Subdomain Competition, Cooperativity, and Topological Frustration in the Folding of CheY

The folding of multidomain proteins often proceeds in a hierarchical fashion with individual domains folding independent of one another. A large single-domain protein, however, can consist of multiple modules whose folding may be autonomous or interdependent in ways that are unclear. We used coarse-grained simulations to explore the folding landscape of the two-subdomain bacterial response regulator CheY. Thermodynamic and kinetic characterization shows the landscape to be highly analogous to the four-state landscape reported for another two-subdomain protein, T4 lysozyme. An on-pathway intermediate structured in the more stable nucleating subdomain was observed, as were transient states frustrated in off-pathway contacts prematurely structured in the weaker subdomain. Local unfolding, or backtracking, was observed in the frustrated state before the native conformation could be reached. Nonproductive frustration was attributable to competition for van der Waals contacts between the two subdomains. In an accompanying article, stopped-flow kinetic measurements support an off-pathway burst-phase intermediate, seemingly consistent with our prediction of early frustration in the folding landscape of CheY. Comparison of the folding mechanisms for CheY, T4 lysozyme, and interleukin-1β leads us to postulate that subdomain competition is a general feature of large single-domain proteins with multiple folding modules.