described herein is a new tool for biophysical and structural analysis of NOS enzymes, regulatory events and other homologous reductase-containing enzymes. 3 A note to the readerThis manuscript has over the past several years been submitted to and rejected by several journals, usually on the basis of reviewer opinion that it was not an important enough result to merit inclusion in the journal. Owing to the passing of the first author and the loss of his expertise in fluorescence lifetime spectroscopy, it has become too onerous a task to continually revise the manuscript to suit the whims of reviewers who nevertheless still reject the work. We are thus simply releasing the final form of the manuscript to BioRxiv in the hopes that it finds a readership who will find, as we do, that the results are of value to the field.

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Elucidating nitric oxide synthase domain interactions by molecular dynamics

Cited by 17 publications

References 53 publications

A cross-domain charge interaction governs the activity of NO synthase

A cross-domain charge interaction governs the activity of NO synthase

Nitric oxide synthase and structure-based inhibitor design

Control of eNOS and nNOS through regulation of obligatory conformational changes in the reductase catalytic cycle

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