Lactate dehydrogenase (LDH) is a tetrameric enzyme that catalyzes the last step of anaerobic glycolysis through the reversible conversion of pyruvate to lactate via coupled oxidation of NADH cofactor. The LDH protein has a distinct activity pattern across normal tissues and its protein level gets increased in response to tissue injury, necrosis, hypoxia, hemolysis, and myocardial infarction. In anaerobic glycolysis, the LDH type A (LDHA) plays an important role in regulating glycolysis by catalyzing the final step of the process. Therefore, in cancer cells increases in the expression level of LDHA, may facilitate the efficiency of anaerobic glycolysis. Recently, the structure of the LDH has been determined and five active LDH isoenzymes are known yet. Each of these isoenzymes has a tetrameric structure that composed of two different subunits called M and H subunits (formally A and B) which are encoded by Ldh-A and Ldh-B genes, respectively. The isoenzymes that there have more A chains than B chains, the conversion of pyruvate to lactate is more efficient (LDH5, LDHA) and regenerate NAD+ from NADH. Conversely, the increase of B chains favors the conversion of pyruvate to Acetyl-CoA. Previous immunohistochemistry (IHC) assays showed that the LDHA mostly is expressed in cancer cells, and accordingly the LDHA level is often utilized as a biomarker for cancer diagnosis.
Detection of serum LDH-A is a key step in the diagnosis of many types of cancers. Nanomaterials are known to interact with this protein and influence its functions including its enzymatic activity. Achieving detailed information about the molecular mechanism of nanoparticle interactions with biological macromolecules would be required for designing efficient nanomaterials for medical technologies including medical diagnostic imaging and cancer therapies.
Molecular dynamics (MD) simulations is a powerful method for analyzing and prediction of significant details of atomic motions in proteins. The method is extensively applied for analyzing the structure and dynamics of protein-ligand complexes. The present work analyzes pristine graphene and COOH-Graphene sensitivity on LDH adsorption and introduces the graphene specially COOH-graphene, as favorable to adsorb the LDH. In this study, by using theoretical techniques, we found that COOH-Graphene and PG decrease enzyme activity with change the active site conformation and also, significantly influence the overall structure of the enzyme. The MD simulations results found in this study can provide a good perspective on the interaction of LDH with carboxylic-functionalized and no functionalized graphene.
Materials and methods
The initially reported structure of LDH was obtained from rcsb databank. COOH-Graphene was modified from the same PG. The carbon atoms were modeled as uncharged Lennard-Jones particles and described using parameters for the atoms of aromatic carbon.
Initially, PG and COOH-Graphene were placed at the edge of a 5.0 × 6.5 × 6.5 nm−3 rectangular box, with their basal plane parallel to the XY-plane. Carbon atoms of PG and COOH-Graphene were restrained by a harmonic potential with a spring constant of 1000 kJmol−1 nm−2 during the simulation. The LDH segments were approximately parallel to the basal plane, and the center of mass (COM) distance between the protein and PG or COOH-Graphene was 1nm (figures 2(a) and (b) with snapshots at t = 0ns). All MD simulations were performed using the Gromacs package 5.0.7 with the OPLS/AA force field. Water molecules were achieved using the TIP3P model and constrained by SETTLE algorithms (Miyamoto and Kollman 1992). Bond lengths within the protein and graphene were constrained using the LINCS method. Periodic boundary conditions were applied in all three directions. The particlemesh Ewald (PME) method was used to treat the long-range electrostatic interaction, and Van der Waals (vdW) interactions were calculated with a cut off of 1.0nm. After energy minimization, the solvated systems were equilibrated in an NVT ensemble at 298K for 500 ps.
The Berendsen thermostat is used with a coupling time of 0.1 ps for all MD simulations. Protein structure was then released and production simulation continued in an NPT ensemble at a constant pressure of 1 bar and a temperature of 298K for 30ns using a velocity rescaling thermostat and Berendsen barostat.
Binding free energy analyses
The binding free energies of the complexes between LDH withPG and COOH-Graphene, during the MD simulation analyses, were computed using g_mmpbsa tool of GROMACS based on the molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) method.
The binding energy calculations were performed for 250 snapshots taken at an interval of 40 ps during the 20-30 ns (equilibrium phase) of each trajectory of MD simulation.
Results and discussion
To provide information about the LDH adsorption on the PG and COOH-Graphene surface, we calculated the center of mass (COM) distance between the LDH with PG and COOH-Graphene during MD simulations. As shown, during the early steps of the simulations the distances between PG and LDH decreased drastically, indicating the early adsorption of the LDH on the surface of PG and this distance are stable until the end of the simulation. At the COOH-Graphene system, during the early steps of the simulation, the distance is increased slightly until 3000 ps and after this time the distance is decreased.