Activation Mechanism of a Signaling Protein at Atomic Resolution

Abstract

The interconversion between the inactive and active state is the heart of signaling. This process has traditionally been described by the two corresponding structures, sometimes complemented with kinetic data. However the question of how these folded states interconvert is largely unknown due to the inability to experimentally observe the transition pathways.Here we present a recent investigation of the full free energy landscape of the receiver domain of the response regulator NtrC (NtrC ) by combining several computational methods including the string method, Markov state models of massive unbiased MD simulations, and long MD simulations on ANTON, with new NMR structural data.The results unveil several unexpected features underlying efficient signaling: The active and inactive states have to be considered purely in kinetic terms. The functional need of attaining a stable and well-defined conformer, crucial to the active form of the protein, is absent in the inactive state. The inactive state comprises a structurally heterogeneous collection of sub-states that interconvert on timescales shorter than the transition to the active state. The transitions between the two functional states occur through multiple pathways characterized by transition states with dramatically different structural features. In addition to this entropic lowering of the transition barrier, a number of polar side-chains engage in unspecific transient interactions during the barrier crossing and thus make the activation mechanism flexible, efficient and robust.These novel findings challenge the structural paradigm of signaling and may represent general features for functional conformational transitions within the folded state.