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

International Journal of Biological Macromolecules 130 (2019) 125–138

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Thermodynamic analysis of interactions of the Hsp90 with adenosine nucleotides: A comparative perspective Karine Minari a,b, Érika Chang de Azevedo c, Vanessa Thomaz Rodrigues Kiraly a, Fernanda Aparecida Heleno Batista a, Fábio Rogério de Moraes d, Fernando Alves de Melo d, Alessandro Silva Nascimento c, Lisandra Marques Gava b, Carlos Henrique Inácio Ramos e, Júlio César Borges a,⁎ a

São Carlos Institute of Chemistry, University of São Paulo, São Carlos, SP 13566-590, Brazil Center of Biological and Health Sciences, Federal University of São Carlos, São Carlos, SP 13560-970, Brazil c São Carlos Institute of Physics, University of São Paulo, São Carlos, SP 13560-970, Brazil d Biosciences, Languages, and Exact Sciences Institute, Multiuser Center for Biological Innovation (CMIB), São Paulo State University, São José do Rio Preto, SP 15054-000, Brazil e Institute of Chemistry, University of Campinas UNICAMP, 13083-970 Campinas, Brazil b

a r t i c l e

i n f o

Article history: Received 18 December 2018 Received in revised form 20 February 2019 Accepted 20 February 2019 Available online 20 February 2019 Keywords: Hsp90 Protein-ligand interaction ITC STD-NMR Molecular dynamics

a b s t r a c t Hsp90s are key proteins in cellular homeostasis since they interact with many client proteins. Several studies indicated that Hsp90s are potential targets for treating diseases, such as cancer or malaria. It has been shown that Hsp90s from different organisms have peculiarities despite their high sequence identity. Therefore, a detailed comparative analysis of several Hsp90 proteins is relevant to the overall understanding of their activity. Accordingly, the goal of this work was to evaluate the interaction of either ADP or ATP with recombinant Hsp90s from different organisms (human α and β isoforms, Plasmodium falciparum, Leishmania braziliensis, yeast and sugarcane) by isothermal titration calorimetry. The measured thermodynamic signatures of those interactions indicated that despite the high identity among all Hsp90s, they have specific thermodynamic characteristics. Specifically, the interactions with ADP are driven by enthalpy but are opposed by entropy, whereas the interaction with ATP is driven by both enthalpy and entropy. Complimentary structural and molecular dynamics studies suggested that specific interactions with ADP that differ from those with ATP may contribute to the observed enthalpies and entropies. Altogether, the data suggest that selective inhibition may be more easily achieved using analogues of the Hsp90-ADP bound state than those of Hsp90-ATP bound state. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Hsp90s (heat shock proteins of 90 kDa) are ubiquitous molecular chaperones involved in many cellular processes, such as cell cycle control, signaling, maturation and activation of client proteins, and protein translocation [1–4]. Due to their importance in such a large variety of metabolic pathways, Hsp90s are considered potential targets for treating diseases, such as cancer and protozoa diseases [5–8]. Structurally, Hsp90s are homodimers formed by two identical protomers, which can be divided into three domains: The N-terminal domain (NTD – ~25 kDa) contains the adenosine nucleotide-binding site, undergoes transient cross-dimerization upon ATP binding and is responsible for the Hsp90 ATPase activity. The middle domain (~35 kDa) contains binding sites for co-chaperones and client proteins and is critical for the ATPase activity executed by the NTD. A charged ⁎ Corresponding author at: Instituto de Química de São Carlos, Universidade de São Paulo – USP, P.O. Box 780, 13560-970 São Carlos, SP, Brazil. E-mail address: [email protected] (J.C. Borges).

https://doi.org/10.1016/j.ijbiomac.2019.02.116 0141-8130/© 2019 Elsevier B.V. All rights reserved.

linker connects the N-terminal and middle domains, and its length and dynamics influence the ATPase activity and protein dynamics [9,10]. The C-terminal domain (~20 kDa) is responsible for protein dimerization and interaction with client proteins and co-chaperones [2,4,5,11–15]. Despite the high structural conservation across species, the dynamics and cycle mechanism of Hsp90 have peculiarities from protein to protein and from organism to organism. For instance, many conformational states of Hsp90 exist in equilibrium, and the functional cycle of Hsp90 is affected by adenosine nucleotides, co-chaperones and posttranslational modifications [3,13,15–19]. Initially, the open state of Hsp90 (a homodimer dimerized by the C-terminal domain of each protomers) binds to ATP molecules, which drives Hsp90 to closed states where NTD dimerizes transiently [20,21]. Afterward, a compact state responsible for ATP hydrolysis is reached. ATP hydrolysis, which can be considered low compared with that of other classical ATPases, and ADP/Pi release drive Hsp90 to the open state. Afterward, the client protein is released after folding, stabilization/maturation and activation or both, and the cycle restarts [2–4,15].

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Curiously, the conformational plasticity discussed above allows Hsp90 to interact with a large range of client proteins and cochaperones [3,15,22]. Apparently, random conformational fluctuations exist because these fluctuations are isoenergetic [14,19], but the fluctuations are also not irreversibly determined by nucleotides. This hypothesis is supported by recent studies showing that the Hsp90 machinery evolved and became less dependent on nucleotides. For instance, ADP or ATP can bind to the nucleotide-binding site in both the open and closed states without forcing the protein into a specific conformational state [18]. Additionally, for some organisms, the Hsp90 interaction with ATP or ADP results in an equilibrium of different conformational populations [5,13–16,23]. Another investigation [24] showed that the Hsp90 cycling time is critical for optimal action with client proteins, i.e., Hsp90 spends more time in the open conformations than in the closed conformations to process client proteins more efficiently. For humans, several Hsp90 studies focused on the prospect of inhibitors that are less toxic and more specific for cancer cells than geldanamycin, radicicol and analogues, which are the Hsp90 inhibitors first used in clinical trials [7,8,25–27]. However, the lack of solubility, the collateral clinical effects and the accumulation of toxic substances are bottlenecks for the development of a specific Hsp90 inhibitor for cancer treatment [28]. In other organisms, such as Leishmania braziliensis and Plasmodium falciparum (involved in the neglected diseases leishmaniasis and malaria, respectively), Hsp90s are involved in growth and differentiation at different stages of the cell cycle [29–33]. Some studies showed that the Hsp90 content increases under thermal stress and in different parasite development stages, which is interesting since the parasite transits through two hosts and undergoes a temperature change during the lifecycle [30,33,34]. When targeting Hsp90 in infectious diseases, one challenge that must be addressed is the high sequence identity between Hsp90 orthologues (60% to 70%) [5,7,35]. As clearly stated above, a detailed comparative analysis of several Hsp90s is relevant to the overall understanding of its activity. This work reports on a comparative thermodynamic analysis of the ATP and ADP interactions with Hsp90s of different organisms, as well as various NTD constructs. The findings indicated that the interactions of all Hsp90 orthologues with ATP were similarly driven by both enthalpy and entropy, while their interactions with ADP were variable and largely driven by enthalpy but opposed by entropy. Additionally, when adenosine nucleotide interactions were evaluated using NTD constructs, no significant differences were observed compared with the full-length protein, indicating that the measured thermodynamic signature reports events that are mainly located at the NTD and not on the entire Hsp90; many of these events are relevant to its molecular cycle [2,4,13,36]. Results from nuclear magnetic resonance (NMR) experiments indicated that both nucleotides interacted similarly through their adenine and ribose moieties with the NTD constructs of Hsp90 orthologues of human, yeast and L. braziliensis, indicating that the observed differences should not be related to those moieties. However, the results from molecular dynamics (MD) simulations suggested that the phosphate groups of ADP and ATP have different mobilities and that these groups tested different microstates, thus providing ATP with a more dynamic interaction profile and resulting in ATP having more entropy in the bound state than ADP. Therefore, for the first time, a comparative analysis of the interaction of adenosine nucleotides with Hsp90s of different organisms using the same methodology indicates that Hsp90s should allow selective inhibition. The results also imply that this inhibition might be more easily achieved using analogues of the Hsp90-ADP bound state. 2. Material and methods 2.1. Sequence analysis The sequences of Hsp90s from L. braziliensis (LbHsp90 XP_001567804.1), P. falciparum (PfHsp90 - XP_001348998.1), yeast

