E. Coli Adenylate Kinase Exhibits Inter-Domain Coupling

Abstract

Protein molecules are dynamic and conformationally heterogeneous, and these properties contribute to their functions. Ensemble-based equilibrium measurements are not always sensitive to low-population conformations or the kinetics that govern their fluctuations. We have applied single-molecule force spectroscopy to study the free energy landscape of adenylate kinase (AK), a three domain protein known to visit excited states defined by local unfolding of its peripheral domains (LID and AMPbd). These states have previously been shown to modulate both the binding affinity and catalytic rate of this essential enzyme. Using entropy-enhancing mutations, we selectively lower the free energy of locally unfolded states and find that LID stability is highly correlated with AK's mechanical stability. Our results reveal tight coupling between the LID and CORE domains. What's more, the coupling is LID domain specific, as destabilizing the AMPbd results in no detectable effect. Our results further suggest that AK exists in at least two states, with very different mechanical stabilities, interconverting with very slow kinetics. The heterogeneity in AK's conformational landscape is greater than currently appreciated and may play additional uncharacterized roles in the protein's function and regulation. What's more, these adaptations are likely not restricted to AK, and may reflect a more general mechanism of evolutionary tuning. In the future, they may be leveraged in new strategies for the altering of protein function via novel therapeutics or through de novo design.