Protein structures determined in high resolution by

Protein structures determined in high resolution by X-ray crystallography and NMR techniques provide wealth of structural information on intra-domain and inter-domain protein interactions of small sized proteins. In addition, cryo-EM often provides low resolution structures of multi-component protein complexes which lack the intricate details of protein interactions involved in the cross talk of adjacent domains [18]. Despite the great advances in experimental methods of protein structure determination, the characterization of multi-domain protein complexes remains a challenging and often unreachable task [17]. Unavailability of a comprehensive sGC holoenzyme structure makes it difficult to elucidate the domain interactions and activation mechanism of the protein. The current study aims to determine the entire structure of hsGCαβ complex using computational protein structure modelling approaches. We applied knowledge based integrative protein structure modelling approach in which we utilized already reported experimental information that is available through X-ray crystallography and cryo-EM studies of hsGC homologs to predict full length multi-domain hsGCαβ heterodimer. Conserved orientations of adjacent domain partners in our query structure were predicted through template based (single or multiple chain threading) and template free (docking) methods. Biochemical constraints such as interaction residues at interface of neighboring domains were used as a benchmark to confirm their best possible relative orientations whereas cryo-EM fitting techniques (rigid and flexible map fitting) were applied on R. norvegicus sGC cryo-EM reconstruction to achieve quaternary structure organization of multi-domain hsGCαβ complex.
Methodology In the current study we have integrated comparative modelling, ab-initio protein modelling, protein-protein docking and pdgf receptor density map fitting techniques to determine the three dimensional (3D) domain organization of the hsGC quaternary structure.
Results
Discussion Soluble guanylate cyclase (sGC) is the primary receptor of cGMP signaling pathway involved in Nitric-oxide mediated production of cGMP molecules and activate a repertoire of downstream signaling pathways that induces smooth muscles relaxation and vasodilation as well as regulates proliferation of smooth muscle cells and platelet aggregation [1,2]. From 30 years of research, significant information has been reported on individual sGC domain structures but little has been known on the domain architecture in an active hsGC structure. Along with this, structural events involved in NO signal perception, regulation and propagation by sGC molecule in higher organism are still elusive. It has been reported in various studies that NO diffuse through the cell membrane and binds to a ferrous heme-iron meioty of hsGCβ-HNOX domain to form a transient 6 coordinate heme-NO complex [54]. This intermediate complex is highly unstable and immediately breaks the proximal histidine bond with ferrous ion (Fe2+) to produce a 5-coordinate heme complex state [55]. This bond breakage triggers slight movement of helix-F in βHNOX domain which brings about conformational change that is transmitted to the C-terminal cyclase domains [56]. This bond breakage between histidine and heme iron is thought to activate N-terminal cyclase domains but how this N-terminal to C terminal domains crosstalk/communication takes place and how this heme (iron)-histidine bond breakage signal propagates from βHNOX domain and directs the activation and folding of entire structure is still unclear [6]. The molecular architecture of full length hsGCαβ heterodimer predicted in the current study is strengthened by validating it through experimental studies. Structural similarities can be present even in proteins that share little sequence similarity, as structure is more strongly conserved than sequence. Relative position of the multiple domain decides the inter domain communication as well as the folding behavior of the proteins. Complex topology of hsGCαβ heterodimer was achieved by borrowing the conserved key residues at adjacent domain interfaces of homologs that influence their correct orientation during hsGCαβ folding mechanism (Fig. 7). Use of dimeric domain pairs as rigid bodies during density map fitting in cryo-EM actually narrow down the degree of freedom during manual adjustment. To propose the complete structure of hsGC, we tried to link the biological information with different structural and functional aspects of multi-domain protein.