Thermodynamic analysis of interactions of the Hsp90 with adenosine nucleotides: A comparative perspective

Aspergillus fumigatus Hsp90 interacts with the main components of the cell wall integrity pathway and cooperates in heat shock and cell wall stress adaptation

“…As a first approach to assess structural properties of the rAfHsp90, circular dichroism (CD) spectropolarimetry and intrinsic tryptophan fluorescence spectroscopy were used to analyse secondary and local tertiary structures of the protein, respectively. The CD spectra presented a similar profile to that reported for different Hsp90 orthologues, with secondary structure content estimated as 33% of α‐helix, 17% of β‐sheets, 17% of turns and 33% of random coils (Figure 5b), also similar to previous characterizations (Minari et al, 2019; Silva, Seraphim, & Borges, 2013). The intrinsic emission fluorescence spectra for Hsp90 (Figure 5c) indicated a maximum emission wavelength (λ max ) at around 334 nm, suggesting that the five Trp residues in the polypeptide are buried or partially protected from the solvent.…”

“…In spite of that, the apparent Gibbs energy change (ΔG app ) was around −6.3 to −6.8 kcal mol −1 which means binding affinities in the same order reflecting in dissociation constants (K D ) of 12 ± 2 μM to ADP and 24 ± 2 μM to ATP. These values are in the same range of those determined to Hsp90 orthologues in the same conditions (Minari et al, 2019). To mention some orthologues previously studied, AfHsp90 is 61, 64 and 75% identical to Hsp90 of Plasmodium falciparum , human (alpha isoform) and yeast, respectively (Minari et al, 2019).…”

“…These values are in the same range of those determined to Hsp90 orthologues in the same conditions (Minari et al, 2019). To mention some orthologues previously studied, AfHsp90 is 61, 64 and 75% identical to Hsp90 of Plasmodium falciparum , human (alpha isoform) and yeast, respectively (Minari et al, 2019). rAfHsp90 showed low ATPase activity as evaluated by the kinetic parameters obtained by the Michaelis–Menten fitting of the ATPase activity rate (Figure 6d).…”

“…It is known that the low ATPase activity of Hsp90 is required for the chaperone cycle and this enzymatic activity relies on the N‐domain dimerization as well as the synergistic interaction with different co‐chaperones and resulting conformational changes (Schopf et al, 2017). Based on these features, the ATP hydrolysis rate varies in different species (Chatterjee & Tatu, 2017; Minari et al, 2019). To evaluate the functionality of the rAfHsp90, we determined its binding affinities to ADP and ATP by isothermal titration calorimetry (ITC), which also allowed obtaining the thermodynamic parameters of the nucleotide binding.…”

“…ITC experiments were used to measure the interaction of Hsp90 and its natural ligands ATP or ADP in the presence of Mg 2+ . The assays were conducted as previously reported (Minari et al, 2019) in a MicroCal ITC200 microcalorimeter (GE Healthcare).…”