Identification and Validation of Reaction Coordinates Describing Protein Functional Motion: Hierarchical Dynamics of...

While adequately chosen reaction coordinates are expected to reveal the mechanism of a dynamical process, it proves to be notoriously difficult to model the complex structural rearrangements of a macromolecule by a low-dimensional collective coordinate. Adopting the hinge-bending motion of T4 lysozyme (T4L) as a prominent example and performing a 50 μs long unbiased molecular dynamics (MD) simulation of T4L, a general strategy to identify reaction coordinates of protein functional dynamics is developed. As a systematic method to reduce the dimensionality of the dynamics, first various types of principal component analyses are employed, and it is shown that the applicability and outcome of the approach crucially depends on the type of input coordinates used. In a second step, prospective candidates for a reaction coordinate are tested by studying the molecule's response to external pulling along the coordinate, using targeted MD simulations. While trying to directly enforce the open-closed transition does not recover the two-state behavior of T4L, this transition is triggered by a locking mechanism, by which the side chain of Phe4 changes from a solvent-exposed to a hydrophobically buried state. The mechanism is found to stabilize the open and closed states of T4L and thereby causes their relatively long lifetime of ∼10 μs. In extension of the usual two-state picture, a four-state model of the functional motion of T4L is proposed, which describes a hierarchical coupling of the fast nanosecond opening-closing motion and the slow microsecond locking transition.