Effect of Crystal Packing on Cro Dimer Conformation

Abstract

X-ray crystallography is the primary method in structural biology for providing information about protein conformation. However, artificial packing forces in the crystal lattice select just a snapshot of a protein’s conformational ensemble, whereas proteins are flexible and may adopt different conformations in order to function. This raises the question, how accurately do X-ray structures describe the solution state of a protein? To address this critical issue, we have established protocols for performing Molecular Dynamics (MD) simulation in solution as well as the crystalline environment and for using network analysis to study the conformational ensembles1-3.As a model system, we consider the λ Cro repressor, whose solved X-ray structures range from a closed to an open global conformation. The fully open form is observed both bound and unbound to DNA. Network analysis and a free energy surface constructed from Replica Exchange MD reveal that closed and semi-open conformations are stable in solution, with a modest barrier separating these two states3. Yet the fully open conformation, while accessible, lies higher in free energy, indicating it requires stabilization by DNA or crystal contacts. Since a semi-open state is among the low energy conformations sampled in simulation, we propose that this form may initiate DNA recognition and only minor adjustments are needed to achieve the fully open conformation as observed in the functional complex. Subsequent crystal MD simulations estimated the strength of packing interfaces in the lattice, showing the influence of crystal form and mutation in stabilizing different dimer conformations. Our quantitative results will aid analysis of X-ray data in establishing protein structure-function relationships. [1] Vorontsov, I. I. and Miyashita, O. (2009) Biophys. J., 97, 2532-2540. [2] Campbell, Z. T., et al. (2010) Biophys. J. 99, 4012-4019. [3] Ahlstrom, L. S. and Miyashita, O., submitted.