(yHsp82 - NP_015084.1), human α isoform (hHsp90α NP_005339.3), human β isoform (hHsp90β - NP_031381.2) and sugarcane (ScHsp90 - AGC60019.1), as well as of the NTD of L. braziliensis (LbHsp90N - residues 1 to 221), yeast (yHsp82N - residues 1 to 220) and human β isoform (hHsp90βN – residues 1 to 223), were aligned using the Clustal Omega program (http://www.ebi.ac.uk/Tools/msa/ clustalo/). Sequence identity tables were generated using the MUSCLE software (http://www.ebi.ac.uk/Tools/msa/muscle/). 2.2. Expression and purification The Hsp90 recombinant proteins of L. braziliensis, P. falciparum, human, yeast and sugarcane were obtained using different Escherichia coli Bl21(DE3) strains, as shown in Table S1 (Supporting information). Bacteria cells were transformed with the following expression vectors: pET28a::PfHsp90 [37], pET28a::LbHsp90 [5], pProEx-HTa::hHsp90α (a gift from Prof. Walid A. Houry, University of Toronto, CA), pProExHTa::hHsp90β (a gift from Prof. Jason C. Young, McGill University, CA), pET28a::ScHsp90 [38], pProEx-HTa::yHsp82 (a gift from Prof. Walid A. Houry, University of Toronto, CA), pET28a::LbHsp90N [5], pET28a:: hHsp90βN (cloned into Nde I and Bam HI restriction sites), p11:: yHsp82N (a gift from Prof. Walid A. Houry, University of Toronto, CA). Cultures were grown in lysogenic broth medium at 37 °C and 200 rpm up to A600nm 0.4–0.6 AU. After induction, cells were harvested by centrifugation, and the pellets were resuspended in 25 mmol L−1 sodium phosphate (pH 7.5) buffer containing 500 mmol L−1 NaCl and 20 mmol L−1 imidazole and incubated on ice for 40 min after the addition of 30 μg mL−1 lysozyme (Sigma) and 5 U DNAse (Sigma). Bacterial cells were lysed by sonication and subjected to centrifugation at 43,000g, and the supernatant was filtered with a 0.45 μm membrane. Proteins were purified according to Silva et al. [5] with minor modifications. Briefly, the purifications were performed in two steps: affinity chromatography using a HisTrap column (GE Healthcare Life Sciences), followed by size exclusion chromatography using a Superdex 200 26/60 column (GE Healthcare Life Sciences) prepared in 40 mmol L−1 HEPES (pH 7.5) buffer containing 100 mmol L−1 KCl. Both purifications were performed with columns coupled to an Akta Prime Plus (GE Healthcare Life Sciences) and using the same buffer as mobile phase. Protein purification was verified by 10% SDS-PAGE. Protein concentration experiments were performed using the molar extinction coefficient at 280 nm according to Pace et al. [39] obtained through the Protparam suite (bo.expasy.org/tools/ protparam.html). 2.3. Isothermal titration calorimetry measurements A MicroCal iTC200 microcalorimeter (GE Healthcare Life Sciences) was used for testing protein-ligand interactions. All experiments were performed in similar conditions for each protein, and the details are presented in Tables S2 and S3 (Supporting information). Briefly, proteins (approximately 15 μmol L−1 for dimeric whole Hsp90s or 50 μmol L−1 for the monomeric NTD constructs) were dialyzed for 3 h in 40 mmol L−1 HEPES (pH 7.5) buffer containing 100 mmol L−1 KCl in the presence of 2 mmol L−1 or 5 mmol L−1 MgCl2. The same buffer was used to prepare the stock solutions of ATP and ADP (Sigma). Adenosine nucleotides (1–2 mmol L−1) were added to the syringe and titrated into protein solutions (19 injections) at 25 °C and 1000 rpm with 120–150 s intervals. Typical ATP and ADP titrations in the aforementioned buffer were performed for controls. The concentrations of both proteins and nucleotides were previously quantified spectrophotometrically. The isothermograms were analyzed with the Microcal Origin software supplied with the device, using the fitting model for one set of sites to calculate the binding stoichiometry (n), apparent binding enthalpy change (ΔHapp), and association constant (KA). The heat of injectant dilution was determined from the baseline at the end of titration

K. Minari et al. / International Journal of Biological Macromolecules 130 (2019) 125–138

and subtracted from the data. The dissociation constant (KD) was calculated as the inverse of the KA value. The apparent Gibbs energy (ΔGapp) and apparent binding entropy change (ΔSapp) were calculated by the following equation: ΔG ¼ −RT lnKA ¼ ΔH−TΔS

ð1Þ

where T is the absolute temperature (in Kelvin) and R is the gas constant (in cal K−1 mol−1). Data are presented as the average of at least three independent experimental titrations. 2.4. NMR experiments The interactions of ADP and ATP with the NTD constructs from hHsp90βN, yHsp82N and LbHsp90N in the presence of 800 μmol L−1 MgCl2 were investigated by the saturation transfer difference (STD), which is an NMR-based technique [40]. STD experiments were performed on a 600 MHz 1H Bruker AVANCE III HD spectrometer (Bruker, Germany) equipped with a triple-resonance, pulsed-field, z-gradient cryoprobe. NTD constructs (20 μmol L−1) and adenosine nucleotides (800 μmol L−1) were prepared as described above. The standard Bruker pulse sequence STDDIFFESGP.3 was used to acquire data using excitation sculpting to suppress the water signal. An off-resonance at 20 ppm was used, and selective protein saturation was achieved by irradiating protein signals at 4 ppm for 2 s with a recycle delay of 3 s. Protein suppression was achieved by a spin-lock filter of 30 ms. Each 1D NMR spectrum was Fourier transformed using Bruker TopSpin 3.2, after applying a line broadening of 2 Hz. The same phasing factors were used for each pair of off- and on-resonance spectra. STD amplification factors (STD-AF) were calculated using Eq. (2), where IOFF and ION are the integrals for each ligand signal in the off- and on-resonance experiments, respectively. STD−AF ¼ ðIOFF −ION Þ

