Low Resolution Structural Studies Indicate that the Activator of Hsp90 ATPase 1 (Aha1) of Leishmania braziliensis Has an Elongated Shape Which Allows Its Interaction with Both N- and M-Domains of Hsp90

Abstract

The Hsp90 molecular chaperone is essential for protein homeostasis and in the maturation of proteins involved with cell-cycle control. The low ATPase activity of Hsp90 is critical to drive its functional cycle, which is dependent on the Hsp90 cochaperones. The Activator of Hsp90 ATPase-1 (Aha1) is a protein formed by two domains, N- and C-terminal, that stimulates the Hsp90 ATPase activity by several folds. Although the relevance of Aha1 for Hsp90 functions has been proved, as well as its involvement in the desensitization to inhibitors of the Hsp90, the knowledge on its overall structure and behavior in solution is limited. In this work we present the functional and structural characterization of Leishmania braziliensis Aha1 (LbAha1). This protozoan is the causative agent of cutaneous and mucocutaneous leishmaniasis, a neglected disease. The recombinant LbAha1 behaves as an elongated monomer and is organized into two folded domains interconnected by a flexible linker. Functional experiments showed that LbAha1 interacts with L. braziliensis Hsp90 (LbHsp90) with micromolar dissociation constant in a stoichiometry of 2 LbAha1 to 1 LbHsp90 dimer and stimulates 10-fold the LbHsp90 ATPase activity showing positive cooperativity. Furthermore, the LbHsp90::LbAha1 complex is directed by enthalphy and opposed by entropy, probably due to the spatial freedom restrictions imposed by the proteins’ interactions. Small-angle X-ray scattering data allowed the reconstruction of low resolution models and rigid body simulations of LbAha1, indicating its mode of action on LbHsp90. Western blot experiments allowed Aha1 identification (as well as Hsp90) in three Leishmania species at two temperatures, suggesting that Aha1 is a cognate protein. All these data shed light on the LbAha1 mechanism of action, showing that it has structural dimensions and flexibility that allow interacting with both N-terminal and middle domains of the LbHsp90.

Identification of two p23 co‐chaperone isoforms in Leishmania braziliensis exhibiting similar structures and Hsp90 interaction properties despite divergent stabilities

“…Interestingly, the thermodynamic parameters suggested that the Lbp23 proteins share a similar mechanism for interaction with LbHsp90; the interaction for both proteins is enthalpically driven but has unfavorable entropy. This entropic penalty is most likely a result of restriction of the LbHsp90 freedom, similar to that observed for the interaction between LbAha1 and LbHsp90 , and is consistent with the dimerization of the Hsp90 N‐domains . Considering all of the structural results reported above, the similarity of the mechanism for the interaction of the Lbp23 proteins with LbHsp90 is not unexpected, despite the low amino acid sequence identity.…”

“…Additionally, the apparent enthalpic and entropic contributions were almost the same for both cases (ΔH app = À13.6 AE 0.9 kcalÁmol À1 , ΔS app = À16.6 calÁ mol À1 Ádeg À1 for Lbp23A and ΔH app = À13.4 AE 0.8 kcalÁ mol À1 , ΔS app = À16.8 calÁmol À1 Ádeg À1 for Lbp23B), with the enthalpic component driving both interactions. As recently reported for the L. braziliensis Aha1 co-chaperone [34], the interaction of both Lbp23 proteins with LbHsp90 leads to a negative entropic change, suggesting a restriction in the freedom of the formed complex. Taken together, thesethermodynamicdataindicatethattheinteraction mechanism of the Lbp23 proteins with LbHsp90 is essentially the same, despite their low amino acid sequence identity.…”

“…aSEC experiments and R S determination were performed as described previously [34]. SV-AUC experiments were conducted in a Beckman Optima XL-A analytical ultracentrifuge (Beckman Coulter, Fullerton, CA, USA) at Lbp23 concentrations ranging from 100 to 800 lgÁmL À1 in 25 mM Tris-HCl (pH 7.5) containing 100 mM NaCl and 1 mM b-mercaptoethanol at 20°C at 129 024 g, with data acquisition at 280 nm.…”

Hsp90 inhibitors radicicol and geldanamycin have opposing effects on Leishmania Aha1-dependent proliferation

“…1). Seraphim et al (2013) observed an interaction of recombinant L. braziliensis Aha1 and Hsp90 by ITC. Our pull-down analysis of 3HA::Hsp90 and endogenously coded Aha1 failed probably due to the N-terminal localisation of the HA-tag interfering with the interaction of these two proteins (Fig.…”

“…Previous studies (Rosenzweig et al 2008;Seraphim et al 2013) showed that Leishmania Aha1 is not a heat-inducible protein. We could confirm this in our experiments.…”