Investigation of structural differences between wild-type and mutant forms of mutsα by molecular dynamics simulations

Abstract

Cancer is the most common cause of death after cardiovascular diseases. Among the cancer types, colon cancer, which is very common in our country and the world, endangers human health. The aim of the studies in this thesis is to determine the changes in the structure and dynamics of the MutSα protein due to mutations in the protein complex associated with colon cancer. Molecular Dynamics (MD) Simulations provide important information about protein dynamics by providing the opportunity to observe changes in the structures and functions of proteins. Computational methods have been a remarkable field in recent years because they provide energetic, structural and dynamic information at the atomic level that cannot be obtained using experimental techniques. In this study, the effects of mutations in MutSα on protein structure and dynamics were analyzed using computational and theoretical methods. MutSα complex, formed by the MSH2-MSH6 heterodimer is responsible for the recognition and repair of mismatches. Some mutations in MutSα have been associated with cancer studies. Certain mutations that occur on protein complex cause the accumulation of mutations in the cell and eventually causing cancer. Only some of the mutations in protein complex have been associated with cancer, but the effect of some mutations, defined as pathogenic variation, on protein activity is not yet known. The MSH2 and MSH6 proteins that make up the MutSα complex are divided into five major regions: the mismatch binding domain, the connector domain, the lever domain, the clamp region, and ATPase domain. The mismatch binding domain recognizes mismatched bases and binds to these bases. The connector domain is responsible for allosteric signaling, while the lever region is thought to be responsible for signaling between the mismatch binding domain and the ATPase domain. The clamp domain stabilizes the DNA by interacting and bonding with the DNA backbone. The ATPase domain is the region where the energy required for the protein to move along the DNA is obtained by hydrolysis of ATP. MutSα complex detects mismatches by scanning the DNA and binds to mismatch region to start repair mechanisms. In a healthy cell, DNA repair mechanisms work smoothly, DNA damages are repaired, and apoptosis occurs in case of irreparable damage. However, mutations in the proteins in the DNA repair mechanism prevent the correct functioning of the DNA repair mechanism, causing the accumulation of damaged/mutant DNA in the cell and causing tumor cells. Studies have revealed that specific mutations in MutSα, one of the DNA mismatch repair proteins, can be hereditary and cause cancer. Some mutations obtained as a result of genetic screening tests by Prof. Dr. Levent Doğanay and his research group, with samples taken from individuals with a family history of colon cancer have been reported to our study group. Ala733Thr, Arg577Cys and Ser127Asn mutations were detected, and the effects of these mutations on MutSα without DNA were investigated by performing all-atom MD simulations. In order to look at the changes that these mutations may cause in protein function, first of all, systems were prepared for the MD simulations of the MutSα protein using structures with PDB ID: 2O8B. Two replica systems were created for each construct to compare the simulation results for wild-type and mutant proteins. While simulating the proteins, the required ion concentration was provided by considering the cell's physiological conditions. After the structures were dissolved in water, short minimization and equilibrium simulations were performed. The equilibrated structures were simulated for 200ns. Two replicate simulations were carried out for wild-type and three mutant proteins, and totaling 1600ns MD simulations were performed. Root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), heat capacity, interaction analysis and principal components analysis (PCA) were performed to investigate the effect of the mutations on the MutSα complex. The RMSD analysis gives information about whether the simulation has reached equilibrium and whether there has been a conformational change. The average RMSD values of mutant proteins were higher than the wild-type protein. Therefore, these mutations resulted in changes in protein conformation and dynamics. RMSF analysis is used to provide insight about structural flexibility, mobility and thermal stability. In this thesis, fluctuations were investigated for five domains separately and it was observed that the mutant proteins had similar or higher fluctuations than the wild-type protein in general. For the wild-type, the region with the highest fluctuations was observed to be the clamp region. Heat capacity can provide information about the thermodynamic properties of the protein. The findings obtained as a result of the analysis showed higher heat capacities for all mutants compared to that of the WT protein and show that the thermodynamic properties of the protein may have been differentiated. As a result of the interaction analysis, it was observed that the mutants had changes in hydrogen bond and salt bridge interactions compared to the WT protein. With PCA analysis, the two most dominant movements in the protein were obtained. Conformations were projected onto principal components and based on the conformational space sampled by the proteins during the MD simulations heat map surfaces were constructed. In this study, the effects of mutations in the DNA-free MutSα protein structure and dynamics were investigated and it was concluded that mutant structures showed different thermodynamic and structural properties compared to wild-type.