Computational protocol: The molecular mechanism of DNA damage recognition by MutS homologs and its consequences for cell death response

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Protocol publication

[…] The simulation of the G/T mismatch is based on the X-ray structure of Escherichia coli MutS in complex with heteroduplex DNA (). Hydrogen atoms were added using the hbuild facility of CHARMM (). The CHARMM force field was used for the entire complex with additional parameters based on pre-existing cisplatin parameters (,). This force field has been extensively parameterized for a wide range of biologically important molecules, including nucleic acids, amino acids, lipids and some small-molecule ligands. The platinated DNA structure was built using the mismatch as a template. The cross-linked structure was fitted into the binding pocket to maximize the structural overlap with the mismatched DNA structure, followed by rotations and translations to minimize the energy of the unrelaxed structure using the coordinate manipulation and energy minimization facilities of CHARMM (). The structure was fully solvated with TIP3P water in a cubic box using the visual molecular dynamics (VMD) package (). The fully solvated simulation was performed with a 2.0 fs timestep using SHAKE on all bonds to hydrogen atoms, and with Particle Mesh Ewald as implemented in NAMD (). The simulation protocol consisted of 100 ps of free solvent equilibration with fixed solute, followed by 400 ps of full unrestrained equilibration, and 1.6 ns of production. During the unrestrained equilibration and the production simulation, all atoms in the complex and the solvent are allowed to move freely. As a result, for example, the DNA can bend freely and the protein side chains can reorient. The angles were calculated with the trajectory analysis tools in VMD using the backbone atoms in the terminal and central amino acids after the equilibration period (). The conformational analyses were performed using VMD and CHARMM. After 2 ns, the unplatinated mismatch interaction scheme was essentially unaltered from the X-ray, and is not discussed. As a control, an additional simulation was performed of a platinated G/T mismatch using the same protocol. The conclusions from both simulations are similar, although the magnitude of distant conformational changes is smaller in the G/T simulations. Modeling alone at present does not reveal any significant differences between the binding in the G/C and G/T interactions. In the G/T simulations no structure (sampled every 2 ps) has an RMSD of more than 2.1 Å from the X-ray structure, excluding those residues that are undefined in the crystal structure. All the platinated structures are within 2.8 Å of all the structures sampled in the mismatch simulation, including the residues undefined in the X-ray structure (data not shown). The simulation of the mutant MutS-E38A structure was performed using the same methodology after the computational mutagenesis was performed. The mutation was introduced using the wild-type structure with hydrogen atoms as a template. The alanine side chain was built upon the original glutamate side chain, and the entire structure was subjected to 500 steps of steepest descent minimization with an r-dependent dielectric to locally minimize the structure. […]

Pipeline specifications

Software tools CHARMM, VMD, NAMD
Application Protein structure analysis
Diseases Genetic Diseases, Inborn, Drug-Related Side Effects and Adverse Reactions
Chemicals Adenosine Triphosphate, Cisplatin