Dynamic Protein-Protein Complexes: How Alternative Interactions Create Ensembles and How Solution NMR and MD Simulations can Characterize them

Abstract

It is now recognized that protein-protein interactions in solution are dynamic, especially if the binding affinities are only moderately strong. Dynamics, originate in part from the population of alternative protein complex structures, e.g. one bound state that is in equilibrium with one or several alternative configurations. Here we present two protein complexes that exhibit fluctuations in solution: the plexin RBD:Rac1 and the EphA2:SHIP2 SAM:SAM complexes. A number of techniques are used to shift the equilibrium and/or to demonstrate the population of alternate complex configurations, including ion-pair swap mutagenesis, disulphide cross-linking and Paramagnetic Relaxation Enhancement (PRE) NMR. An ensemble approach is required to calculate the structures even when a wide collection of NMR restraints (chemical shifts, PREs, NOEs and RDCs) are available. The origin of the alternate structures is revealed by the different patterns of sidechain contacts that are possible in the complexes. These interactions and the dynamics of the complexes are further analyzed by microsecond unrestrained molecular dynamics simulations carried out on the MD optimized supercomputer Anton. The functional significance of protein complex dynamics is discussed.