Adding Dynamical Insight to X-Ray Images by Solution and Crystal Molecular Dynamics Simulation

Abstract

X-ray crystallography is the most robust technique for protein structure determination. However, this method is still largely a trial-and-error process of changing solution conditions and relies on unpredictable protein-protein interactions. Consequently, crystal packing may select a conformation unrepresentative of the physiologically relevant form of a protein and there are many examples in which multiple solved structures exist for the same protein. Here we compare solution and crystal lattice molecular dynamics (MD) simulations1 to add dynamical insight to the interpretation of X-ray images. As a model system we consider the dimeric λ Cro transcription factor whose crystal structures range from a closed DNA-free conformation to an open DNA-bound form. Free energy profiles reporting on the conformational space sampled by the dimer in solution reveal that both states are accessible but that the closed form may be slightly more stable2. Subsequent crystal MD simulations were performed to establish how mutation within the dimer could have stabilized a DNA-free open conformation in contrast to the wild-type apo closed form. Both structures were simulated with wild-type as well as mutant neighbors to study a variation of crystal environment. Applying the MMPBSA approach we calculated the relative stabilities of crystal contact regions in the mutant and wild-type lattices. Moreover, the packing arrangement in the mutant lattice may have prevented the formation of an intersubunit salt bridge that stabilizes the closed conformation. These results suggest that differences in crystal packing due to mutation affected λ Cro dimer conformation in the lattice. Our methods of performing crystal MD simulations may also improve the interpretation of other protein X-ray images to aid in establishing structure-function relationships. [1] Vorontsov, I.I. and Miyashita, O. (2009) Biophys. J., 97, 2532-2540. [2] Ahlstrom, L.S. and Miyashita, O., in progress.