Conformational Dynamics And Modulation Mechanism Of Neuronal Calcium Sensor-1 Protein

Objective: Physical activity and the brain have a dynamic, bi-directional relationship. Molecular mechanisms of exercise and brain health may involve neuronal growth factors, immune factors and stress hormones, and many other signaling proteins like neuronal calcium sensor(NCS) proteins. Physical exercise enhances cognitive function, and one of the important protein linking the physical activity and cognitive function is the Neuronal calcium sensor-1(NCS-1) proteins,which has been shown to render different effects of training on cognitive function.Neuronal calcium sensor-1 is an important factor controlling the animal’s response to temperature change. It is unclear that NCS-1 inhibits the locomotion of the C. elegant following temperature elevation. To probe the molecular mechanism of NCS-1restraining the motion and to reveal the conformational dynamic and the structural changed key factors of NCS-1 protein, we investigated the global structural properties and the conformational dynamics of NCS-1 protein in the solvent environment change and the local structural change by extensive all-atom molecular dynamics(MD)simulations, which afford theoretical basis for the future research.Methods: All-atom molecular dynamics(MD) simulations was used in the thesis.MD simulations were carried out using the GROMACS 4.5.3 software package and the CHARMM27 force field with CMAP corrections. The simulation box type was rhombic dodecahedron and the protein located in the center with the distance to the box border of 1.0 nm. The TIP3 P water model was used. Additional NaCl was added to the system with a concentration of 0.1/0.3 M. The solute and solvent were separately coupled to external temperature bath using velocity rescaling method and pressure bath using Parrinello-Rahman method. The temperature and pressure were maintained at 310/316 K and 1 bar using coupling constants of 0.1 and 1.0 ps,respectively. Bond lengths with hydrogen atoms were constrained by the LINCS and SETTLE algorithms, allowing an integration time step of 2 fs. The particle mesh Ewald(PME) method was used to calculate the electrostatic interaction with a real space cutoff of 1.0 nm, and the van der Waals interactions were calculated using a cutoff of 1.4 nm.Results:(1) Human NCS-1 protein was sensitive to the solvent temperature.With the temperature elevated, the secondary structure content at 316 K was similar tothat at 310 K, whereas the global protein structure was expanded and the free energy landscape was changed. The elevated temperature resulted in loop 3(L3) adopting an extended state that more occupied the hydrophobic crevice(HC), thereby blocking the binding of NCS-1 protein to its receptors. The number of suboptimal communication paths starting at residue D176 and ending at V190 in the L3 C-terminal tail was reduced with the elevation of temperature. The dynamic community network analysis suggested that the correlation between the N- and C-domain was weakened and the intradomain coupling was strengthened at 316 K. The elevation of temperature reduces the number of the salt bridges, especially in the C-domain, which may induce the conformational dynamic changes.(2) NCS-1 protein was also sensitive to the small variation of NaCl concentration. With the NaCl concentration increased, the secondary structure content was conserved well, whereas the global protein structure and HC was expanded simultaneously. Loop 3 collapsed in HC and exposed the binding sites of the hydrophobic crevice at 0.3 M NaCl concentration. The number of suboptimal communication paths starting at residue D176 and ending at V190 in the C-terminal tail L3 was reduced with the elevation of NaCl concentration. The correlation between the N- and C-domain is weakened and the intradomain coupling is strengthened at 0.3 M NaCl concentration. The elevation of NaCl concentration reduced the number of the salt bridges, especially in the C-domain. This study suggests that the small variation of NaCl concentration results in the correlation between the residues, the dynamic community network and salt bridges altered. This may be the principal reasons for the conformational dynamic changes of the human NCS-1 protein.(3) Our cumulative 2-μs MD simulations demonstrated that the secondary structure content of the wild type NCS-1 was similar to that of the NCS-1Δ176-190 variant. L3 deletion increased the structural flexibility of the C-domain and the distant N-domain. The community network analysis illustrated that C-terminal tail truncation weakened the inter-domain correlation. Moreover, our data showed that the variant significantly disrupted the salt-bridges network and expanded simultaneously the global structure and HC. These conformational changes caused by C-terminal tail truncation may affect the regulation of target interactions. Our study provides atomic details of the conformational dynamics effects of the C-terminal tail on human wild type NCS-1.(4) R102 Q mutation in NCS-1 protein dramatically reduces the flexibility of loops L2 and L3, and facilitates L3 in a more extended state to occupy the hydrophobic crevice to a larger extent, and significantly affects theinter-segment salt-bridges, and changes the subspace of the free energy landscape of NCS-1 protein. Analysis of salt bridge network in both WT and the R102 Q variant demonstrates that the R102Q-mutation-induced salt bridge alternations play a critical role on the reduced flexibility of L2 and L3. These results reveal the important role of salt bridges on the structural properties of NCS-1 protein and that R102 Q mutation disables the dynamics relocation of C-terminus, which may block the binding of NCS-1 protein to its receptors. This study may provide structural insights into the autistic spectrum disorder associated with R102 Q mutation.Conclusions:(1) Human NCS-1 protein was sensitive to the small variation of the solvent temperature and NaCl concentration. The correlation between the residues and altered salt bridges may be the key factors on protein conformation. The main reasons affecting protein function is the positional relation between L3 and HC.(2)L3 covered the binding sites inside the HC and may affect the protein function, which may protect the body not to be hurt by the high temperature.(3) NCS-1Δ176-190 variant is still able to maintain a well-organized structure, albeit with a higher flexibility, the largely opened hydrophobic crevice may make binding of incoming ligand possible, indicating a functionally active NCS-1Δ176-190 variant.(4) Our study suggests the possible way to restore the function of R102 Q mutant of NCS-1protein need to make the C-terminus more flexible to allow NCS-1 to interact with its binding ligands.