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

Rachel J Jones,Susan M.E Smith,Ying Tong Gao,Bradley S Demay,Kevin J Mann,Kathleen M Salerno,John C Salerno

doi:10.1074/jbc.m402808200

Rachel J Jones, Susan M.E Smith + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m402808200

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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 beta 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.

Highlights

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
We reasoned that the standard alanine mutant would not be informative in this case because Gly and Ala both have small side chains associated with sharp turns
Identified regions involved in the control of nitric oxide (NO) synthesis by Ca2ϩ/CaM have either been CaM binding sites or elements involved in suppressing activity in the absence of Ca2ϩ/CaM

Summary

EXPERIMENTAL PROCEDURES

Materials—All chemicals used for purification were obtained from Sigma. The genes for eNOS and nNOS were gifts from Professor B. Expression and Purification of Wild Type and Mutant Bovine eNOS and Rat nNOS—Expression and purification of bovine eNOS and rat nNOS were performed using procedures similar to those described previously (33). NO production was assayed using the Griess assay as adapted for microtiter plates (34); calcium dependence was measured using EDTA as a Ca2ϩ buffer system. NO production by NOS isoforms was routinely assayed with different buffering systems because eNOS is much more active in MOPS, whereas iNOS and nNOS work well in Tris. The 500-␮l reaction mixture contained 50 mM Naϩ/Tris buffer, pH 7.5, 50 ␮M EDTA, 50 ␮M NADPH, 50 ␮M horse heart cytochrome c, and ϳ10 nM nNOS. The BioRad DC protein kit, an adaptation of the method of Lowry et al (35), provided the primary measurements; a correction for tyrosine and tryptophan content was applied. Mutants were sequenced at the State University of New York at Albany Center for Functional Genomics sequencing facility

RESULTS

Small Insertion Function in the Control of NO Synthases

TAL deletion mutant

DISCUSSION