Molecular dynamics studies on proteins involved in genetic variation and metabolism: DMC1 and lipase

Abstract

Both homologous recombination and enzymatic processes, such as those catalyzed by lipases, play essential roles in biological systems, with wide-reaching applications in science and industry. Scientists have studied homologous recombination for a long time to understand its evolution, mechanics, and biological significance. The involvement of the Dmc1 protein in the context of homologous recombination is the focus of this study's thorough investigation of these features. Recombinases, such as Rad51 and Dmc1, which are found in eukaryotes, are essential for homologous recombination and DNA repair. Despite significant sequence similarities, Rad51 and Dmc1 serve different purposes; although Rad51 is essential for DNA repair, Dmc1 participates in homologous recombination during meiosis. More details about its structure and activities are revealed, including the involvement of ATP binding sites and the precise amino acids required for ssDNA binding. Loop areas have been found to be essential for DNA binding. Dmc1 has been characterized by several crystal structures showing an octameric ring configuration in the absence of ATP and DNA. Additionally, it has crystal structures in filament form. These different structures provide valuable insights into the structural flexibility of Dmc1 and its mechanism of interaction with DNA. Molecular dynamics simulations were performed on human Dmc1 protein in various oligomeric states to investigate its structural and dynamic behavior. This study aimed to explore the effects of nucleotide binding, protonation states, and peptide bond isomerization on the stability and conformational dynamics of Dmc1's N- and C-terminal domains. Key simulations included standard molecular dynamics, thermodynamic integration for pKa calculations, and umbrella sampling for free energy profiling. Protonation states of residues E162 and H295, cis-trans isomerization of the D223-S224 peptide bond, and nucleotide-binding states (ATP, ADP, or nucleotide-free) were systematically examined. Root mean square deviation (RMSD) analyses showed distinct equilibration dynamics for the C-terminal domain, while the N-terminal domain displayed significant mobility. Structural analysis revealed the connection between the protonation of E162 and its influence on DNA-binding residue R230. Besides, when E162 is protonated, the ring structure of the protein remains stable. when the D223-S224 peptide in a cis configuration, ATP binds to the Walker A and Walker B motifs in a canonical manner, similar to how ATP binding occurs in other ATPases. However, when the peptide bond is in the trans configuration, the interactions between ATP, Mg²⁺, and the Walker A and Walker B motifs are disrupted, weakening the binding. In the nucleotide-free state, the trans configuration with E162 appears more stable. Upon ATP binding, the structural behavior changes, and multiple configurations become possible. Simulations indicate that trans isomer with protonated E162, trans isomer with unprotonated E162, and cis isomer with unprotonated E162 likely exist in comparable amounts, suggesting a dynamic equilibrium driven by ATP binding and associated conformational flexibility of the protein. Additionally, molecular dynamics simulations on lipase enzymes offered comparative insights into structural flexibility and catalytic efficiency. Lipase dynamics highlighted the role of active site flexibility in substrate binding and enzymatic activity, providing a broader perspective on protein behavior. Overall, the simulations enhanced the understanding of Dmc1's dynamic behavior, interdomain interactions, and potential DNA-binding mechanisms, contributing to deeper molecular-level insights into homologous recombination processes.