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. 2013 Jun 24;8(6):e66822.
doi: 10.1371/journal.pone.0066822. Print 2013.

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

Thiago V Seraphim  1 , Marina M Alves, Indjara M Silva, Francisco E R Gomes, Kelly P Silva, Silvane M F Murta, Leandro R S Barbosa, Júlio C Borges
Affiliations

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

Thiago V Seraphim et al. PLoS One. .

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Structural features, acquisition and spectroscopic analysis of the LbAha1.
A) Through homology-modeling, the LbAha1 domains were modeled using as templates the N- and C-terminal domains of the yAha1 (PDB: 1USV) and hAha1 (PDB: 1X53), respectively. The bar scheme represents the domain organization of LbAha1 and the regions highlighted in gray are represented by the structural domains shown. The first residues of the LbAha1 N-terminal domain, as well as the linker region and the last residues of the C-terminal domain, are represented by dotted lines. LbAha1 has 9 Trp residues along its structure (4 in the N-terminal domain and 5 in the C-terminal domain), which are shown as blue sticks, and at the beginning of the N-terminal domain, an absent and unstructured region in the template, the Trp residues are represented by circles. B) The LbAha1 protein was expressed in E. coli cells and purified by three chromatographic steps. All procedures were followed by SDS-PAGE, and the final purity of the target protein was higher than 95%. M: MM marker; 1 and 2: cell lysate before and after induction; 3, 4 and 5: LbAha1 after the anionic exchange, calcium affinity chromatography and preparative SEC, respectively. C) The CD spectra of LbAha1 were collected in 25 mM sodium phosphate (pH 7.0), 50 mM NaCl, 2 mM EDTA, 1 mM β-mercaptoethanol. The LbAha1 CD spectrum was compatible with those for proteins constituted by α-helix and β-sheet conformation, as estimated by the DichroWeb server (see text for details). Inset: Intrinsic fluorescence spectra of the LbAha1 at native (solid line) and denatured (dashed line) condition were acquired in the same buffer of the CD analysis, but the latter contained 6 M of GndHCl. Moreover, the recombinant LbAha1 protein was purified in the folded state.
Figure 2
Figure 2. Chemical stability studies of the LbAha1.
A) The chemical-induced unfolding experiments for LbAha1 were carried out using GndHCl as the denaturing agent and changes in the secondary structure of the protein were monitored by the CD220nm signal, normalized to [θ]. Two defined transitions were observed with Cm-values centered at 1.0±0.1 and 2.8±0.1 M of GndHCl. B) Changes in the tertiary structure of the LbAha1 during its chemical-induced unfolding by GndHCl were monitored by fluorescence. The samples were excited at 280 nm, the fluorescence emission spectra were normalized to <λ>. The Cm-values determined for each transition were 1.0±0.1 and 2.8±0.1 M of GndHCl. Altogether, these results showed that LbAha1 has two relatively independent domains with different chemical stabilities. C) Chemical-unfolding model pathway proposed for LbAha1 based on its structural organization and chemical stabilities (see text for details).
Figure 3
Figure 3. Characterization of the hydrodynamic properties of the LbAha1.
A) Analytical SEC experiments showed that LbAha1 eluted as one peak between 45 kDa and 67 kDa. The MM of the standard proteins is displayed by arrows. Inset: estimation of the LbAha1 Rs, which was 32±2 Å. B) Sedimentation velocity experiments showing the c(S) distribution of LbAha1, which behaved as a monomeric species with s020,w of 2.62±0.02 S, MM of 42±2 kDa and ƒ/ƒ 0 of 1.65±0.04 also at higher concentrations. Inset: determination of the s020,w of the LbAha1 by linear regression analysis. In summary, the LbAha1 protein behaved as an elongated monomer in solution.
Figure 4
Figure 4. LbAha1 interaction and ATPase activity stimulation of LbHsp90.
