Diverse conformational ensembles define the shared folding-allosteric landscapes of protein kinases

Sequence variation across members of an enzyme family contributes to diverse ensemble behaviors, which subtly influence substrate affinity, selectivity, and regulation. A classic example is the family of eukaryotic protein kinases (EPKs), which regulate numerous cellular processes and serve as important drug targets. Here, we dissect the consequences of sequence variation on the folding-conformational landscapes by performing a meta-analysis of 274 EPKs through a structure-based statistical mechanical framework. We find that EPKs populate several partially structured states in their native ensemble with a hierarchy of structural order in the N-terminal lobe that is critical for catalysis and activation. Despite this, the (un)folding mechanism is uniquely conserved across the majority of kinases, with the N-terminal lobe unfolding first. Kinase activation modulates the local stability and thermodynamic connectivity in a nonconserved manner and across the entire structure, due to the strong coupling between the active site residues to distant sites, including the established allosteric pockets. We further show how activation drives the Abl kinase ensemble toward a more folded and thermodynamically coupled system in a graded manner. Our work uncovers the thermodynamic design principles of kinases with insights into allostery, while shedding light on the extents to which ensemble behaviors are impacted by sequence variations in paralogs.