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.

Claudine Mayer,Thiago V Seraphim,Leandro R S Barbosa,Indjara M Silva,Júlio C Borges,Kelly P Silva,Marina M Alves,Silvane M F Murta,Francisco E R Gomes

doi:10.1371/journal.pone.0066822

Claudine Mayer, Thiago V Seraphim + Show 7 more

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https://doi.org/10.1371/journal.pone.0066822

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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.

Highlights

The molecular chaperones of the Hsp90 family are essential for the growth of many organisms [1,2], including protozoans such as Plasmodium falciparum, Leishmania donovani and L. amazonensis where they work in the heat stress response for the cellular differentiation in the parasite life cycle [3,4,5,6,7]
The low conservation between Leishmania braziliensis Aha1 (LbAha1) and orthologous proteins suggests that the mechanism of interactions of LbAha1 with L. braziliensis Hsp90 (LbHsp90) can present some peculiarities
The modeled domains of LbAha1 are shown in Figure 1A, where the linker between the N- and C-terminal domains is presented by a dotted line

Summary

Introduction

The molecular chaperones of the Hsp family are essential for the growth of many organisms [1,2], including protozoans such as Plasmodium falciparum, Leishmania donovani and L. amazonensis where they work in the heat stress response for the cellular differentiation in the parasite life cycle [3,4,5,6,7]. The Hsp ND has an ATP binding site and presents a weak ATPase activity [1,2] This domain can bind to client proteins, Hsp cochaperones and some Hsp inhibitors, such as geldanamycin, 17-AAG and radicicol. It is suggested that the binding of some Hsp cochaperones, such as Aha (Activator of Hsp ATPase 1), can reverse, in vivo, the Hsp inhibitor effects [13]

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