A) The interaction of LbAha1 with the LbHsp90 was investigated by using ITC. The LbAha1 protein was titrated in the LbHsp90, generating a characteristic thermogram of an exothermic reaction. The raw data (upper panel) was treated and the points were adjusted by a One Set of Sites curve fitting (lower panel). These results revealed a LbAha1:LbHsp90 stoichiometry of 2∶1, with a KD of 1.0±0.1 µM and ΔHapp of −18,000±400 cal.mol−1. B) The calorimetric values of ΔHapp, ΔGapp and TΔHapp of the binding interaction between LbAha1 and LbHsp90 as a function of temperature. The binding ΔCpapp was determined from the slope of ΔHapp in dependence on temperature using Equation 3 (see text for details). The black lines represent the linear fitting. C) The analysis of the activator effect of LbAha1 on the ATPase activity of LbHsp90 was verified by enzyme kinetics. LbHsp90 (1 µM of dimer) was incubated with LbAha1 at various concentrations (0–16 µM) in 40 mM HEPES buffer (pH 7.5), containing 5 mM KCl and the Pi released from ATP hydrolysis was measured spectrophotometrically . The data obtained, treated as relative ATPase activity, presented a sigmoidal behavior and was adjusted using the Hill equation. The results revealed that LbAha1 increased around 10-fold the LbHsp90 ATPase activity in a mechanism of positive cooperativity, with a Hill coefficient of 1.7±0.2.
Figure 5
Figure 5. LbAha1 SAXS curves and data analysis.
A) The final scattering curve (open circles) of the LbAha1 was the average of the SAXS curves collected at various protein concentrations. The solid line represents the SAXS curve fitting until q = 0 Å−1 performed by the GNOM program during the distance distribution function generation. Inset: Guinier analysis of the LbAha1 SAXS curve showing its linearity. B) The particle distance distribution function of the LbAha1 was constructed by the GNOM program using the SAXS curve shown in A). LbAha1 showed a Dmax of 140±10 Å and the p(r) revealed a prolate shape. Inset: Kratky plot showing that LbAha1 is a compact protein.
Figure 6
Figure 6. Low resolution structure and rigid body modeling of the LbAha1.
A) DAM model and B) LbAha1 EOM conformer (superposed to the DAM model) in different views displaying its domain arrangement and the relative independence between them. This EOM conformer, which is one of the 18 selected conformers (Figure 6E and Figure S3– Supporting Information) of the best ensemble simulated for LbAha1, was selected based on the best superposition with the shape of the DAM model. C) Rg and D) Dmax relative distributions of the LbAha1 conformers calculated by the EOM approach considering the experimental SAXS pattern. The solid lines represent the distribution within the pool of 10,000 conformers and the dotted lines, the Rg- and Dmax-values observed for the best ensemble conformers. E) Representative LbAha1 conformers present in the best ensemble obtained by the EOM. The first conformer was used for superposing to the DAM model in the panel B. The N-terminal and C-terminal domains are shown in green and red, respectively; the reconstructed missing regions are displayed in blue.
Figure 7
Figure 7. Western blotting analysis of Aha1 and Hsp90 molecular chaperones.
The expression levels of LbAha1 and LbHsp90 proteins were analyzed in three Leishmania species: L. braziliensis, L. guyanensis and L. chagasi cultured at 26°C or 37°C during 2, 4 or 6 h. Rabbit polyclonal antibodies anti-Aha1 and anti-Hsp90 recognized the native and recombinant proteins. Monoclonal anti-α-tubulin antibody was used as loading control. The arrows indicate the protein bands.
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This work was supported by Fundação de Amparo à pesquisa do Estado de São Paulo (http://www.fapesp.br/): grants #2007/05001-4; #2008/09025-8; #2010/19242-6; #2011/23110-0; #2012/50161-8; #2012/01953-9; and Conselho Nacional de Pesquisa e Desenvolvimento (http://www.cnpq.br): grants #303792/2009-4 and #479229/2011-2. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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