Nucleotide docking: prediction of reactant state complexes for ribonuclease enzymes

Abstract

International audienceRibonuclease enzymes (RNases) play key roles in the maturation and metabolism of all RNA molecules. Computational simulations of the processes involved can help to elucidate the underlying enzymatic mechanism and is often employed in a synergistic approach together with biochemical experiments. Theoretical calculations require atomistic details regarding the starting geometries of the molecules involved, which, in the absence of crystallographic data, can only be achieved from computational docking studies. Fortunately, docking algorithms have improved tremendously in recent years, so that reliable structures of enzyme-ligand complexes can now be successfully obtained from computation. However, most docking programs are not particularly optimized for nucleotide docking. In order to assist our studies on the cleavage of RNA by the two most important ribonuclease enzymes, RNase A and RNase H, we evaluated four docking tools--MOE2009, Glide 5.5, QXP-Flo+0802, and Autodock 4.0--for their ability to simulate complexes between these enzymes and RNA oligomers. To validate our results, we analyzed the docking results with respect to the known key interactions between the protein and the nucleotide. In addition, we compared the predicted complexes with X-ray structures of the mutated enzyme as well as with structures obtained from previous calculations. In this manner, we were able to prepare the desired reaction state complex so that it could be used as the starting structure for further DFT/B3LYP QM/MM reaction mechanism studies

Distinct Roles of Catalytic Cysteine and Histidine in the Protease and Ligase Mechanisms of Human Legumain As Revealed by DFT-Based QM/MM Simulations

“…The theoretical tools that have been used in these calculations are discussed in the methods section (see the Supporting Information (SI)) and in prior publications. ,− Important to the discussion here is that they are applied to a system containing the protein/substrate complex solvated in aqueous background. To develop a reaction mechanism for the cleavage step, it is necessary to have a reliable initial structure (RS structure) that is based on a good resolution X-ray structure of the legumain/cystatin complex (PDB code: 4N6O ) followed by system preparation and optimization, as discussed in the “Computational Details and Methods” section of the SI.…”

QM/MM Simulation (B3LYP) of the RNase A Cleavage-Transesterification Reaction Supports a Triester AN + DN Associative Mechanism with an O2′ H Internal Proton Transfer

“…The theoretical tools that have been used in these calculations are discussed in detail in the methods section in the SI (Section 2) and in prior publications. − In this study these are applied to the cleavage-transesterification reaction in the enzyme system containing the enzyme/RNA complex fully solvated in a discrete water representation of the solution. The analysis implements a comprehensive effort to use all available structural data to define the reactive enzyme substrate complex; a QM/MM type calculation , implementing an accurate first-principle-based electronic structure calculation (B3LYP) to estimate the interactions in the catalytic cell represented by a large QM region ((117 atoms) required to reliably describe the enzyme/substrate complex), see SI, Section 2; a minimally biased search method to identify reaction mechanisms; thermal equilibration and nudged elastic bend (NEB) structural optimization with full flexibility of the protein and substrate, including all Coulomb interactions between the QM and MM regions; and free energy calculations along the reaction path.…”

“…For the RNase A enzyme this type of information is available only for the hydrolysis step. , To provide a starting structure for the transesterification reaction we used information from these structures as much as possible and in addition carried out an extensive structural search using a combination of docking algorithms and QM/MM optimizations with validation from the structures of analogous systems (RNase A–DNA single strand (CA) complex (Section 3, SI)) …”