Mapping the Intersubunit Interdomain FMN-Heme Interactions in Neuronal Nitric Oxide Synthase by Targeted Quantitative Cross-Linking Mass Spectrometry.

Nitric oxide synthase (NOS) in mammals is a family of multidomain proteins in which interdomain electron transfer (IET) is controlled by domain-domain interactions. Calmodulin (CaM) binds to the canonical CaM-binding site in the linker region between the FMN and heme domains of NOS and allows tethered FMN domain motions, enabling an intersubunit FMN-heme IET in the output state for NO production. Our previous cross-linking mass spectrometric (XL MS) results demonstrated site-specific protein dynamics in the CaM-responsive regions of rat neuronal NOS (nNOS) reductase construct, a monomeric protein [Jiang et al., Biochemistry, 2023, 62, 2232-2237]. In this work, we have extended our combined approach of XL MS structural mapping and AlphaFold structural prediction to examine the homodimeric nNOS oxygenase/FMN (oxyFMN) construct, an established model of the NOS output state. We employed parallel reaction monitoring (PRM) based quantitative XL MS (qXL MS) to assess the CaM-induced changes in interdomain dynamics and interactions. Intersubunit cross-links were identified by mapping the cross-links onto top AlphaFold structural models, which was complemented by comparing their relative abundances in the cross-linked dimeric and monomeric bands. Furthermore, contrasting the CaM-free and CaM-bound nNOS samples shows that CaM enables the formation of the intersubunit FMN-heme docking complex and that CaM binding induces extensive, allosteric conformational changes across the NOS regions. Moreover, the observed cross-links sites specifically respond to changes in ionic strength. This indicates that interdomain salt bridges are responsible for stabilizing and orienting the output state for efficient FMN-heme IET. Taken together, our targeted qXL MS results have revealed that CaM and ionic strength modulate specific dynamic changes in the CaM/FMN/heme complexes, particularly in the context of intersubunit interdomain FMN-heme interactions.

Probing calmodulin-NO synthase interactions via site-specific infrared spectroscopy: an introductory investigation.

Calmodulin (CaM) binds to a linker between the oxygenase and reductase domains of nitric oxide synthase (NOS) to regulate the functional conformational dynamics. Specific residues on the interdomain interface guide the domain-domain docking to facilitate the electron transfer in NOS. Notably, the docking interface between CaM and the heme-containing oxygenase domain of NOS is isoform specific, which is only beginning to be investigated. Toward advancing understanding of the distinct CaM-NOS docking interactions by infrared spectroscopy, we introduced a cyano-group as frequency-resolved vibrational probe into CaM individually and when associated with full-length and a bi-domain oxygenase/FMN construct of the inducible NOS isoform (iNOS). Site-specific, selective labeling with p-cyano-L-phenylalanine (CNF) by amber suppression of CaM bound to the iNOS has been accomplished by protein coexpression due to the instability of recombinant iNOS protein alone. We introduced CNF at residue 108, which is at the putative CaM-heme (NOS) docking interface. CNF was also introduced at residue 29, which is distant from the docking interface. FT IR data show that the 108 site is sensitive to CaM-NOS complex formation, while insensitivity to its association with the iNOS protein or peptide was observed for the 29 site. Moreover, narrowing of the IR bands at residue 108 suggests the C≡N probe experiences a more limited distribution of environments, indicating side chain restriction apparent for the complex with iNOS. This initial work sets the stage for residue-specific characterizations of structural dynamics of the docked states of NOS proteins.

Probing Protein Dynamics in Neuronal Nitric Oxide Synthase by Quantitative Cross-Linking Mass Spectrometry.

