Investigation of the mechanism of domain closure in citrate synthase by molecular dynamics simulation

Six, 2 ns molecular dynamics simulations have been performed on the homodim eric enzyme citrate synthase. In three, both monomers were started from the open, unliganded X-ray conformation. In the remaining three, both monomers started from a closed, liganded X-ray conformation, with the ligands remov ed. Projecting the motion from the simulations onto the experimental domain motion revealed that the free-energy profile is rather flat around the ope n conformation, with steep sides. The most closed conformations correspond to hinge-bending angles of 12-14 degrees compared to the 20 degrees that oc curs upon the binding of oxaloacetate. It is also found that the open, unli ganded X-ray conformation is situated at the edge of the steep rise in free energy, although conformations that are about 5 degrees more open were sam pled. A rigid-body essential dynamics analysis of the combined open traject ories has shown that domain motions in the direction of the closed X-ray co nformation are compatible with the natural domain motion of the unliganded protein, which has just two main degrees of freedom. The simulations starti ng from the closed conformation suggest a free-energy profile with a small barrier in going from the closed to open conformation. A combined essential dynamics and hinge-bending analysis of a trajectory that spontaneously con verts from the closed to open state shows an almost exact correspondence to the experimental transition that occurs upon ligand binding. The simulatio ns support the conclusion from an earlier analysis of the experimental tran sition that the beta -hairpin acts as a mechanical hinge by attaching the s mall domain to the large domain through a conserved main-chain hydrogen bon d and salt-bridges, and allowing rotation to occur via its two flexible ter mini. The results point to a mechanism of domain closure in citrate synthas e that has analogy to the process of closing a door. (C) 2001 Academic Pres s.