Computer Aided Study of Ligand Binding With Catalytic Domain of Avian Sarcoma Virus Integrase and Its Ligand Binding Loops

Abstract

We have carried out 1 nanosecond (ns) Molecular Dynamics (MD) simulations of the drug Y3 (4-acetylamino-5-hydroxynaphthalene- 2, 7-disulfonic acid) complexed with catalytic domain of Avian sarcoma virus Integrase (ASV-IN), both in vacuum and in the presence of explicit solvent. Starting models were obtained on the basis of PDB co- ordinates (1A5X) of ASV-IN-Y3 complex. Mn(2+) cation was present in the active site. To neutralize the positive charge in the presence of explicit solvent, eight Cl(-)anions were added. Energy Minimization (EM) and MD simulations, for both the systems, were carried out using Sander's module of AMBER5.0 with all atom force field. We also carried out 1 ns MD simulation on two flexible loops--L1 (Gly54-Gln62) and L2 (Trp138-Met155) playing crucial role in interaction of IN with the drug, under differing environmental conditions (vacuum, aqueous and organic solvent methanol). Comparison of the conformational changes in the loops, monomer and dimer is presented in the paper. Our results showed that the conformation of the loop region was closest to crystallographic data in case of monomer and constrained loops in aqueous environment. However, the dimer in vacuum was more stable than monomer. The beta sheet structure of the monomer in aqueous environment was unstable. Latter also took long time for equilibration. The box formed by loops L1 and L2 from two sub units (IINA and INB) of the dimer satisfies prerequisites for ligand recognition site and seems to be the functional biological unit.

Activation of Phenylalanine Hydroxylase: Effect of Substitutions at Arg68 and Cys237

“…Because of the large size of the protein system to be simulated, solvent water molecules were not included in the calculations, and a constant dielectric constant of 4 and a distance-dependent dielectric function were used. At these conditions, the rigid part of the macromolecule provides a realistic electrostatic environment for the part that moves, and this procedure has been found to satisfactorily reproduce the X-ray crystal structures of soluble proteins without including water molecules (30), and to maintain stability during MD simulations, notably for dimeric systems (31). The cutoff radius for nonbonded interactions was 12 Å, with a secondary cutoff radius of 15 Å.…”

“…However, some significant differences can be noticed in the structure of the mutants as compared with the wild-type form short after the start of the MD simulations. As seen in Figure 4B,C, showing the averaged structures at the time span 200-300 ps for the wt-hPAH and the representative mutant C237D-hPAH, changes were observed in the conformation of the core of the regulatory domain, especially in the loop 68-75, and in the N-terminal autoregulatory sequence (residues [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33]. This sequence stretches along the entrance of the active site in the wt-hPAH and is displaced from this position in the mutant C237D-hPAH providing an open active site.…”

“…As shown by deletion mutagenesis (15) and by the crystal structure (8)(9)(10), PAH has a three-domain structure. Each subunit contains (i) an N-terminal regulatory domain (up to Ser110) that contains an R-β sandwich core and an extended N-terminal autoregulatory sequence (residues [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33] that stretches over the active site of the catalytic domain; (ii) a catalytic domain (Asp111-Phe410), containing the active site iron and the substrate and BH 4 binding sites; and (iii) an oligomerization domain, with a dimerization motif (Ser411-Thr427) and a helical tetramerization motif at the C-terminus that promotes oligomerization through antiparallel helix-helix interactions (9). The displacement of the Nterminal autoregulatory sequence away from the active site is supposed to be an important activating conformational change that accompanies the transition from the T-to the R-state (19,20).…”