Team:CUHKSZ/pages/dry-lab
Dry Lab
Docking
We first tried to dock the cofactors and the ligands to their domains respectively, namely NAD+ and ethanol to ADH domain, and NAD+ and aldehyde to ADLH domain. However, in the initial trial, we mistook the whole tetramer structure of the enzyme for docking, which caused NAD+ and ligand to be separated into site within distant monomers. After revising the technical misconducts, we docked in the order of “first NAD+ then ligand” to their domain respectively in their monomer. This generated several possible conformations. Here we first chose the conformation with the highest rank for the following process.
However, further investigation could be done on the other conformations that ranked after the first. These conformations would also undergo molecular dynamics simulation to discover which to be a proper conformation.
Molecular Dynamics (MD)
The MD process were first conducted in a mistaken procedure due to the misconduct as is mentioned in docking part. Due to time limit, the primary MD had only run for 200 picoseconds for the reaction in ALDH domain, which was not long enough to capture a frame that suits the reaction as is proposed in previous literature. The two main problems of the MD trajectory were: First, the oxidation site of NAD+ was at the opposite of ethanol, which was too distant for the oxidation to occur. Second, the ethanol was not close to a cysteine residue, which should be the nuclearphile to attack the carbonyl on aldehyde. It might be the reason either that the docking conformation, though highly ranked, was not the most optimized one for the reaction to occur, or that 200 picoseconds were too short for the reaction to witness a conformational change.
However, the MD result also provided new insight into the mechanism. Instead of moving around the cysteine residue, the aldehyde were moving around a methionine residue. This indicated the sulfur on methionine had the probability of being the nuclearphile to attack aldehyde. Based on this discovery, we moved on to quantum chemistry calculations parallel to extra MD processing on other possible conformations from docking.
Molecular Dynamics (MD)
At the first stage of QM, the optimization of the clustered frame from MD turned out to be compelling. Although it might be the reason that clustered structure lacked residue to resist the movement of NAD+, NAD+ in fact bent itself turned its oxidative site towards aldehyde. This discovery could support the mechanism proposed by ourselves, and need further justification of whether the NAD+ would also bend inside the enzyme and why.
