Selective allosteric regulation of Leishmania braziliensis Hsp90 through a parasite-specific regulatory hub.

Heat shock protein 90 (Hsp90) is a central biological macromolecule and essential molecular chaperone in protozoan parasites, whose functional cycle relies on tightly regulated conformational dynamics and allosteric communication between domains. Despite its relevance as an antiparasitic target, the high conservation of Hsp90 across species complicates the identification of selective regulatory mechanisms. Here, we investigate the allosteric regulation of Leishmania braziliensis Hsp90 (LbHsp90) using a celastrol-derived modulator, named SS2, as a mechanistic probe. We identify a previously unrecognized, parasite-specific allosteric regulatory hub at the interface between the N-terminal and middle domains (NTD-MiD), which acts as a key node integrating ATPase regulation with client-processing functions. An integrated biochemical, biophysical, and computational analysis, including proteolysis-protection assays, solution NMR spectroscopy, docking, molecular dynamics simulations, and targeted mutagenesis, demonstrates that this regulatory hub couples conserved allosteric elements with parasite-specific structural determinants, and is stabilized by a cysteine residue (Cys230) absent in the human homolog. Functional and kinetic analyses show that perturbation of this hub modulates the conformational landscape of LbHsp90, stabilizing inactive states, enhancing intrinsic negative cooperativity, and selectively impairing chaperone activity. These findings establish a mechanistic framework for the allosteric regulation of kinetoplastid Hsp90 and highlight regulatory hubs as structurally and functionally defined elements for the selective modulation of macromolecular machines.