. I OFF

ð2Þ

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the simulation protocol was based on the energy minimization of the initial structure in 500 steps of a steepest descent algorithm and 500 steps of a conjugated gradient, followed by the heating of the system to 300 K in 50 ps while keeping the system volume constant. Afterward, the density of the system was equilibrated to 1 g cm−3 by 50 ps of equilibration at constant pressure (1 atm). During the energy minimization, heating and density equilibration, harmonic restraints were applied simultaneously to the protein structure, the ligand (ADP or ATP) and the Mg2+ ions (when present), with a weight of 2 kcal mol−1 Å−2. Finally, the system was equilibrated for 500 ps without restraints, and productive simulations were conducted for 100 ns for each complex. The equilibrium simulations were analyzed using CPPTRAJ [46] and AmberEnergy (http://github.com/alessandronascimento/ amberenergy), a program developed in-house for interaction energy calculations based on MD simulations. 3. Results and discussion 3.1. Sequence analysis The amino acid sequences of LbHsp90 (L. braziliensis Hsp90), PfHsp90 (P. falciparum Hsp90), ScHsp90 (sugarcane Hsp90), yHsp82 (yeast or Saccharomyces cerevisae Hsp90), hHsp90α (human Hsp90α isoform) and hHsp90β (human Hsp90β isoform) were aligned and analyzed by the Clustal Omega software. The sequence alignment analysis indicated identity values between 60% and 70% for both full-length and NTD proteins (Tables S4 and S5, Supporting information). Such high values represent a challenge for developing selective inhibitors since the inhibitors may end up interacting with many Hsp90 orthologues with similar affinities due to the few differences in the structures of these proteins. Accordingly, the amino acid residues involved in the interaction with adenosine nucleotides are conserved among all proteins, as depicted in Fig. 1, which shows an alignment of their NTD amino acid sequences. Therefore, Hsp90 inhibitors targeting the nucleotidebinding site are highly toxic to humans, precluding their therapeutic use [5,12,27,35,47].

2.5. Interaction map construction

3.2. Interaction study by isothermal titration calorimetry

The LIGPLOT program [41] was used to provide the interaction map between proteins and ligands and its conformational changes upon binding. LIGPLOT generates a 2D schematic diagram based on the three-dimensional coordinates from a PDB file and shows the amino acid residues that interact with the ligand by hydrogen bonds or hydrophobic contacts. The diagram also shows the amino acid residues that are equivalent when two structural models are superimposed, suggesting which residues are more likely to occupy the same coordinates in the three-dimensional space. The PDB structures used to analyze conformational changes were PDB ID: 1BYQ and PDB ID: 3T0Z of the human Hsp90Nα isoform in complex with ADP and ATP, respectively. We also evaluated the PDB ID: 3U67 and PDB ID: 3H80 of L. major Hsp90 NTD in complex with ADP and AMPPNP, respectively.

The thermodynamics of the interaction of nucleotides with Hsp90 has been puzzling the investigators involved with this study. Nilapwar et al. reports that the yeast Hsp82 NTD interaction with ADP has both a higher enthalpy contribution and a higher entropic cost than the ATP analogue [48]. However, from a molecular dynamics perspective, ATP is more rigid and longer than ADP; thus, the binding of ATP should have a higher entropic cost than that of ADP [21,49], rather than the opposite, which is observed in the experimental data [48]. Therefore, this work aims to further investigate the thermodynamic details of the interaction between nucleotides and Hsp90s. For such deep thermodynamic analysis, ATP and ADP were titrated into six different Hsp90 proteins and three NTD constructs from these proteins. For comparative purposes, all proteins were expressed and purified using the same methodology, as described in the Methods section, which provided 95% purity, as confirmed by SDS-PAGE (Fig. S1 – Supporting information). All purified recombinant had approximately 35% α-helices, as shown by circular dichroism analysis (Fig. S2 – Supporting information), similar to the findings reported in the original publications for LbHsp90N [5], LbHsp90 [5] and for ScHsp90 [38]. Based on the elution profile of the preparative size exclusion chromatography (data not shown), the full-length proteins were dimeric and NTD constructs were produced and purified as monomers, as described for LbHsp90N [5], LbHsp90 [5], ScHsp90 [38] and yHsp82 [50]. To obtain comparable data, all experimental parameters were maintained at the same values as much as possible for all assays (see Tables S2 and S3 - Supporting information), and all tested samples were fresh. Additionally, before titrations, all protein samples were dialyzed, and the final dialysis buffer was used to prepare the

2.6. Molecular dynamics simulations The crystal structures of NTD from either the human Hsp90α isoform, S. cerevisiae Hsp90 or Leishmania major Hsp90 were simulated in complex with either ADP or ATP (see Table 5 for details). Since there is no available crystallographic structure for LbHsp90N, we generated a molecular homology model using the MODELLER software [42] with the crystal structure from L. major Hsp90 NTD (PDB ID: 3H80) as a template. The model was validated by the DOPE-score [43] and SAVES programs (http://services.mbi.ucla.edu/SAVES/). In the MD simulations, all protein structures were parametrized according to the AMBER FF14SB force field [44], and the ligands were parametrized with the AMBER force field parameters as previously described [45]. For every complex,

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Fig. 1. Sequence alignment of the NTDs from the Hsp90 orthologues studied here. Gray, NTD. Yellow, residues directly interacting with adenosine nucleotides. Green, residues interacting with adenosine nucleotides through a water molecule. Blue, conserved Asp residues (which is Glu in yHsp82). Orange, the lid region.

ligand solutions, avoiding potential experimental mismatches in the ITC experiments. First, it is important to address the choice of using ATP instead of a non-hydrolyzing analogue since an isothermal titration calorimeter may also be used to monitor enzyme kinetics [2,51,52]. When used in this way, the heat (Q) involved in conversion of n moles of substrate (S) into product (P) can be expressed as Q = nΔH = [P]TotalVcelΔH, where ΔH is the enthalpy of the reaction (in cal.mol−1 of S). Therefore, the amount of Q generated can represent the presence of enzyme catalysis. However, for this calculation, the enzymatic activity needs to be N0.007 μmol L−1 s−1 (0.42 μmol L−1 min−1) at 10% Vmax [51], which is much higher than the ones reported for Hsp90 due to its low ATPase activity [5,14,29,37]. Importantly, an extensive search in the literature indicated that Hsp90 orthologs present ATPase activity, at 37 °C, varying from 0.089 up to 1.3 min−1 (in average of 0.5 min−1 – Table S4 – Supporting information), which means 10 times less than the necessary for being monitored by ITC. Besides, we found some published results for hHsp90β and yHsp82 at 25 °C, the same temperature we used in our ITC experiments, and it was 15 and 12 times b37 °C, respectively. These results suggest that, in conditions similar to those used in the ITC experiments, the ATPase activity is negligible. Furthermore, ITC experiments with Hsp90s and NTD constructs also indicated that under similar conditions, there was a greater heat release in titrations with ADP than in those with ATP (see below). Furthermore, ATP titrations with NTD constructs, which lack ATPase activity, yielded similar thermodynamic values to those of full-length proteins (see below). For an

