Theoretical Studies On The Interactions Between Ligands And SGC Heme Binding Domain

As a pivotal protein in NO-cGMP signaling pathway, sGC regulate many physiological phenomena sucu as the cardiovascular system, nervous system and the apoptosis. By means of data mining and bioinformatics, the heme binding domain of sGC were classified as new conserved family, named H-NOX family. Several crystal structures of prokaryotic H-NOX had been resolved and they share a strong sequence identity with sGC. Some of prokaryotic H-NOX proteins could bind oxygen tightly and some of them, like sGC did not have the ability of oxygen binding.In the absence of the crystal structure of sGC, We employ the structures of prokaryotic H-NOX Tt H-NOX and Ns H-NOX as template, use multiple sequence alignment technology to construct the homologous model of sGC heme binding domain with MODELLER package. WHAT CHECK package was used to evaluated out model, the results indicated that our sGC H-HOX model were in good conformation. In order to optimize and equalize our structure, We used sGC H-NOX as a initial structure, the NAMD molecular dynamics package and CHARMM force field was employed for perform the optimization. The RMSD value of 4ns MD trajectories showed that the sGC H-NOX model was stable during our simulations. In addition, many experimental results could be reproduced in our MD simulations such as the non-planar conformation of heme group in the H-NOX family, the conserved YSR motif stabilize the heme group via the hydrogen network interactions.By means of Locally Enhanced Sampling molecular dynamic simulation, the ligand migration pathways in H-NOX proteins had been investigated in the dissertation. Although Tt H-NOX and Ns H-NOX are great similar in fold, the ligand migration pathways in the two proteins are quite different. In Tt H-NOX, the distal heme pocket is defined by N74, W9 and Y140. The hydrogen network contributed by these three amino acids make a very narrow volume of distal pocket about 21 ?3. The small volume of distal packet increases the rebind process of oxygen molecule, therefore facilitates the oxygen binding. In Ns H-NOX, the heme distal pocket is hydrophobic and has a relatively large size of distal pocket, 75 ?3. By inspection of our sGC model, we found the distal pocket of sGC has a more hydrophobic environment due to lacking of the polar residues and an even larger cavity volume in distal pocket. Compared with the LES MD results of the Tt H-NOX and Ns H-NOS, we found that the gas escape rate is very fast in Ns H-NOX. Therefore, we speculate that the residues constitution of distal pocket and the ligand migration pathways make great contributions to the ligand discrimination. In order to verify the liagnd migration modulated by tunnel systems, we conducted several mutate LES MD simulations. M140 in Ns H-NOX play a key role in gating the ligand migration and the W74 in Ns H-NOX had a strong steric effect in blocking ligand migration. We introduced Y to the site 140 and N to the site 74. The M140Y. The M140Y results showed that the introduction of Y block the Ns H-NOX short pathway while the W74N opened a new channel which existed in Tt H-NOX. The Tt H-NOX Y140F and N74L dual mutant did not change the ligand migration pathway but speed up the rate of ligand escaption.In order to interpretation the sGC activation mechanisms by RNO on the point of view of molecular orbital theory and coordination chemistry, we calculated the interactions between NO, CO, RNO(R = H, CH3, CH3CH2, CH2CH) and the FeP(Im) model by means of B3LYP through Density functional theory (DFT). After the structure optimization, we found that as the different nature of the ligand molecule, the bond length of the iron atom and the imidazole group are also different.The longest distance between the iron and the imidazole was identified in the model of FeP(Im)-NO complex due to the trans effect of NO. We also calculated the FeP (Im) - ligand vibration frequency of Raman spectra, compared with the experimental data, our results more accurately matched the experimental data, the vibrational modes of porphine ring were well assigned. Our calculations can provide good theoretical models to perform further experiments.