The Function of the Small Insertion in the Hinge Subdomain in the Control of Constitutive Mammalian Nitric-oxide Synthases

Abstract

Control of nitric oxide (NO) synthesis in the constitutive nitric-oxide synthases (NOS) by calcium/calmodulin is exerted through the regulation of electron transfer from NADPH through the reductase domains. This process has been shown previously to involve the calmodulin binding site, the autoinhibitory insertion in the FMN binding domain, and the C-terminal tail. Smaller sequence elements also appear to correlate with control. Although some of these elements appear well positioned to function in control, they are poorly conserved; their role in control is neither well established nor defined by available information. In this study mutations have been induced in the small insertion of the hinge subdomain, which has been shown recently to form a ␤ hairpin in structural studies of the neuronal NOS reductase domains adjacent to the calmodulin site and the autoinhibitory element. Modification of the small insertion in neuronal NOS tends to increase cytochrome c reduction but not NO synthetic activity; some modifications or deletions in the corresponding region in endothelial NOS modestly increase activity under some conditions. Unexpectedly, some minor changes in the sequence introduce a loss in the content of heme relative to flavin cofactors. Taken together, these results suggest that the small insertion protects the calmodulin binding site and that it may be a modulator of NOS activity.Nitric-oxide synthases (NOS) 1 are a growing family of modular enzymes, including three mammalian isoforms, as well as a number of related eukaryotic and more distantly related prokaryotic enzymes. Because of the discovery of the function of nitric oxide (NO) as a molecular messenger in a variety of signal transduction pathways (1-4), the endothelial and neuronal isoforms (eNOS and nNOS) have been shown to function as signal generators controlled by calcium/calmodulin (Ca 2ϩ / CaM) (5). The other mammalian isoform, iNOS, is induced during immune response (6 -9); it is constitutively active and binds CaM at basal Ca 2ϩ levels.NO synthesis from arginine and molecular oxygen requires 3 mol of electrons supplied by NADPH/mol of NO formed (10). The reductase domains in eukaryotic NOS are homologous to NADPH P450 reductase (11). These domains contain one flavin mononucleotide (FMN) and one flavin adenine dinucleotide (FAD) cofactor (11-18), which function in electron transfer from NADPH to the heme-containing active site (13,17,19,20).Ca 2ϩ /CaM control in NOS is exerted through the regulation of electron transfer between NADPH and heme (5). This regulation is primarily a function of the reductase domains (21). The canonical CaM binding site was recognized in the sequence region connecting the oxygenase and reductase regions (11). A major insertion in the FMN binding region, correlating with Ca 2ϩ /CaM control, was identified as a control element with autoinhibitory character (autoinhibitory insertion, or AI) (22,23). This has been confirmed by a number of groups (e.g. 24,25).Recently the C-terminal region has been shown to be necessary ...

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

“…The 25 residues, unresolved in the crystal structure, are most likely ordered in the CaM-stabilized reconstructions evidenced by the unoccupied density present in this region that can account for all of the missing residues. Another structural element clearly affected by CaM binding is the β-finger (or SI, small insertion, residues 831–844) in the hinge region (Zhang et al, 2001; Knudsen et al, 2003; Jones et al, 2004), possibly weakening contacts within the FMN domain. Thus, the effect of CaM binding is allosteric in nature, stabilizing a conformation of eNOS that primes the release of the FMN domain from the FAD-binding sub-domain by weakening autoinhibitory interactions (Salerno et al, 1997; Roman et al, 2000a; Roman and Masters, 2006; Salerno et al, 2013).…”

“…NOS homodimers are joined by an extensive protein-protein surface interface (~3000Å 2 ), as well as a tetrahedrally coordinated zinc atom, in the oxygenase domain dimer interface (Raman et al, 1998). In addition to the CaM-binding site, at least three CaM-responsive sequences reside in the reductase domains – an autoregulatory region (AR), originally identified as a ~40 residue sequence in the FMN-binding subdomain (Salerno et al, 1997; Daff et al, 1999; Lane and Gross, 2000; Montgomery et al, 2000), a C-terminal tail sequence (CT), which differs in length among the three isoforms (Roman et al, 2000; Roman and Masters, 2006), and a small insertion (β-finger) in the hinge region (Zhang et al, 2001; Knudsen et al, 2003; Jones et al, 2004). …”

Electron Transfer by Neuronal Nitric-oxide Synthase Is Regulated by Concerted Interaction of Calmodulin and Two Intrinsic Regulatory Elements

“…In this case, an additional CaM-sensitive element in the connecting region, originally identified by Zhang et al (51) as the "␤-finger," may play a role in either stabilizing the open conformation or in destabilizing a conformationally restricted state (52). This 15-residue ␤-finger structure, called the SI for "small insertion," is adjacent to the edge of the FMN-binding subdomain, proximate to the AR and, by inference, the CaM-binding site (52). This element, like AR, is inhibitory for electron transfer and NO production (52,53) in the absence of CaM.…”

“…This 15-residue ␤-finger structure, called the SI for "small insertion," is adjacent to the edge of the FMN-binding subdomain, proximate to the AR and, by inference, the CaM-binding site (52). This element, like AR, is inhibitory for electron transfer and NO production (52,53) in the absence of CaM. Its function appears to be masked in the presence of the AR, and it is only when AR is deleted that the regulatory effects of SI become apparent (53).…”

Nitric-oxide Synthase Output State

“…CaM has little or no effect on the thermodynamics of redox processes in NOS, suggesting that regulation is accomplished through modulation of electron transfer (15-18). A 40 -50-residue autoinhibitory insertion in the FMN binding domain marks the major difference between cNOSs and iNOS (19), but electron transfer modulation appears also to involve a much smaller insertion present in the subdomain region as well as C-terminal tail differences (20,21). Although no solved structure of the holoenzyme exists, both partial structures and structures of close homologs provide considerable information (22-24).…”