additional control, we titrated AMPPNP into hHsp90β samples (Fig. S3 – Supporting information) and a very similar thermodynamic signature to titrations with ATP was observed (Table 1 and Fig. 2B). Our results indicated that ATP and AMPPNP are, therefore, equivalent. McLaughlin et al. [53] reports the AMPPNP titration into hHsp90β and, despite no thermodynamic data is presented, a very similar profile is shown. Hence, the ATP hydrolysis performed by Hsp90s in the tested conditions would be nonexistent or would yield a heat similar to that at baseline [54,55]. Fig. 2 shows representative isothermograms of the ADP and ATP titrations into hHsp90β samples. Similar isothermograms were observed for other full-length proteins and NTD constructs (Fig. S4 – Supporting information). Tables 1 and 2 summarize all experimental parameters obtained for the protein-ATP and protein-ADP interactions, respectively. On average, ADP titration (Fig. 2A) released more heat than did the titrations with ATP (Fig. 2B). The titrations of adenosine nucleotides into buffer preparations were endothermic with no clear transition (Fig. 2C), whereas the titrations with the proteins were exothermic (Fig. 2). Transitions were not observed in the experiments in the absence of Mg2+ (data not shown), indicating that this divalent cation is critical for the occurrence of interactions, which was also reported by others for yHsp82N [12] and for full-length porcine Hsp90 [56]. Nonetheless, adenosine nucleotide interactions in the absence of Mg2+ have been reported for steady-state measurements such as static fluorescence suppression [5], indicating that the ligand binding/dissociation rate constants probably influenced the ITC experiments.

Table 1 Experimental parameters obtained by ITC for protein-ATP interactions. Data represent the average of at least three independent titrations at 25 °C. Protein:ATP

n

hHsp90α hHsp90β yHsp82 LbHsp90 PfHsp90 ScHsp90 hHsp90βN yHsp82N LbHsp90N

3.5 ± 0.3 2.8 ± 0.8 3.5 ± 0.5 3.5 ± 0.5 2.9 ± 0.4 4.0 ± 0.9 1.8 ± 0.4 2.3 ± 0.3 1.7 ± 0.4

ΔHapp (kcal mol−1)

KA (10−3.mol L−1)

ΔGapp (kcal mol−1)

KD (μmol L−1)

−2.5 ± 0.4 −2.3 ± 0.1 −2.5 ± 0.4 −0.9 ± 0.2 −4.8 ± 0.2 −3.8 ± 0.3 −2.2 ± 0.1 −2.5 ± 0.4 −1.1 ± 0.3

65 ± 8 72 ± 3 90 ± 7 52 ± 5 90 ± 20 47 ± 3 19 ± 5 24 ± 5 40 ± 20

−6.6 ± 0.1 −6.6 ± 0.1 −6.8 ± 0.1 −6.4 ± 0.1 −6.6 ± 0.2 −6.4 ± 0.1 −5.8 ± 0.2 −6.0 ± 0.1 −6.2 ± 0.3

15 ± 2 14.0 ± 0.5 11.7 ± 0.4 19 ± 2 12 ± 5 21 ± 1 60 ± 10 40 ± 10 30 ± 10

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Fig. 2. Representative isothermograms for the adenosine interaction with Hsp90 orthologues. hHsp90β (15 μmol L−1 dimer concentration) in the presence of 2 mmol L−1 Mg2+ titrated with ADP (A) or ATP (B). All samples were prepared in 40 mmol L−1 HEPES (pH 7.5) buffer containing 100 mmol L−1 KCl and 2 mmol L−1 MgCl2. Assays were performed using an iTC200 device (GE Healthcare Life Sciences) at 25 °C. C) Representative titration of ATP into the aforementioned buffer. The goodness of fit was evaluated by the reduced chi2 value.

The results found for the ADP titrations notably differed from those for ATP (compare Tables 1 and 2). As expected, the stoichiometry for ADP interactions was approximately 2 to the whole Hsp90s, which are dimers, and was ~1 to the NTDs, which are monomers. Curiously, for ATP interactions, the stoichiometry with the full-length proteins was N3, suggesting the possible existence of a second type of ATP-binding site in the Hsp90 structure. There are some proposals for the existence of such an ATP-binding site at the C-terminal domain of Hsp90, as indicated by observations at low ionic strength [56,57]. Nevertheless, we cannot rule out the hypothesis that our results were due to the experimental titration set, which involved a high concentration of ATP (see Table S1), thus causing potential non-specific binding. First and foremost, our ATP titrations with the NTD constructs, which are monomers, also showed a stoichiometry of approximately 2, instead ~1 (Table 1). Second, our ITC curve fitting attempts with a model of two independent binding sites did not result in additional improvements in the fitting parameters (data not shown). Third, the experiments performed by Garnier et al. [56] were performed at a lower ionic strength, which could favor the interaction of ATP with the Hsp90 C-terminal domain. Further, the ITC experiments reported by Garnier et al. [56] indicate that the ΔHapp for the ATP interaction with the C-terminal binding site

was 4 times lower than that with the full-length protein. Therefore, if such interaction occurred in our experiments, it would not yield enough heat to change the curve profile in the calorimetric titrations performed here; thus, the detected ΔHapp data arose mainly from the nucleotidebinding site at the NTD, allowing our additional analysis as follows. Our results showed that the interaction of each Hsp90 with ADP had exothermic ΔHapp values at least twice those with ATP. In general, the ATP interaction yielded ΔHapp values from −1 to −5 kcal mol−1, while ADP yielded ΔHapp values from −8 to −20 kcal mol−1. Such results allowed the conclusion that the ADP interaction with Hsp90 orthologues released more heat than the ATP interaction when the same experimental conditions were considered. Similar results were reported for the interaction of yHsp82N with ADP and AMPPNP (non-hydrolyzable ATP analogue), where the former was more exothermic than the latter [48]. As a further support for this conclusion, deleting the Cterminal dimerization domain did not alter the trend. A higher heat release for ADP titrations was found for both the full-length and NTD proteins. The values of KA were also higher for interactions with ADP than for those with ATP, indicating that Hsp90 orthologues have higher affinity for ADP than ATP (Tables 2 and 3), as previously shown for yHsp82

Table 2 Experimental parameters obtained by ITC for protein-ADP interactions. Data represent the average of at least three independent titrations at 25 °C. Protein:ADP

n

hHsp90α hHsp90β yHsp82 LbHsp90 PfHsp90 ScHsp90 hHsp90βN yHsp82N LbHsp90N

1.5 ± 0.1 2.0 ± 0.5 2.4 ± 0.3 2.4 ± 0.2 2.4 ± 0.2 2.7 ± 0.5 1.0 ± 0.2 0.9 ± 0.1 1.1 ± 0.1

ΔHapp (kcal mol−1)

KA (10−3.mol L−1)

ΔGapp (kcal mol−1)

KD (μmol L−1)

−14.4 ± 0.4 −15 ± 2 −9.2 ± 0.8 −10.0 ± 0.3 −9.4 ± 0.3 −8.0 ± 0.9 −21 ± 2 −14 ± 1 −9.2 ± 0.9

200 ± 30 180 ± 10 200 ± 9 45 ± 4 470 ± 50 120 ± 20 200 ± 40 230 ± 70 90 ± 10

−7.2 ± 0.1 −7.2 ± 0.1 −7.2 ± 0.1 −6.3 ± 0.1 −7.7 ± 0.1 −6.9 ± 0.1 −7.2 ± 0.2 −7.3 ± 0.2 −6.8 ± 0.1

