from the GenBank, accession number AAC83551.1. The sequence and 3D structure of PFV IN was extracted from the Protein Data Bank , and only residues 51– 374 covering the three functional Paclitaxel domains were luded. According to the secondary structure information of the template, the alignment was adjusted manually to obtain a more reasonable matching. And then, based on the sequence alignment result, a 3D model of the HIV 1 IN was generated and refined with the Program Prime from Schrodinger Suite 2008. Construction for the HIV 1 IN–vDNA complex and INSTIs binding model The structure based alignment of PFV IN and HIV 1 IN was used to guide the construction of HIV 1 IN–DNA complexes.
The 19 base pair mimic of pre processed terminal vDNA from the PFV structures and the Mg2þ ions from the inhibited PFV structures were introduced to the HIV 1 IN model by program Swiss Pdb Viewer using the Ca positions of active site residues Asp64, Asp116, and Glu152 from the Procollagen C Proteinase CCD. Similarly, RAL, vDNA, and Mg2þ ions from the PFV structure were fitted into the modeled HIV 1 IN to get the HIV 1 IN–vDNA–RAL complex. Here, it should also be pointed out that vDNA from the PFV structure was mutated to HIV 1 vDNA based on the DNA sequence alignment. Molecular dynamics simulations The MD simulations of the HIV 1 IN–vDNA and HIV 1 IN– vDNA–RAL complexes described above were preformed using the AMBER10.0 program. The strand AMBER force field was used to describe the HIV 1 IN and vDNA. The force field parameter for the inhibitor generated using the Antechamber program in AMBER10.
0 were described by the General objectified Amber Force Field . The partial charges of the inhibitor was determined using the RESP fitting technique based on the optimized structures and the electrostatic potentials calculated using HF/6 31G* in Gaussian09 program. The hydrogen atoms and the missing atoms were added using the Leap module, 29 Na þ counter ions were added to electrically neutralize each systems. Then the two starting structures were immersed in a rectangular prism preequilibrated TIP3P waters with at least 10 A˚ distance around the complex, and about 23351 and 23341 TIP3P waters were added in the HIV 1 IN–vDNA and HIV 1 IN–vDNA–RAL complex, respectively. Prior to MD simulations, two steps of minimization were carried out. In the first stage, we kept the protein fixed with a position restraint of 2.
0 kcal/mol/A˚ 2, and we just minimized the positions of the water molecules. In the second stage, we minimized the entire system by releasing all the restrains. The two minimization stages consisted of 5000 steps in which steepest descent method was used in the first 1000 steps and conjugated gradient method was used in the last 4000 steps. MD trajectories were run using the minimized structure as a starting input. After 50 ps NVT ensemble heating process, the systems were gradually raised from 0 to 300 K using the Langevin dynamics method, and the density of the systems quickly reached 1 g/cm3 during this heating stage. Then, 150 ps equilibrating process was executed at 1 atm and 300 K using the NPT ensemble in three steps of 50 ps. During the three periods of this second stage, the Ca was restrained with harmonic force constant of 2.0, 1.0, and 0 kcal/mol/A˚ 2, respectively.