LIFE SCIENCES

The process of protein-protein complex formation is of fundamental importance for a better understanding of a variety of biological processes. In a cellular environment the high concentration of surrounding proteins can influence the association process between proteins. Aim of the research project was to simulate the formation of specific and non-specific protein-protein complexes and to investigate the effect of additional protein molecules (crowding) on complex formation in atomic detail. 

The project also involved methodological advancements to accurately calculate the free energy profiles of biomolecular interactions. So far protein association has been studied mostly using simplified coarse-grained protein models without an explicit inclusion of solvent molecules. Alternatively, Brownian Dynamics simulations using rigid partner proteins can also be used to study protein-protein association and encounter [1]. However, such techniques largely neglect the conformational flexibility of proteins and often approximate the aqueous environment poorly. Both conformational adaptation of protein partners and hydration effects are of critical importance for protein-protein interactions and need to be accounted for accurately in order to realistically simulate and understand complex formation events. In the present study atomistic Molecular Dynamics (MD) simulations of proteins in the presence of explicit solvent and ions were used.

As a model system the complex formation of two well known small proteins, Colicin E9 (E9) and the Immunity protein 9 (Im9), known to form very specific high-affinity complexes [2, see Fig. 1]. The E9 protein is a DNA hydrolyzing enzyme and Im9 is a high-affinity protein inhibitor that blocks the E9 activity upon binding. Both proteins consist of ~100 residues and structures of the complex and the isolated partners are known [2]. Specific binding of protein partners may occur directly from the separate fully solvated unbound states of the partner molecules. However, it is also possible that the process involves several intermediate states (encounter complexes) and initial non-specific binding followed by diffusion on the protein surfaces towards the specific complex structure. Since in MD simulations each protein molecules is flexible it will be possible to also directly investigate conformational changes associated with binding events at an unprecedented resolution in time and space. In addition, the role of solvent and dehydration (de-wetting) during binding can be studied at a high level of detail during multiple binding events. The simulation model system contained multiple proteins and allowed studying whether the crowding environment in a cell (high protein density) has a direct influence on the association of protein molecules.

Results

The simulations can help to answer several fundamentally important questions on the mechanism of protein-protein complex formation. How do transient non-specific encounter complexes look like? Do they still have a wet (solvated) or partially dry interface structure? This question is also related to the possibility of anomalous diffusion that may play a role for protein-protein binding in an in vivo situation.

Are there intermediates on the path towards a specific protein-protein complex? Does the complex formation process involve the diffusion of proteins on the surface of the partner (initially bound at a nonspecific site) before reaching the native binding site? Are there trapped incorrectly bound states competing with specific binding? These questions cannot be answered accurately using simpler coarse-grained protein models or rigid protein partners and a rough implicit description of the surrounding solvent.

MD simulations of multiple copies of the colicin E9 and Im9 protein were started from random initial placements in simulation box which contained up to 27 copies of each protein. The simulation system also contained surrounding explicit water molecules and ions (200 mM) resulting in a total number of atoms of 1.2·106. The mean distance of ~3-5 nm between proteins corresponded to a dense protein solution similar to the crowded environment of a cell. After equilibration of the simulation system several production runs using the GROMACS software [3] were performed resulting in an aggregate simulation time of almost 1 us (990 ns). This was possible due to the excellent scaling of the program for the large simulation system on multiple cores. Typically, between 512-2048 cores were used.