Change in protein flexibility upon complex formation: analysis of Ras-Raf using molecular dynamics and a molecular framework approach.

Changes in flexibility upon protein-protein complex formation of H-Ras and the Ras-binding domain of C-Raf1 have been investigated using the molecular framework approach FIRST (Floppy Inclusion and Rigid Substructure Topology) and molecular dynamics simulations (MD) of in total approximately 35 ns length. In a computational time of about one second, FIRST identifies flexible and rigid regions in a single, static three-dimensional molecular framework, whose vertices represent protein atoms and whose edges represent covalent and non-covalent (hydrogen bond and hydrophobic) constraints and fixed bond angles within the protein. The two methods show a very good agreement with respect to the identification of changes in flexibility in both binding partners on a local scale. This implies that flexibility can be successfully predicted by identifying which bonds limit motion within a molecule and how they are coupled. In particular, as identified by MD, the beta-sheet in Raf shows considerably more pronounced orientational correlations in the bound state compared to the unbound state. Similarly, FIRST assigns the beta-sheet to the largest rigid cluster of the complex. Interestingly, FIRST allows us to identify that interactions across the interface (but not conformational changes upon complex formation) result in the observed rigidification. Since regions of the beta-sheet of Raf that do not interact directly with Ras become rigidified, this also demonstrates the long-range aspect to rigidity percolation. Possible implications of the change of flexibility of the Ras-binding domain of Raf on the activation of Raf upon complex formation are discussed. Finally, the sensitivity of FIRST results with respect to the representation of non-covalent interactions used as constraints is probed.