Molecular dynamics simulations of chk2 kinase domain mutants

Abstract

ancer remains to be a key cause of death globally and remains one of the most dangerous diseases of our century. Breast cancer, in particular, is the most diagnosed malignancy in women worldwide. In recent years, developments in molecular biology have much contributed to the understanding of the molecular basis of cancer and have resulted in notable advancements in both diagnosis and treatment. These developments and studies highlight the key importance of resolving the complex molecular mechanisms that drive the progression of cancer in the body. However, despite these significant developments, the prevalence of cancer continues to increase because of changing lifestyles, persistent environmental risk factors, and greater exposure to carcinogens. A persistent increase in its prevalence in recent years has been globally observed. This increase can be accredited to changing lifestyles, dietary habits, and frequent exposure to carcinogenic substances from both internal and external sources. Various studies have evidently shown that breast cancer is the most commonly diagnosed cancer type among women and remains to be one of the leading causes of cancer related deaths. In this thesis, the serine/threonine checkpoint kinase 2 (CHK2) protein, which plays a crucial role in the DNA repair mechanism and is thought to be significantly involved in breast cancer, was examined. The primary aim of this study is to investigate the potential effects of variants of uncertain significance (VUS) in the kinase domain (KD) of the CHEK2 gene on the structure and dynamics of the CHK2 protein in individuals with a familial history of breast cancer. All analyses in this study are based on molecular dynamics (MD) simulations. MD simulations are a powerful computational method that enables the atomic-level understanding of biomolecular systems. Complementary to experimental techniques, this approach provides a detailed characterization of chemical systems and aids in optimizing experimental designs. MD simulations play a crucial role in various fields, including the investigation of disease mechanisms, drug discovery, and the modeling of biological processes. They offer insights into a wide range of biomolecular interactions, from protein-ligand binding to membrane structure and permeability, lipid-protein interactions, and protein dynamics. Supported by advancements in computational power and sophisticated algorithms, MD simulations continue to be an essential tool in scientific research and discovery. CHEK2 gene is a critical target in cancer prevention due to its essential role in maintaining genomic stability by regulating DNA repair processes and cell cycle checkpoints. Although CHK2 mutations have been associated with various cancers, many aspects of its catalytic cycle remain unclear and require further investigation. Understanding the molecular details of CHK2 function and dysregulation could potentially open the way for novel targeted therapeutic interventions and improved cancer management strategies. As a key tumor suppressor gene, CHEK2 encodes the CHK2 protein, which is vital for cell cycle checkpoint control and DNA repair pathways. CHK2 protein is a tumor suppressor protein that is critical in cell division and regulates cell growth and division. CHK2 protein interacts with many proteins involved in DNA damage repair mechanisms and regulations, such as ATM, BRCA1, CDC25, and P53. The fundamental structural components of CHK2 include the N-terminal SQ/TQ cluster domain (SCD), a central forkhead-associated (FHA) domain, and the C-terminal serine/threonine KD. The SCD contains phosphorylation sites essential for CHK2 substrate recognition, while the FHA domain enables CHK2 to detect and respond to DNA damage signals. The KD is responsible for phosphorylating target proteins, playing a pivotal role in CHK2's function as a regulator of cellular responses to genomic stress. In a separate study, genetic screening tests were performed at CHK2 using samples taken from people with a family history of breast cancer to identify VUSs. Among these we selected G306A, E351D, S356L, D409V, and V415I mutants, located at the KD of CHK2, for in silico investigation. Phosphorylated ADP-bound structure of CHK2 was subjected to all-atom MD simulations to examine the possible effects of these mutations on protein function. The CHK2 protein crystal structures with PDB IDs 2CN5 and 3I6U were utilized to build up the systems for the MD simulations. Two runs of MD simulations were performed for each of the five mutant proteins and the WT protein, totaling 3600 ns in length. Various analyses, such as heat capacity, root-mean-square deviation (RMSD), and root-mean-square fluctuation (RMSF), were conducted to evaluate the effect of mutations in the CHK2 protein and to analyze the results of the MD simulations performed. The stability of the protein structure and conformational changes during simulations were evaluated using RMSD, RMSF, and heat capacity analyses. The results showed that the RMSD values of three mutant proteins (G306A, D409V, and V415I) were higher compared to those of the WT CHK2 protein. This result suggests that mutations had an impact on conformational stability and protein dynamics. To investigate the source of high RMSD fluctuations in CHK2, the flexible FHA domain was excluded from the analysis, focusing solely on the more stable KD region (residues 213–501). This resulted in lower and smoother RMSD values across all mutations, indicating that the FHA domain contributes significantly to conformational variability. Notably, the sharp RMSD increase seen with the S356L mutation disappeared in the KD-only analysis, confirming the FHA domain's role in that fluctuation. These findings highlight the importance of considering regional structural effects when evaluating the impact of mutations. Mutant proteins show lower RMSF values in the glycine-rich loop in the CHK2 KD and higher RMSF values in the A-loop region compared to the WT. Moreover, it was shown that some mutant constructs have lower heat capacities than the WT protein. The analysis and results of this thesis show that the thermodynamic stability and dynamics of the CHK2 are altered due the investigated mutations.