5.2 ± 0.8 5.6 ± 0.4 5.0 ± 0.4 22 ± 2 2.0 ± 0.2 9±2 5±1 5±1 12 ± 2

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[12,48]. Consistent with the conclusions when the other parameters were analyzed, deleting the C-terminal domain did not alter this observation. It is important to mention that despite the higher affinity for ADP, the in vivo cellular concentrations of ATP/ADP suggest that Hsp90 is mainly bound to ATP and not to ADP [12]. It is well known that ATP binding, ATP hydrolysis and the ADP release cycle are important events for directing the Hsp90 molecular mechanism [18], as well as for Hsp90s to populate several conformational states that are important for its molecular cycle timing [24]. Therefore, a strategy to develop an inhibitor based on the difference in affinity between ATP and ADP will have to consider the high intracellular concentration of ATP. Other thermodynamic parameters have also been taken into account when considering the binding of ATP and ADP (Fig. 3). The results found herein suggest that ΔGapp remained approximately similar for all ADP interactions (−6.3 to −7.7 kcal mol−1 – Table 2), as well as for all ATP interactions (−5.8 to −6.8 kcal mol−1 – Table 1). For ATP interactions (Fig. 3A and C), the thermodynamic signatures showed negative values for both the ΔHapp and -TΔSapp parameters, which contribute to the spontaneous nature of the interactions of both the full-length and NTD constructs. Among Hsp90 orthologues, one can notice some differences in the ΔHapp values for ATP interactions; these values became larger and more evident for the ADP interactions. Since ΔGapp values lie in a narrow window as argued above, the TΔSapp values also present an inherent compensatory performance. Such thermodynamic signature

dissimilarities can suggest the existence of subtle differences in the interaction mechanism among the Hsp90 proteins tested here. It is interesting to note that the similar thermodynamic signatures for ATP interactions with NTD constructs and with hHsp90β, yHsp82 and LbHsp90 proteins (Fig. 3A and C) indicate that the events measured might be intimately related solely to the interaction of ATP with the NTD. Therefore, the conformational changes involved with the Hsp90 cycle, which are triggered by ATP binding to Hsp90 and lead to intermediate states, probably did not produce enough heat during the time of the calorimeter measurements or were negligible once ligand binding occurred at the nucleotide-binding site. As a matter of fact, it is well documented that the Hsp90 molecular cycle is directed by nucleotide binding and hydrolysis and that the conformational changes during this cycle are time limited [18,20,58]; thus, their contribution to the released heat were likely negligible. The comparison of the magnitude of KD for ATP interaction (Table 1) and KM (Table S4 – Supporting information) also points the existence of a limiting step for ATP hydrolyzing. For example, the rate constant for hHsp90β assuming a compact conformation after ATP binding is of about 250 s [53]. Our ITC experiments indicated that all heat releasing for each initial ATP titration occurred in b50 s (data not shown). Therefore, heat due to conformational changes that drive Hsp90 to closed states, which involves the transient NTD dimerization [20,21], were not populated by ITC data or were in the baseline, being neglected. On the other hand, the thermodynamic signatures for interactions with ADP, but not ATP, were largely driven by enthalpy, although there were entropic penalties for both ADP interactions (Fig. 3B and

Fig. 3. Bar graphs summarizing the thermodynamic signatures for all tested interactions. Thermodynamic signature profile for interactions of full-length Hsp90 orthologues with ATP (A) and ADP (B) and of some Hsp90 NTD constructs with ATP (C) and ADP (D). The ΔGapp values were similar in all cases. The ATP binding shows a similar profile among all proteins and their NTD constructs but differ from those for ADP, whose interactions had unfavorable entropic contributions.

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D). Normally, an entropic penalty arises from one, or even more than one, of the following events: i) water trapping, ii) tight ligand binding, iii) stiffening of side chain amino acids at the binding site, iv) protein conformational changes due to ligand binding, among others [59–62]. Enthalpy contributions arise from formation of electrostatic interactions, protonation/deprotonation events, Van der Waals interactions and hydrogen bound, even those mediated by water molecules. Some of these events can also cause an entropic penalty in an enthalpy/entropy compensation [59–62]. Additionally, ADP binding to Hsp90 orthologues and NTD constructs yielded variable enthalpic and entropic contributions (Table 2), indicating subtle differences in the interaction mechanisms and/or conformational rearrangements upon ADP:Mg2+ binding. This observation is very interesting since it suggests that the ligand selectivity for protozoa Hsp90 over its human orthologues might be most easily achieved using analogues of the Hsp90-ADP bound state. 3.3. Molecular interactions evaluated by STD-NMR NMR spectroscopy has been widely used for the study of proteinligand interactions [63,64], and it plays a pivotal role in both structure-based and fragment-based drug design [65]. One of the main ligand-based techniques used in NMR is the evaluation of STD [40]. In this experiment, protein molecules are selectively irradiated, and due to spin diffusion, the molecules are saturated at the ligand-binding site. When a protein-ligand interaction occurs in the fast chemical exchange regime, magnetization is transferred from the protein to the

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ligand atoms by the nuclear Overhauser effect (NOE); the resulting decrease in intensity is observable in the difference spectrum. The proportion of each ligand signal in the difference spectra compared to its reference intensity gives rise to the so-called epitope mapping [66], i.e., the proximity of each ligand atom to the protein binding site. In the present study, we applied STD-NMR to further understand the molecular basis of the NTD Hsp90 interaction with ATP and ADP, specifically the hydrophobic proton-mediated interactions. First, Fig. 4 depicts the STD-NMR results for the hHsp90βN construct; these results indicate that the technique was capable of monitoring magnetization transfer for the same three protons from both ATP (Fig. 4A) and ADP (Fig. 4B). In these two cases, the hydrogen atoms bound to the C2 (II) and C8 (I) of the adenine base and the C1′ (III) of the ribose received magnetization from the protein (Table 3). Similar results were observed for yHsp82N (Table 3 and Fig. S5 – Supporting information) and LbHsp90N (Table 3 and Fig. S6 – Supporting information). Taken together, these results indicate that the binding mode of ATP and ADP with the nucleotide binding cleft of those three NTD proteins involved similar proton-mediated interactions and adenine and ribose moieties. These results are in accordance with the similar affinities observed in the ITC experiments for each NTD construct with ATP (Table 1) or ADP (Table 2), as well as with the structural identity observed for the amino acid residues in the NTD (Fig. 1). Additionally, as shown below, the crystallographic structures for the NTDs of hHsp90β in complex with ATP or ADP have virtually the same structures and interaction patterns with the adenine and ribose moieties, as indicated by the STD-NMR results for the tested

Fig. 4. Evaluation of the binding mechanism of ATP and ADP to hHsp90βN by STD-NMR. Binding of ATP (A) and ADP (B) to hHsp90βN in the presence of Mg2+. Ligand atoms are assigned to each signal in the NMR spectra by a Roman numeral. The STD-AF (numbers shown near each ligand atom). The on-resonance spectra are shown in the left upper panel and the offresonance spectra is shown the left lower panel.

