Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.

Nitric oxide (NO) produced by NO synthase (NOS) participates in diverse physiological processes such as vasodilation, neurotransmission, and the innate immune response. Mammalian NOS isoforms are homodimers composed of two domains connected by an intervening calmodulin-binding region. The N-terminal oxidase domain binds heme and tetrahydrobiopterin and the arginine substrate. The C-terminal reductase domain binds FAD and FMN and the cosubstrate NADPH. Although several highresolution structures of individual NOS domains have been reported, a structure of a NOS holoenzyme has remained elusive. Determination of the higher-order domain architecture of NOS is essential to elucidate the molecular underpinnings of NO formation. In particular, the pathway of electron transfer from FMN to heme, and the mechanism through which calmodulin activates this electron transfer, are largely unknown. In this report, hydrogendeuterium exchange mass spectrometry was used to map critical NOS interaction surfaces. Direct interactions between the heme domain, the FMN subdomain, and calmodulin were observed. These interaction surfaces were confirmed by kinetic studies of site-specific interface mutants. Integration of the hydrogen-deuterium exchange mass spectrometry results with computational docking resulted in models of the NOS heme and FMN subdomain bound to calmodulin. These models suggest a pathway for electron transfer from FMN to heme and a mechanism for calmodulin activation of this critical step.iNOS | NO signaling | flavin | hemoprotein N itric oxide (NO) has several essential functions in mammalian physiology. NO produced by the neuronal and endothelial nitric oxide synthase isoforms (nNOS and eNOS, respectively) initiates diverse signaling processes including vasodilation, myocardial function, and neurotransmission (1). The eNOS and nNOS isoforms are constitutively expressed and their activity responds to intracellular calcium concentrations. The inducible NOS isoform (iNOS) is transcriptionally controlled and produces NO as a cytotoxin at sites of inflammation or infection. Aberrant NO signaling contributes to a variety of diseases including stroke, hypertension, and neurodegeneration (2).Mammalian NOS isoforms are homodimeric and composed of two principal domains: the N-terminal oxidase domain and C-terminal reductase domain, which are connected by an intervening calmodulin (CaM) binding region (Fig. 1A). The N-terminal oxidase domain contains the heme and tetrahydrobiopterin cofactors and the binding site for the substrate arginine. The reductase domain is further divided into the FMN-binding subdomain and the FAD/NADPH-binding subdomains. This array of cofactors works in concert to catalyze the conversion of arginine to the intermediate N-hydroxyarginine and, ultimately, citrulline and NO. NADPH and oxygen are consumed in the process. During catalysis, electrons are shuttled from the reductase domain of one monomer to the heme domain of the opposite monomer in the homodimer (Fig. 1B) (1, 3). Electron transfer is initiat...

SUMMARY Soluble guanylate cyclase (sGC) is the primary mediator of nitric oxide (NO) signaling. NO binds the sGC heme cofactor stimulating synthesis of the second messenger cyclic-GMP (cGMP). As the central hub of NO/cGMP signaling pathways, sGC is important in diverse physiological processes such as vasodilation and neurotransmission. Nevertheless, the mechanisms underlying NO-induced cyclase activation in sGC remain unclear. Here, hydrogen/deuterium exchange mass spectrometry (HDX-MS) was employed to probe the NO-induced conformational changes of sGC. HDX-MS revealed NO-induced effects in several discrete regions. NO binding to the heme-NO/O2-binding (H-NOX) domain perturbs a signaling surface implicated in Per/Arnt/Sim (PAS) domain interactions. Furthermore, NO elicits striking conformational changes in the junction between the PAS and helical domains that propagate as perturbations throughout the adjoining helices. Ultimately, NO-binding stimulates the catalytic domain by contracting the active site pocket. Together, these conformational changes delineate an allosteric pathway linking NO-binding to activation of the catalytic domain.

