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  • To solve the atomic structures of several individual

    2021-10-16

    To solve the atomic structures of several individual domains and a complete molecular architecture of sGC, tangible experimental research work has been done which resulted in a cryo-electron density map of R. norvegicus. However, the full structure of the human sGC remains to be determined. In the present study, we employed comparative modelling approaches to build individual models for the individual protein domains and then assembled them into their correct relative orientation using the single chain threading approaches. As hsGC is a multi-domain protein complex, after getting the plausible orientation of adjacent domains, we used protein-protein docking to figure out the orientation of successive domains in a folded state. To validate the aforementioned multi-domain assembly approach, we also performed sequence based ab inito multi-domain assembly of hsGC and generated a model of α and β subunits. Assembled structures of both subunits were then used for flexible fitting by MVP fit tool [29] in which COTH and SPRING [24] generated orientations of two adjacent domain structures (αβ-PAS, αβ-coiled-coil, αβ-cyclase), as well as protein-protein docking based conformation of multiple domains (α-HNOX/β-HNOX, αβ-PAS & αβ-PAS/-β-HNOX) was retained. We predicted a model of the hsGC heterodimer by fulfilling the spatial restraints reported in already available cross-linking studies, crystal structures and cryo-EM density maps of homologous proteins. The proposed model is consistent with the biochemical structure to function data because of the incorporation of physical and biochemical constraints of sGC. This model will become the starting point for understanding the dynamic characterization of sGC, to disclose the signal transduction mechanism between domains.
    Conclusion The structure of the enzyme is important to understanding its role in performing vital functions it plays in controlling blood pressure and Dacarbazine sale relaxation. Current study is primarily focused on understanding the structural organization of the human soluble Guanylate Cyclase (hsGC). Modelling of hsGC is a very non-trivial study mainly because it involves the assembly of multiple domain structures. We introduced a new protocol to build a high-resolution complex structural model of hsGC by the combination of the state-art-the-art tertiary and quaternary protein structure prediction algorithms (using I-TASSER and SPRING), followed by the cryo-EM density map fitting (using MVP-fit). The detailed model analyses unveil close consistency of the overall topology and domain orientation of the predicted model with the experimental data of ligand binding and peptide docking, which demonstrate the reliability and robustness of the hybrid structural model. This study would help to unveil the inter-domain communication upon nitrogen oxide binding, which is believed to play a significant role in the hsGC associated human diseases.
    Author contributions
    Funding The authors would also like to thank the International Research Support Initiative Program (IRSIP), National Research Program for Universities (NRPU-4050) by Higher Education Commission (HEC) Pakistan and 2216 Research Fellowship Program for International Researchers by TUBITAK. This research was supported in part by National Institute of General Medical Sciences [GM083107, GM116960], National Institute of Allergy and Infectious Diseases [AI134678], and National Science Foundation [DBI1564756].
    Acknowledgments Support by the High Performance Computing facility of “Zhang Lab” University of Michigan is acknowledged.
    Soluble guanylate cyclase (sGC) is an enzyme that catalyzes the conversion of guanosine 5′-triphospate (GTP) to the second messenger guanosine 3′,5′-cyclic monophosphate (cGMP). sGC carries a prosthetic heme groups that serves as a nitric oxide (NO) sensor, which activates sGC activity by up to 400-fold. Increased levels of cGMP have been shown to lead to smooth muscle relaxation, inhibition of platelet aggregation, anti-apoptotic and anti-inflammatory effects. On the other hand, overactivation of the sGC/cGMP signaling pathway has been shown to occur in several pathological conditions such as during sepsis and neurodegenerative disorders. Compounds that inhibit the activity of sGC could serve as potential therapeutic agents as well as chemical biology tools that will aid in unravelling the role of sGC-regulated pathways in vivo.