From productive coupling to catalytic commitment: An evolutionarily tuned interdomain connector in Leishmania Aha1...

Hsp90 activation depends on cochaperones that couple binding to catalytic commitment, yet some structural elements mediating this transition remain poorly understood. Here, we investigate the molecular mechanism by which the cochaperone Aha1 from Leishmania braziliensis activates Hsp90 and present evidence supporting a linker-mediated disorder-to-order transition associated with this process. Thermodynamic analyses show that, while the Aha1 N-domain mediates Hsp90 binding, only the full-length Aha1 containing its wild-type interdomain linker exhibits a distinct enthalpy-dominated interaction and robust ATPase stimulation. Structural and kinetic measurements indicate that the wild-type linker modulates the conformational distribution of the Aha1 N-domain, promoting productive docking with Hsp90. Structural modeling suggests that the Aha1 linker adopts an α-helical conformation in the activated complex, whereas the corresponding synthetic linker peptide displays inducible helicity under structure-promoting conditions, consistent with a binding-coupled disorder-to-order transition. This transition appears evolutionarily tuned, as linker properties vary across species. Collectively, these findings support a model in which the Aha1 linker actively couples binding to activation, providing a new framework for Hsp90 regulation and highlighting a potential target for selective intervention in protozoan Aha1:Hsp90 systems.