Nitric oxide (NO) signaling pathways mediate diverse physiological functions, including vasodilation and neurotransmission. Soluble guanylate cyclase (sGC), the primary NO receptor, triggers downstream signaling cascades by producing the second messenger cGMP. NO binds the sGC heme cofactor to stimulate cyclase activity, yet the molecular mechanisms of cyclase activation remain obscure. Although structural models of the individual sGC domains are available, the structure of the full sGC heterodimer is unknown. Understanding the higher-order domain architecture of sGC is a prerequisite to elucidating the mechanisms of NO activation. We used protein footprinting to map interdomain interaction surfaces of the sGC signaling domains. Hydrogen/deuterium exchange mass spectrometry revealed direct interactions between the Per/Arnt/Sim domain and the heme-associated signaling helix of the heme-NO/O 2 binding (H-NOX) domain. Furthermore, interfaces between the H-NOX and catalytic domains were mapped using domain truncations and full-length sGC. The H-NOX domain buries surfaces of the α1 catalytic domain proximal to the cyclase active site, suggesting a signaling mechanism involving NO-induced derepression of catalytic activity. Together, our data reveal interdomain interactions responsible for communicating NO occupancy from H-NOX heme to the catalytic domain active site.HDX-MS | hemoprotein | gas receptor | nucleotide cyclase

Soluble guanylate cyclase (sGC) is the primary nitric oxide (NO) receptor in mammals and a central component of the NO-signaling pathway. The NO-signaling pathways mediate diverse physiological processes, including vasodilation, neurotransmission, and myocardial functions. sGC is a heterodimer assembled from two homologous subunits, each comprised of four domains. Although crystal structures of isolated domains have been reported, no structure is available for full-length sGC. We used single-particle electron microscopy to obtain the structure of the complete sGC heterodimer and determine its higher-order domain architecture. Overall, the protein is formed of two rigid modules: the catalytic dimer and the clustered Per/Art/Sim and heme-NO/O 2 -binding domains, connected by a parallel coiled coil at two hinge points. The quaternary assembly demonstrates a very high degree of flexibility. We captured hundreds of individual conformational snapshots of free sGC, NO-bound sGC, and guanosine-5′-[(α,β)-methylene]triphosphate-bound sGC. The molecular architecture and pronounced flexibility observed provides a significant step forward in understanding the mechanism of NO signaling.N itric oxide (NO) has emerged as an integral signaling molecule in biology. Soluble guanylate cyclase (sGC), the primary receptor of NO in mammals, binds NO via an Fe II heme cofactor leading to a several hundred-fold increase in 3,5-cyclic guanosine monophosphate (cGMP) synthesis. cGMP then acts as a second messenger, targeting phosphodiesterases, ion-gated channels, and cGMP-dependent protein kinases. These target proteins go on to regulate many critical physiological functions including vasodilation, platelet aggregation, neurotransmission, and myocardial functions (1, 2). Disruptions in NO signaling have been linked to hypertension, erectile dysfunction, neurodegeneration, stroke, and heart disease (3, 4). sGC has been the focus of small-molecule modulators of activity for therapeutic advantage. Riociguat, which is a stimulator of sGC, has recently been approved for treatment of pulmonary hypertension (5). However, the mechanistic details underlying the modulation of sGC catalytic activity by NO and other small molecules remain largely unknown. Determining the structure of the full-length sGC, free and in complex with NO, is therefore a prerequisite to understanding its function and for the design and improvement of therapeutics for treatment of diseases involving the NO/cGMP pathway.The most extensively studied and physiologically relevant isoform of sGC is the 150-kDa heterodimer containing one α1 and one β1 subunit. Each subunit is comprised of four modular domains: the N-terminal heme-NO/O 2 -binding (H-NOX), the Per/Arnt/Sim (PAS), the helical, and the C-terminal catalytic domain (Fig. 1). No structure of the complete holoenzyme is available to date, and its absence precludes answering key questions such as how NO occupancy of the N-terminal β H-NOX sensor domain is communicated to the C-terminal cyclase domain. Atomic models of isola...