In Silico Study On The Regulation Mechanism Of Soluble Guanylate Cyclase

Soluble guanylate cyclase (sGC) is an important heme protein for production of the intracellular second messenger cyclic guanosine monophosphate (cGMP). The increase of intracellular cGMP mediated by sGC is related to many physiological processes including cellular growth and contractility, cardiovascular homeostasis, inflammation, sensory transduction, and neuronal plasticity and learning [2]. The C-terminal regions of the two subunits make up the cyclase domain, while the N-terminal region of theβsubunit has been identified as the heme binding domain; both subunits are necessary for enzyme activity. Mammalian sGC is a heterodimeric heme enzyme composed of oneαsubunit and oneβsubunit. In the native enzyme, the heme prosthetic group is bound to the sGC heme binding domain (or heme nitric oxide and/or oxygen binding domain, HNOX domain) of theβsubunit via the coordinated bond withβ105-His and is stabilized through the interaction of its propionic acid groups with Y135, S137 and R139 residues of theβsubunit. Nitric oxide (NO) bound to the distal heme coordination site can induce breaking the iron-histidine (Fe-Nhis) bond, which is accompanied by a 200-400-fold enhancement of sGC catalytic ability. Carbon monoxide (CO) can activate sGC as well (4-6-fold enhancement), and the activation level can be boosted to a degree similar to that of NO by further addition of an allosteric modulator, 3-(5'-hydroxymethyl- 2'furyl)-1-benzylindazole (YC-1). Interestingly, recent reports indicate that the synergistic activation of sGC by CO and YC-1 also involves cleaving the Fe-Nhis bond. This evidence suggests that cleavage of the Fe-Nhis bond is the key event in the sGC activation process.The function of sGC in vivo is the major interest of scientist. The study on the structure and function of sGC should be helpful to understand the mechanism of sGC-cGMP signaling pathway. Elucidating the regulation mechanism of sGC is important not only for understanding the activation mechanism of sGC but also for future drug development. However, it is difficult to obtain the crystal structure or solution structure data of sGC, the mechanism related to detail structure is still unclear. This made hypothesis related to activation of sGC hard to be validated. Investigating the structure information of sGC should be helpful to the research of the regulation mechanism of sGC. A novel HNOX (Heme-NO and oxygen-binding) family of ligand binding domains were identified as homologous to the heme binding domain of sGC in bacteria and animals. Recently, two members of the family were crystallized. The secondary structure prediction also suggests that sGC will have the same HNOX domain folding pattern. These data make it possible to gain further insight into structural details related to sGC. Spectroscopic and biochemical methods have been widely used to collect information on the sGC heme binding domain. The sGC-HNOX domain possesses heme characteristics similar to those of the full-length enzyme during the binding process of gaseous activators. Studies exploring the heme binding domain remain a heated topic investigating the activation mechanism of sGC.In this work, the structural models of the sGC-HNOX domain were constructed using the MODELLER package, by homology with the crystal structure of the Ns-HNOX domain and Ts-HNOX domain. The models were screened from thousands of models by taking the DOPE score, Ramachandran plot profiles and G-scores into consideration. The well-defined models (AT,4H,7H,8H) were then selected for application in MD simulations with explicit water solvent. The objective of our study was to explore the regulation mechanism of sGC. The simulations were carried out aimed to solve the following issues:(1) the function of Y-S-R motif in sGC-HNOX domain; (2) the cleavage mechanism of Fe-His bond; (3) the primary response of sGC-HNOX domain to the cleavage Fe-His bond.Our results could be ranked as three parts: (1) In the simulation of structural models of sGC-HNOX domain with D1, D2 and D3 restrained, the RMSD, RMSF, Cαmotions along the eigenvector, the secondary RMSF and the relative distance from center mass of different structural elements to the center mass of protein were analysed. All data indicated that the structural models of the sGC-HNOX domain become stable with the restraints of D1, D2 and D3. Additionally, the restraints have no influence on the bending extent of heme plane. This evidence suggest that the role of Y-S-R motif is to make the heme moiety and protein more like a integrality. (2) In the family of sGC, Leu104 and Leu108 are conserved as the LHxxL motif. Homology modeling results show that Leu104 and Leu108 are located between the heme pocket and the water solvent. MD results suggest that the motions of the two residues form a gate, with open and closed states that regulate the transit of the water molecule to the heme pocket. The quantum mechanical single-point energy calculations indicated that the water molecule docking on the heme proximal side can further weaken the Fe-His bond by making it thermodynamically unstable. This is possible related to the cleavage of the Fe-His bond. (3) To expand the primary response of the sGC-HNOX domain to the cleavage event, the Fe-His bond was released at 6 ns of a 10-ns molecular dynamics simulation. An instant increment of Cα-RMSD over L2 (Loop2, residues 124-130) was found after the cleavage of the Fe-His bond, which was consistent with the principle component analysis. The energy analysis results suggest that the motions of L2 are energetic. Based on the results, energetic conformational transformation of L2 is identified as the primary response of the sGC HNOX domain to the cleavage of the Fe-His bond. The expression of the domains of sGC has suggested that there is a direct interaction between the HNOX domain and the cyclase domain. The activation information from HNOX domain upon the cleavage of the Fe-His bond has been possibly propagated to the cyclase domain by the conformational transformation of L2.The function of Y-S-R motif in sGC-HNOX domain, the cleavage mechanism of Fe-His bond and the primary response of sGC-HNOX domain to the cleavage of Fe-His bond is the main problems related to the regulation mechanism of sGC. In this work, the structural information related to the issues above was collected based the homology models of sGC-HNOX domain. Our results were expected could facilitate future theory and experimental study on regulation mechanism of sGC.