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Table 3 Summary of the magnetization signals in the STD-NMR experiments. Protein

hHsp90βN

Ligand

MgATP

MgADP

MgATP

MgADP

MgATP

MgADP

8.119 2.740

8.117 1.677

8.119 0.097

8.117 0.040

8.119 0.085

8.117 0.063

8.396 0.433

8.402 0.027

8.396 0.011

8.402 0.020

5.999 0.579

5.994 0.031

5.999 0.015

5.994 0.022

Signal I

Signal II

Signal III

δ (ppm) STD-AF % AF δ (ppm) STD-AF % AF δ (ppm) STD-AF % AF

yHsp82N

−39% 8.402 0.569

−59%

−23% 5.994 0.621

LbHsp90N

−26% 8.396 0.013

−59%

−7%

−35% 5.999 0.019

−52%

−14%

% AF: difference in the STD-AF in % due to the presence of ATP in relation to presence of ADP %AF ¼ 100 

ðSDT−AF MgADP −SDT−AF MgATP Þ . STD−AF MgATP:

NTD constructs. Therefore, the thermodynamic signature differences observed for NTD interactions with ADP and ATP (Fig. 3) might be not due to their binding mechanism, suggesting that other features may have important roles. Interestingly, the magnetization transference was more efficient for ATP than ADP as indicated by the negative values for %FA (Table 3). Similar results were registered for the respective full-length proteins (data not shown). Such data are, apparently, in contrast to the relative higher affinity of Hsp90 for ADP, as determined by ITC (Tables 1 and 3) in the same experimental conditions. A possible explanation for these data is the higher flexibility of ATP in the binding site that might somehow favor the magnetization transfer. For ADP, the non-favorable entropy observed by ITC may indicate that ADP has less freedom degree that led to a relative smaller magnetization transfer efficiency. 3.4. Structural analysis of Hsp90N-ATP/ADP crystallographic complexes deposited in PDB database To investigate whether the thermodynamic signatures of the Hsp90 NTD interactions with ADP and ATP are related to structural differences in the binding site of the complexes, we searched for crystal structures in the Protein Data Bank. We found crystal structures for the NTD constructs of hHsp90αN and LmHsp90N (which is highly identical to LbHsp90N) in complex with ADP or ATP nucleotides (or AMPPNP non-hydrolysable analogue) with a resolution of at least 2.2 Å (Table 4). With these structures, we calculated the RMSD between the complexes with both adenosine nucleotides, taking into account (i) all amino acid residues, (ii) the backbone, (iii) the residues participating in the nucleotide-binding site and (iv) the residues forming the lid segment (Table 3). The calculated RMSD values were lower than 0.4 Å in all cases, indicating that the protein structures and nucleotide-binding site of hHsp90αN and LmHsp90N in complex with ADP and ATP are pretty similar. These results agree with the analysis reported by Li et al. for hHsp90αN, which indicated that the only difference between the ATPand ADP-bound states is the additional γ-phosphate moiety in ATP [67]. Taken together, these observations also indicate that the divergences in thermodynamic signatures between the ADP and ATP interactions are not related to differences in the NTD structure since the protein structure and nucleotide-binding site are virtually the same with both adenosine nucleotides [67]. It is worth mentioning that the RMSD between the apo form of the hHsp90α (PDB ID 5J2V) with ATP- and ADP-bound complexes are about 0.7. Considering only the lid segment, the RMSD were about 1.2 (data not shown). Such data indicate that large conformational changes occur induced by ATP or ADP binding (Fig. S7 – Supporting information). However, such complexes are similar, since their RMSD are b0.4 (Table 4), indicating that the thermodynamic signature differences should not be related to the protein structure or its flexibility. Several water molecules are present in the Hsp90 NTD crystal complexes with adenosine and inhibitor ligands [12,67,68]; thus, water

behavior could explain the differences in the thermodynamic signature shown in Fig. 3, since those water molecules might affect the entropic factor. Furthermore, a previous study reported that water arrest is probably involved in the ANPPNP interaction with the yHsp90N construct while ansamycin-based inhibitors result in water release from the nucleotide-binding site [48]. To monitor the water molecules and amino acid residues involved in the interactions in the crystal structures, we used the Ligplot program. Fig. 5 depicts the interaction maps of the NTD of hHsp90α and LmHsp90 with adenosine nucleotides, indicating that these NTDs use virtually the same set of interactions to bind ATP or ADP. Interestingly, the γ-phosphate moiety in ATP establishes electrostatic interactions in both Hsp90:ATP complexes (Fig. 5A and C) that are not observed in the Hsp90:ADP complexes (Fig. 5B and D). It would be expected that those additional electrostatic interactions would cause an entropic cost, but we observed that the interaction of the Hsp90 NTD constructs with ATP was also entropically driven, while ADP presented an entropic cost (Fig. 3). Most likely, an entropic-enthalpic compensation occurs in such interaction systems. Fig. 5B shows the interaction map for hHsp90αN:ADP:Mg2+. One can see that seven water molecules are close to the ADP and participate in

Table 4 Structural comparisons of hHsp90αN and LmHsp90N in complex with ATP or ADP. RMSD compared between the crystal structures of hHsp90αN and LmHsp90N in complex with ATP or ADP and number of water molecules interacting with different portions of the nucleotides or/and Mg2+ ion. For the hHsp90αN and LmHsp90N proteins, the table indicates the PDB ID, ligand, crystal resolution and which portion of the water molecules in the binding site participates in the interaction. The numbers in parentheses indicate the number of water molecules that indirectly interact with each moiety. The total number of water molecules does not account for interaction redundancies. Parameter

Protein complex hHsp90αN

PDB ID Ligand Resolution (Å) RMSD

Number of water molecules interacting with

All residues Backbone Binding site residues Lid (all residues) Lid α-helix 2 (all residues) Adenine moiety Ribose moiety Pi moiety Mg2+ ion Total

a b c d

G108-Y139. I128-G135. G93-Y124. D112-F119.

LmHsp90N

1BYQ 3T0Z 3U67 3H80 ADP ATP ADP ANPPNP 1.5 2.19 1.77 2.0 0.336 0.174 0.297 0.144 0.241 0.165 0.344a 0.350b 4 (6) 4 (2) 8 (10) 3 (1) 23

0.195c 0.290d

3 (1) 0 (0) 6 (6)

3 (8) 4 (6) 8 (6)

3 (1) 2 (1) 5 (1)

1 (1) 16

3 (1) 23

2 (6) 18

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Fig. 5. Interaction map of the LmHsp90N and hHsp90αN isoforms with adenosine nucleotides. Hydrogen bonds are indicated by green dashed lines, and hydrophobic contacts are represented by an arc with spokes radiating toward the ligand atoms. Water molecules are represented by light blue spheres, and the Mg2+ ions are shown in green spheres. A) and B) 2D view of the NTD of hHsp90α with ATP and ADP, respectively, in the binding site pocket. C) and D) 2D view of NTD of LmHsp90 with AMPPNP and ADP, respectively, in the binding site pocket. Green circles depict the additional water molecules observed in the NTD:ADP:Mg2+ complexes.

