Initially, the systems were energy-minimized using the steepest descent (500 steps) and conjugate gradient (2000 steps) methods and placing positional restraints with a force constant of 5 kcal mol -1 Å -2 on all protein atoms; the restraints were reduced in a second energy minimization to a force constant of 1 kcal mol -1 Å -2 (for 2000 steps of steepest descent followed by 8000 steps of conjugate gradient), and removed in a third one (for 1000 steps of steepest descent followed by 4000 steps of conjugate gradient).