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Title: Structural and Functional Studies of Nitric Oxide Signaling Pathways
Authors: Khalid, Rana Rehan
Keywords: Biological & Medical Sciences
Issue Date: 2020
Publisher: COMSATS University, Islamabad.
Abstract: Structural and Functional Studies of Nitric Oxide Signaling Pathways Cardiovascular dysfunction (CVD) is the major cause of mortality and morbidity in Pakistan with a prevalence rate in 2/3rd of the population, and globally, 30% of all deaths are associated with it. To cope with these alarming facts, research on cellular signaling related to CVDs has gained remarkable importance. Soluble guanylate cyclase (sGC) is a key molecule involved in the regulation of the cardiovascular system. sGC is a large heterodimer protein consisting of α and β monomer each spanning over 600 residues. In each monomer, 4 domains termed as H-NOX (Heme containing NO and Oxygen binding), PAS, CC (coiled-coil), and cyclase domains are present. In β H-NOX, the event of NO binding with a histidine borne 5 coordinate heme cofactor generates a molecular cascade that activates C-terminal cyclase domain to catalyze guanosine-5'- triphosphate (GTP) into cyclic guanosine monophosphate (cGMP). cGMP being a second messenger controls downstream signaling cascades related to vasodilation. Thus, sGC has long been a hot target to improve the endothelial blood vascular tone. Despite decades of research, the mechanism of NO-mediated sGC activation and signal transduction remains a topic of debate. Since the structure of the complete molecule of sGC could not be solved owing to its size and instability, its different domains have been studied through various experimental methods. Recently a 30Å resolution cryo-electron microscopy (EM) map of the protein has been reported but that could merely ascertain the contours of quaternary structure content. This study improvises the aforementioned information about various structural attributes of sGC to come up with a complete heterodimeric structure of the molecule. Moreover, an extensive dynamic analysis was employed to delineate the activation mechanism of the human sGC (hsGC) regulatory domain under the effect of NO and other diatomic molecules known to bind with and activate its H-NOX domain such as O2 and CO. The study outcomes revealed that the bond between iron and histidine dilated more in the NO bound H-NOX complex compared to the CO, O2 ligated systems. H-NOX signaling helix-f and loops α, β, and γ were observed to be critical regions, which exhibited structural transformations functionally crucial for xi signal transduction. Further, we also explored the structural dynamics and binding mode patterns of sGC modulators, the BAY compounds, (BAY60-2770, BAY58-2667) ligated with human (hH NOX) and bacterial H-NOX (bH-NOX) domains. Insight into BAY compounds activation mechanism unveiled that both activators have some potential to elicit the signaling across helix-f, loops α and β residues, but these variations are less pronounced compared to NO bound system. The behavior of the BAY bound hH-NOX domain was comparatively different from BAY bound bH-NOX domain due to the difference of amino residues within their respective binding pockets. In BAY bound hH-NOX domain R40, F74, D84, T85, R88, and Y112 residues are significantly contributing in NO and heme independent activation of hH-NOX domain. The explored regulatory domain interaction pattern was taken as a contact footprint filter to screen 4.8 million compounds; this led to extract potential compound by applying a hierarchical docking protocol. Comparative molecular dynamics studies of the cyclase domains of hsGC and human adenylate reveal cyclase revealed the catalytic mechanism and structural rearrangements putatively responsible for the catalysis of GTP to cGMP. This study thus comes up with angstrom details of the entire sGC structure; explores the molecular detail of the activation mechanism of its H-NOX domain on NO, O2, and CO binding; compares the mechanism of NO driven activation with that of sGC modulator BAY compounds; defines a contact footprint filter to screen novel ligands to activate H-NOX and explores in detail the catalysis of sGC cyclase domain whereby GTP is converted to cGMP.
Gov't Doc #: 21812
Appears in Collections:PhD Thesis of All Public / Private Sector Universities / DAIs.

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