hydrogen bonds, which were not observed in the hHsp90αN:ATP:Mg2+ complex (Fig. 5A). Similar features are present for LmHsp90N, whose complex with ADP (Fig. 5D) shows five additional water molecules besides the ones in the complex with ATP (Fig. 5C). These data suggest that water molecules trapped in the ADP interactions could interfere at the thermodynamic signature, since those molecules are losing the freedom degrees, creating new hydrogen bonds and consequently lowering their entropy. However, the structural resolution for each analyzed crystal

structure (Table 3) does not allow such differences to be defined as the determining feature. To better understand these effects, as described below, MD was conducted to further investigate these ambiguities. 3.5. Molecular dynamics simulations NMR data from yHsp82N also indicate that adenosine nucleotide interactions result in widespread structural change in all proteins,

Table 5 Hsp90 NTD crystallographic structures used for structural analysis and MD simulations. PDB IDs and structural resolution for the complexes between Hsp90 NTD constructs with adenosine nucleotides and analogues. Organism

PDB ID

Ligands

H. sapiens (hHsp90αN)a

1BYQ 3T0Z 3U67 3H80 1AMW Homology modela Homology modela

ADP + Mg2+ ATP + Mg2+ ADP + Mg2+ AMPPNP+Mg2+ ADP ADP ATP

L. major (LmHsp90N) S. cerevisiae (yHsp82N) L. braziliensis (LbHsp90N) a

Molecular homology models created using the Modeller software.

Resolution (Å) 1.50 2.19 1.77 2.00 1.85 – –

Reference [61] [60] [6] [6] [12] Not published Not published

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without causing a large structural rearrangement [48]. Therefore, the ligand binding causes conformational changes that are not restricted to the side chains directly involved in the ligand interaction. Such experimental data also indicate that the lid segment of the NTD is the main region that is affected by the ligand interaction [48]. MD simulation data presented by Colombo et al. also indicate that the NTD has intrinsic plasticity and that the lid segment becomes flexible upon ATP binding; however, the NTD-ATP structure becomes more rigid and ordered than the NTD-ADP complex [49]. Nevertheless, such observations are in contrast to the experimental entropy cost found upon ATP/ADP binding in the present study (see item 4.2) and by Nilapwar et al. [48]. Additionally, experimental evidence from the crystal structures of the NTD of hHsp90α bound to ADP and ATPγS (PDB ID: 1BYQ and PDB ID: 3T2S, respectively) suggest that the lid movement might not occur in all Hsp90s [67], as shown by the RMSD of such complexes (Table 5). Considering that the structures of the same Hsp90 NTD construct in complex with ATP or ADP are virtually identical including the ligand and the side chain residues involved in the interactions (Figs. 5 and S7 – Supporting information), we wondered whether the thermodynamic signature differences could be associated with some dynamic interactions in which the ligand tests different modes or microstates of the interaction as a function of time. Therefore, we performed MD simulations with some Hsp90 NTDs in the presence of ATP or ADP in the nucleotidebinding site. Specifically, we investigated the participation of water molecules in the interaction between adenosine nucleotides and Hsp90 proteins and the microstates tested by the ligand and side chains in the nucleotide-binding site. For these tests, we used the hHsp90αN isoform, since there are no crystal structures for hHsp90βN complexed with ATP available in the PDB. We thought this approach was valid because the hHsp90α and hHsp90β isoforms share high sequence identity (91%) between their NTD amino acid sequences and because the observed differences are not in the nucleotide-binding site (see Fig. 1). In addition, their thermodynamic signatures are virtually the same for both the ATP and ADP interactions (see Fig. 3). The complexes formed between Hsp90 NTDs and adenosine nucleotides were simulated for 100 ns, resulting in a total simulation time of N1 μs. Stable interaction conformations were observed during the simulations only when Mg2+ was introduced as a part of the protein:nucleotide:Mg2+ complex. In the absence of this ion, spontaneous unbinding of the nucleotide was observed for some complexes, and in other cases, spontaneous binding of a solvent cation (Na+) in the Mg2+ site was observed (data not shown). These observations indicate the important role of Mg2+ in nucleotide binding, especially for ATP. In addition, this Mg2+ dependence is in agreement with the ITC experiments, in which interactions were observed only in presence of that divalent cation (data not shown). Therefore, the divalent cation was modeled in the nucleotide-binding site, as observed for the crystal structures of hHsp90αN bound to ADP and ATP and was used in all MD simulations. In contrast to previously reported results [49], a nucleotide-dependent lid movement was not observed in our simulations (data not shown), in line with the stable conformation observed for hHsp90N crystal structures [67] (see Fig. S7 – Supporting information). Interaction energies were computed according to the AMBER force field parameters, and the graphical representation of the potential energies between protein:Mg2+ and the nucleotide is given in Fig. S8 for the ADP and ATP complexes (Supporting information). The results indicate that stable conformations of the ligands were achieved within the simulation time, resulting in stable binding energies as well. Interestingly, despite the conservation in the primary structure among the studied proteins, significant differences in the binding energies were observed for the MD data. For ADP:Mg2+:protein complexes, the binding energies followed LmHsp90N b yHsp82N b hHsp90αN b LbHsp90N. For the ATP: Mg2+:protein complexes, the binding energies were ranked as yHsp82N b LmHsp90N b hHsp90αN b LbHsp90N (Fig. S8 – Supporting information).

A more complete evaluation of the binding energies must take into account the role of the solvent in the binding energetics. The average interaction energies were computed with MMPBSA.py [69] using desolvation energies evaluated by the generalized Born method. However, the binding energies were similar (within the estimated error) for all complexes (Table S6). These results are in agreement with the experimental data obtained by ITC, which show that the experimentally determined ΔGapp values are in the range between −6.3 and − 7.7 kcal mol−1 for ADP (Table 2) and in the range between −5.8 and − 6.8 kcal mol−1 for ATP (Table 1). Since the differences are typically b1 kcal mol−1, one would not expect to observe significant differences in the binding energies computed from MD simulations. On the other hand, despite the similar affinities, the thermodynamic signatures observed for ATP and ADP vary significantly among the proteins experimentally evaluated by ITC. Interestingly, the binding of ATP results in favorable entropic contributions, i.e., negative –TΔS (Fig. 3D), while the binding of ADP results in unfavorable entropic contributions (Fig. 3C), leading to very interesting enthalpic/entropic compensatory effects. The reasons for these marked differences were further investigated by means of the MD simulations data. The entropic contribution was investigated by measuring the number of water molecules found in first and second solvation shells of the bound ligand. For all bound nucleotides, a similar feature was observed in our MD simulations: bound ADP had fewer water molecules in its solvation shells than did bound ATP (Table S7), thus suggesting that ligand solvation may actually play a role in the binding entropy. However, this observation is in contrast to the water molecules directly involved in the nucleotide binding complexes, as shown in Fig. 5 and Table 4. To further investigate the solvent entropy of the systems of yHsp82N bound to ADP and ATP, which presented similar numbers of water molecules in both solvation shells, we mapped regions where unfavorable entropy was observed, as computed with GIST (Fig. S9 – Supporting information). Interestingly, the ADP complex had fewer regions of unfavorable entropy around the ligand than the ATP complex did, suggesting that water molecules would tend to have a lower degree of freedom due to the γ-phosphate group. This finding indicates that during the MD simulation, ATP arrested more water molecules than did ADP. Therefore, the NTD:ATP complexes should present a higher entropic cost than the NTD:ADP complexes due to the water molecules, which is in contrast to the experimental data obtained by ITC (Fig. 3). Therefore, despite the possibility of some contribution to the thermodynamic signature, the solvent behavior must not be the critical element that explains the experimental data. Next, we investigated the ligand behavior during the MD simulations. The RMSD distribution for adenosine nucleotides in the NTD complexes of hHsp90αN was computed over the simulation time, and the complexes had a similar distribution profile, with a peak at approximately 1 Å for ADP and 1.5 Å for ATP (Fig. 6A). The main interactions of the ligand with the protein showed that both ADP and ATP interact with Asn41 and Lys102 via the phosphate groups (Fig. 7A). Interestingly, the slight increase in RMSD seems to be associated with the increased mobility of the γ-phosphate group interacting with Lys102. For LbHsp90N, the RMSD distributions computed for ADP and ATP showed a very similar profile, with ligands that were equally mobile in the active site, as depicted in Fig. 6B. For the NTD of yHsp82, in contrast to the L. braziliensis orthologue, notable differences were observed between the ADP- and ATP-binding modes. As depicted in Fig. 6C, ADP was more flexible when bound to the yHsp82N active site than ATP. However, when the RMSD distribution was plotted (Fig. 6C), a bimodal distribution was observed for ATP, with a major peak found at RMSD 1.5 Å and a second peak close to 3 Å. For ADP, a broad, and possibly bimodal, peak was observed at RMSDs of 2.5–3 Å. The increased mobility observed for ADP seemed to be associated with alternating hydrogen bonds between the ribose hydroxyl groups and Glu87 or Lys43, as shown in Fig. 7C. Interestingly, such interaction was observed during the MD simulation only for NTD

