Multilevel molecular modelling of structure-function relationships in enzymes

Proteins are large flexible molecules and conformational dynamics is one of their fundamental properties which correlate the protein’s structure and function [1] [2]. The crystal structure of biomolecular systems such as enzymes reveals the important atomistic details in terms of the ligand binding and possible mechanism albeit providing no information about how conformational flexibility and dynamics influences the protein structure and its key determinants. There is no information regarding the electronic structure and the chemically relevant components of the enzymes and how the protein environment affects the electronic structure. In order to provide understanding of how the conformational flexibility influences structure-function relationships of the enzymes, we applied classical molecular dynamics simulations using Gromacs [3] [4] [5] and Amber [6] [7] packages. The effect of the protein environment on the electronic structure of the active site were studied using Quantum Mechanics and Molecular Mechanics (QM/MM) [8] using ONIOM [9] [10, 11] implemented in Gaussion09 [12] [13]. Tyrosylproteinsulfotransferase (TPST): TPSTs catalyze the transfer of negatively charged sulfate group from 3’-phosphoadenosine 5’-phosphosulfate (PAPS) to the hydroxyl group of a tyrosine residue of polypeptide to form a tyrosine O4-sulfate ester [14]. The binding of the substrate peptide showed more open conformation in Tyrosylproteinsulfotransferase-2 (TPST-2) enzyme in contrast to the crystal structure [15] [16]. There were identification of new hydrophobic interactions responsible for the stabilization of the enzyme dimer [16]. The binding of the substrate and cofactor to the apoenzyme contributed to the stability of the whole active complex, influenced the local interactions in the binding site and importantly, affects the pattern of the correlated motions in the entire molecule [16]. NirE an S-adenosyl-L-methionine dependent Methyltransferase: The NirE enzyme catalyses the transfer of a methyl group from the S-adenosyl-L-methionine (SAM) to uroporphyrinogen III and serves as a novel potential drug target for the pharmaceutical industry. The binding of the substrate contributes to the stabilization of the structure of the full enzyme complex [17]. The conformational changes influence the orientation of the pyrrole rings of the substrate [17]. The mutations of binding and active site residues leads to sensitive structural changes which influence binding and catalysis [17]. Matrix metalloproteinase-1 (MMP-1): The molecular dynamics studies on the Matrix metalloproteinase-1 (MMP-1) were in good agreement with the experimental observation that in the MMP-1•THP (Triple Helical Peptide) X-ray crystallographic structure MMP-1[18] is in a "closed" conformation [19]. The interactions of the THP with both the CAT and HPX domains of MMP-1 are dynamic in nature, and the linker region of MMP-1 influences the interactions and dynamics of both the CAT and HPX domains and collagen binding to MMP-1 [19]. The mutations in the MMP-1 have distinct impact on the correlated motions in the MMP-1•THP. An increased collagenase activity corresponded to the appearance of a unique anti-correlated motion and decreased correlated motions, while decreased collagenase activity corresponded both to increased and decreased anti-correlated motions. Non-heme Fe2+ and 2-oxoglutrate (2OG): The non-heme Fe2+ and 2-oxoglutrate (2OG) dependent dioxygenases such as FTO, AlkB, PHF8 and KIA1718 perform important tasks in homeostasis through the methylation of DNA and histone proteins. The linker region shows increase conformational flexibility and dynamics in PHF8 and KIA1718 and is important for the catalysis. The jelly roll motif structure also showed conformational stability for all demethylases and indicates its vital role in maintaining the iron geometry in the active site. The N domain of the FTO enzyme and the L1 loop region showed increased conformational flexibility and dynamics. The QM/MM optimized structure of reactant complex showed the effect of the conformational flexibility. An important insight into the structure function relationship of different enzymes has been obtained by applying a large number of Atomistic Molecular Dynamics Simulations and Quantum Mechanics/Molecular Mechanics to different enzymes which cannot be gained experimentally. The effect of conformational dynamics, flexibility and important interactions of the active site residues can be used in chemical biology and biotechnology for structure based drug design and in the engineering of novel biocatalyst