18 research outputs found
Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments.
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
Structural basis for ALK2/BMPR2 receptor complex signaling through kinase domain oligomerization.
Upon ligand binding, bone morphogenetic protein (BMP) receptors form active tetrameric complexes, comprised of two type I and two type II receptors, which then transmit signals to SMAD proteins. The link between receptor tetramerization and the mechanism of kinase activation, however, has not been elucidated. Here, using hydrogen deuterium exchange mass spectrometry (HDX-MS), small angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations, combined with analysis of SMAD signaling, we show that the kinase domain of the type I receptor ALK2 and type II receptor BMPR2 form a heterodimeric complex via their C-terminal lobes. Formation of this dimer is essential for ligand-induced receptor signaling and is targeted by mutations in BMPR2 in patients with pulmonary arterial hypertension (PAH). We further show that the type I/type II kinase domain heterodimer serves as the scaffold for assembly of the active tetrameric receptor complexes to enable phosphorylation of the GS domain and activation of SMADs
Single-particle EM reveals the higher-order domain architecture of soluble guanylate 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
A Structural Basis for the Regulation of an H‑NOX-Associated Cyclic-di-GMP Synthase/Phosphodiesterase Enzyme by Nitric Oxide-Bound H‑NOX
Biofilms
are surface-attached communities of bacteria enclosed
in a polysaccharide matrix. Bacteria in a biofilm are extremely resistant
to antibiotics. Several recent reports have linked the signaling molecule
nitric oxide (NO) with biofilm dispersal. We have previously reported
that an H-NOX (heme-nitric oxide/oxygen binding) protein in the biofilm-dwelling
bacterium <i>Shewanella woodyi</i> mediates NO-induced biofilm
dispersal. In <i>S. woodyi</i>, H-NOX (<i>Sw</i>H-NOX) is cocistronic with a gene encoding a dual-functioning diguanylate
cyclase/phosphodiesterase
enzyme, designated here as HaCE (H-NOX-associated cyclic-di-GMP processing
enzyme). Enzymes such as these are responsible for regulating the
intracellular concentrations of cyclic-di-GMP, a secondary signaling
molecule essential to biofilm formation in bacteria. We have demonstrated
that NO-bound <i>Sw</i>H-NOX regulates both enzymatic activities
of <i>Sw</i>HaCE, resulting in decreased cellular cyclic-di-GMP
levels and disruption of biofilm formation. Thus, H-NOX/HaCE represents
a potential drug target for regulating biofilm formation. In this
work, the <i>Sw</i>H-NOX surface residues critical for the
formation of a protein complex with <i>Sw</i>HaCE are identified
using nuclear magnetic resonance, fluorescence quenching, and cosedimentation.
Enzyme assays confirm this protein–protein interface and its
importance for H-NOX/HaCE function