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Fig. 6. Ligand mobility into the nucleotide-binding site during the MD simulations. Superposition of 500 frames from the simulation for the A) ADP and B) ATP, illustrating the ligand mobility. C) Plots of the interaction energies computed between the complex protein:Mg2+ and the ligand.

of yHsp82, which has a longer acid amino acid (Glu87) in that position while the orthologues have the shorter Asp in the same position (Fig. 1). On the other hand, the changes in RMSD for the ATP complex were

associated with new interactions of the ligand with amino acids in the active site. The ATP phosphate groups lost H-bonds with Phe123 and formed new interactions with Lys97 and Lys43 (Fig. 7C). This H-bond

Fig. 7. Main protein-ligand interactions during MD simulations. A) Interaction of the ligand ADP (left) in the hHsp90N complex with Asn41 and Lys102 or ATP (right). B) Interaction of the ligand ADP (left) in the LbHsp90N complex with Arg97 and ATP (right). C) Interaction of the ligand ADP (left) in the yHsp82N complex with Glu87 and Lys43 and of the ligand ATP with Lys97, Phe123 and Lys43 (right).

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Fig. 8. Conformational ensembles. Plots of the RMSD for the ligand and the interaction energy between A) hHsp90αN, B) LbHsp90N and C) yHsp82N and ADP (black) or ATP (blue) as representations of conformational colonies.

exchange seemed to contribute to a favorable entropy for ATP interaction with yHsp82N, which was not observed in the MD simulations with ADP. Since the entropy of a system is directly related to the number of microstates (Ω) sampled, according to the Boltzmann Law, i.e., kS ¼ ln Ω, the favorable entropic contribution for ATP binding, compared to ADP binding, seems to be related to its ability to sample different microstates. An approach to assess the conformational entropy differences of these complexes is an analysis of the plot of the RMSD values versus the binding energies, similar to the analysis of the colony method [70]. As shown in Fig. 8, for hHsp90αN, a more disperse distribution of states is observed for the ATP-bound simulation than for the ADP-bound simulation. These observations are in line with the ITC data where ATP showed a favorable entropic contribution to binding, while ADP had an unfavorable entropic contribution. For LbHsp90N, equally distributed states were observed for ADP and ATP, also in line with the experimental observation of a less divergent thermodynamic profile for the binding of these ligands. Finally, for yHsp82N, ATP clearly sampled a different region of the phase space that did not seem to be accessible to ADP, thus providing a favorable entropic contribution to binding.

4. Conclusions The results presented here involving the adenosine nucleotide interactions with Hsp90 orthologues and NTD constructs showed that the thermodynamic signatures clearly differ between ADP and ATP. Albeit very interestingly, the recorded thermodynamic data for some fulllength Hsp90 and their respective NTD constructions, which were very similar, report only about the interaction with ATP or ADP. In other words, in the tested conditions, our data do not allow any speculation on the Hsp90 ATPase cycle. This observation may be explained by the limiting step for Hsp90 ATPase cycle, which involves several conformational changes triggered by ATP binding that lead the protein to the compact conformation I. The measured affinities were similar for all tested Hsp90s, however, the ADP interactions presented entropic costs, which were compensated by a large enthalpic contribution. This thermodynamic signature was not observed for the ATP interaction since it was driven by both entropy and enthalpy. Therefore, it should be expected that ADP establishes additional interactions with the Hsp90 nucleotide-binding site compared to those for ATP. However, the structural analyses of the Hsp90 NTD in complex with both adenosine nucleotides do not support this conclusion. Such structures also revealed that they are very similar, including the lid segment, suggesting that differences in the structure or flexibility of the NTD also do not support the thermodynamic signature

registered. On the other hand, the MD studies of three NTD constructs indicated that the observed thermodynamic signatures arose from the different microstates tested by each ADP or ATP complex. Therefore, the NTD plasticity and dynamics may explain the Hsp90 behavior upon ADP and ATP binding. Further, MD simulations also indicated that the complexes of orthologues of the Hsp90 NTD have their own microstates. Altogether, our results are very interesting since selective inhibition may be more easily achieved using analogues that populate Hsp90ADP-bound microstates and are capable of discriminating the ATPbound ones. It must be taken into account that cellular ADP concentrations are normally 1–2 orders of magnitude lower than the ATP concentrations; therefore, a compound that binds to a Hsp90-ADP microstate should have higher potential to sequester Hsp90 from the cellular medium. Acknowledgements We are in great debt with FAPESP (2009/53989-4; 2011/23110-0; 2012/50161-8; 2013/25646-0; 2014/07206-6; 2015/26722-8; 2017/ 07335-9 and 2017/18173-0) and CNPq (471415/2013-8 and 303129/2015-8) for financial support. We thank Prof. Walid A. Houry (University of Toronto, CA) and Prof. Jason C. Young (McGill University, CA) for gently providing some of the expression vectors here used. Author contributions K.M., F.A.H.B. and J.C.B. conceived and designed the experiments. K.M. and F.A.H.B. produced and purified all proteins. K.M., F.R.M. and F.A.M. performed the RMN-STD experiments, data analysis and interpreted them together with J.C.B. E.C.A. and A.S.N. performed the MD simulations and interpreted them together K.M., V.T.R.K. and J.C.B. K.M., V.T.R.M. and J.C.B. analyzed the crystallographic structures found in PDB and interpreted the results. K.M., L.M.G., C.H.I.R. and J.C.B. organized the results and wrote the manuscript. All authors read and approved the final version of the manuscript. Competing interests The authors declare no competing interests. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.ijbiomac.2019.02.116.

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