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    • It is extremely difficult to predict a plausible conformer

    2021-10-19

    It is extremely difficult to predict a plausible conformer of hsGCαβ heterodimer with right orientation of multiple domains. Even though the template identities in case of dimeric domains were not high enough but still we modelled the entire heterodimeric structure of hsGCαβ with a reasonable and justifiable multi-domain organization of eukaryotic sGC. It is an established concept that conserved domains interact with more or less similar inter-domain geometry [37]. Keeping in view the relationship between the protein sequence and conservation of geometry among neighboring domains, we predicted the organization of the interaction surfaces in adjacent domain of same subunit as well as dimeric subunits from already known PDB structures and their related literature. Solvent accessibility of βHNOX-PAS (1–385 a.a) in contrast with an individual β-HNOX domain (1–194); results reported that solvent accessibility of signaling helix-F reduces probably due to the burial of signaling helix-F at the interface of β-HNOX-PAS domains [47]. This experimental data were used in this work to find the relative orientation inside the density map. Furthermore, mutagenesis study of specific periphery residues variants of signaling helix-F (D106K, T110R) and adjacent loop R116E drastically decreases the NO simulated cyclase activity. Mutation of single residue from signaling helix (D106K) mutation causes more than 500 fold inhibition of catalytic activity which is involved in cGMPs production. This aspartic Caspase-3/7 Inhibitor residue lies between H-NOX and PAS domains [15]. Histidine Kinase Signal Transduction (HKST) H-NOXA domain has structural similarity with PAS domain. PAS domain fold is commonly found in sensory proteins which are essential to sense the redox potential. PAS sensory domain detects small molecules (oxygen & light) and play a vital role in signal transduction. In sGC, PAS domain maintains the conserved dimeric pattern and it is postulated that they may anchor a secondary allosteric regulatory region in sGC. Being an evolutionary remnant, PAS domain may have role in sensing the Helix-F detachment signal from its preceding βHNOX domain. Dimer conformation of sGC is critical for signal transduction. It is known that seven residues from N-terminal residues of β-PAS domain interface are essential for dimerization with α PAS domain. A study validated the presence of similar NpSTHK-type dimer interface region in sGC by substituting alanines to eight putative PAS domain dimer interface residues. Out of those eight mutations two residues i.e. (F285, Q368) from α subunit two residues i.e. F217, Q309 from β subunit were found conserved [7] (see Fig. 7). Mutations of these residues significantly decrease the dimerization and NO-dependent signaling activity of sGC. Moreover, activity inhibition of sGC may be due to the fact that the overall dimer conformation is disturbed which is a prerequisite to sense the signaling helix [7,57]. Our predicted hsGC αβ-PAS model folds similarly as HKST H-NOXA domain dimerizes with its counterpart. Therefore we subjected the predicted αβ-PAS dimeric model to extend the multi-domain assembly of entire hsGC using protein-protein docking and cryo-EM map fitting. Chemical cross-linking studies confirm a parallel arrangement of coiled-coil domains. Dimer structure has lysine residues, lying parallel to each other and reported to be involved in the dimerization of this region. Alpha coiled-coil has K363, and K365 parallel to corresponding domain K364 these residues were under optimal distance for chemical cross-linking [14]. Furthermore, C-terminal region of coiled-coil domain which is essential for hetero-dimerization is also required for hsGC simulating activity. Adenylate cyclase (AC) domain is structurally homologous to sGC [58,59]. Therefore, activation mechanism of AC might be able to give insight in understanding the catalytic activity of sGC. sGC cyclase domains have conserved homologous motifs with AC. The residues which are required for cyclase catalytic activity are also conserved. For instance, α subunit cyclase D487 and D531 have electrostatic interaction with two Mg2+ ions, which further cyclize the nucleotide ligands (GTP, ATP) by releasing pyrophosphate. These residues are critical for the catalytic activity of cyclase. Moreover, the β subunit cyclase N548 has been proposed to attract the ribose ring. There are some more residues such as E473 and C541 which are thought to be necessary for purine base recognition. In addition, in both α, β subunits R573 and R552 are thought to have robust electrostatic interactions with GTP triphosphate group. The binding pocket residues convert the GTP to cyclic GMP (cGMP) [20], which may act as a secondary message to stimulate various downstream pathways with a wide range of physiological and pathophysiological implications.