Nitric oxide synthase (NOS) is responsible for the biosynthesis of nitric oxide (NO), an important signaling molecule controlling diverse physiological processes such as neurotransmission and vasodilation. Neuronal NOS (nNOS) is a calmodulin (CaM)-controlled enzyme. In the absence of CaM, several intrinsic control elements, along with NADP+ binding, suppress electron transfer across the NOS domains. CaM binding relieves the inhibitory factors to promote the electron transport required for NO production. The regulatory dynamics of nNOS control elements are critical to governing NO signaling, yet mechanistic questions remain, because the intrinsic dynamics of NOS thwart traditional structural biology approaches. Here, we have employed cross-linking mass spectrometry (XL MS) to probe regulatory dynamics in nNOS, focusing on the CaM-responsive control elements. Quantitative XL MS revealed conformational changes differentiating the nNOS reductase (nNOSred) alone, nNOSred with NADP+, nNOS-CaM, and nNOS-CaM with NADP+. We observed distinct effects of CaM vs NADP+ on cross-linking patterns in nNOSred. CaM induces striking global changes, while the impact of NADP+ is primarily localized to the NADPH-binding subdomain. Moreover, CaM increases the abundance of intra-nNOS cross-links that are related to the formation of the inter-CaM-nNOS cross-links. Taken together, these XL MS results demonstrate that CaM and NADP+ site-specifically alter the nNOS conformational landscape.

Differential superoxide production in phosphorylated neuronal nitric oxide synthase mu and alpha variants.

Neuronal nitric oxide synthase (nNOS) is regulated by phosphorylation in vivo, yet the underlying biochemical mechanisms remain unclear, primarily due to difficulty in obtaining milligram quantities of phosphorylated nNOS protein; detailed spectroscopic and rapid kinetics investigations require purified protein samples at a concentration in the range of hundreds microM. Moreover, the functional diversity of the nNOS isoform is linked to its splice variants. Also of note is that determination of protein phosphorylation stoichiometry remains as a challenge. To address these issues, this study first expanded a recent genetic code expansion approach to produce phosphorylated rat nNOSµ and nNOSα holoproteins through site-specific incorporation of phosphoserine (pSer) at residues 1446 and 1412, respectively; this site is at the C-terminal tail region, a NOS-unique regulatory element. A quantitative mass spectrometric approach was then developed in-house to analyze unphosphorylated peptides in phosphatase-treated and -untreated phospho-nNOS proteins. The observed pSer-incorporation efficiency consistently exceeded 80%, showing high pSer-incorporation efficiency. Notably, EPR spin trapping results demonstrate that under l-arginine-depleted conditions, pSer1412 nNOSα presented a significant reduction in superoxide generation, whereas pSer1446 nNOSµ exhibited the opposite effect, compared to their unphosphorylated counterparts. This suggests that phosphorylation at the C-terminal tail has a regulatory effect on nNOS uncoupling that may differ between variant forms. Furthermore, the methodologies for incorporating pSer into large, complex protein and quantifying the percentage of phosphorylation in recombinant purified protein should be applicable to other protein systems.

Cytological dissection and molecular characterization of chromosome 1R derived from 'Burgas 2' common wheat.

Rye chromosome 1R harbors many agronomically important genes such as resistance genes for rusts. Using the gametocidal system, we dissected the 1R chromosome substituted for wheat chromosome 1B in a common wheat cultivar 'Burgas 2'. The gametocidal system induces chromosomal breakage in the 1R chromosome, as well as in wheat chromosomes. We cytologically examined a pool of prescreened common wheat plants that had been shown to have single or multiple rearranged 1R chromosomes and established 95 common wheat lines carrying single 1R segments. We conducted PCR analysis of these lines, termed '1R dissection lines', using 10 PCR-based 1R-specific markers. We mapped the 10 PCR-based markers along the 1R chromosome with the breakpoints of the 1R dissection lines. Based on the PCR result and the positions of the primary and secondary constrictions, we could separate the breakpoints of the rearranged 1R chromosomes into 12 regions along the 1R chromosome. On the other hand, using the breakpoints, we could separate the PCR-based markers from each other except for two markers. These dissection lines are useful in mapping DNA markers and may facilitate the construction of contig maps.