Volume of Hsp90 ProteinâLigand Binding Determined by Fluorescent

Article pubs.acs.org/JPCB

Volume of Hsp90 Protein−Ligand Binding Determined by Fluorescent Pressure Shift Assay, Densitometry, and NMR Zigmantas Toleikis,† Vladimir A. Sirotkin,‡ Gediminas Skvarnavičius,† Joana Smirnoviene,̇ † Christian Roumestand,§ Daumantas Matulis,† and Vytautas Petrauskas*,† †

Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University, Saulėtekio al. 7, LT-10257 Vilnius, Lithuania ‡ A.M. Butlerov Institute of Chemistry, Kazan Federal University, Kremlevskaya Street 18, Kazan 420008, Russia § Centre de Biochimie Structurale, INSERM U1054, CNRS UMR 5048, Universités de Montpellier, 34000 Montpellier, France ABSTRACT: Human heat shock protein 90 (Hsp90) is a key player in the homeostasis of the proteome and plays a role in numerous diseases, such as cancer. For the design of Hsp90 ATPase activity inhibitors, it is important to understand the relationship between an inhibitor structure and its inhibition potential. The volume of inhibitor binding is one of the most important such parameters that are rarely being studied. Here, the volumes of binding of several ligands to recombinant Hsp90 were obtained by three independent experimental techniques: fluorescent pressure shift assay, vibrating tube densitometry, and high-pressure NMR. Within the error range, all techniques provided similar volumetric parameters for the investigated protein−ligand systems. Protein−ligand binding volumes were negative, suggesting that the protein−ligand complex, together with its hydration shell, occupies less volume than the separate constituents with their hydration shells. Binding volumes of tightly binding, subnanomolar ligands were significantly more negative than those of weakly binding, millimolar ligands. The volumes of binding could be useful for designing inhibitors with desired recognition properties and further development as drugs.

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INTRODUCTION Pressure is fundamentally just as important as temperature from the point of view of thermodynamics. However, due to experimental difficulties, pressure studies are relatively rare, and thus the results are limited to temperature dependences, providing an incomplete perspective on a studied subject such as protein folding or protein−ligand binding. Determination of the volumes of protein−ligand binding could be helpful in drug design. The volume of a protein has many contributing factors arising from the volume of the molecule’s atoms, inhomogeneities of protein spatial packing (cavities and clefts), and volume changes related to solvent-mediated interactions and thermal fluctuations.1−7 In addition to the above-mentioned factors, protein volume changes can be a consequence of protein−ligand interaction. Hereafter, we use the term protein−ligand binding volume when referring to the change in system volume observed upon ligand binding to the protein (note that in some papers it is called the reaction or interaction volume). To be more precise, protein−ligand binding volume is defined as the difference between the volume of the hydrated protein−ligand complex and the sum of the volumes of the hydrated protein and hydrated ligand. Protein binding volume is an important but a largely neglected thermodynamic parameter from the perspective of both fundamental science and potential applications in the development of specifically binding protein ligands. © 2016 American Chemical Society

Relatively high controversy persists in the literature regarding the magnitude and even the sign of contributing factors of protein volume.1,3,5,8,9 Such a knowledge gap encourages researchers to develop more accurate experimental techniques and data analysis tools. A detailed analysis of the volumetric data could also yield more knowledge about the compressibility, another important thermodynamic parameter that is difficult to obtain experimentally. A thorough literature survey showed that there exists very little data related to the ligandbinding volume and compressibility changes of proteins.4,10−17 The above-mentioned controversy is a testimony that protein volumetric properties could become more reliable if determined by several independent experimental techniques. The agreement between different methods could also provide more confident reference points when determining other volumerelated thermodynamic parameters such as compressibility or thermal expansion. The most straightforward definition of the protein binding volume, ΔVb, comes from thermodynamics, which says that it is the first partial pressure derivative from the Gibbs energy of binding at constant temperature T Received: July 8, 2016 Revised: August 23, 2016 Published: August 29, 2016 9903

DOI: 10.1021/acs.jpcb.6b06863 J. Phys. Chem. B 2016, 120, 9903−9912

Article

The Journal of Physical Chemistry B

Figure 1. Chemical structures of the Hsp90 ligands used in this work.

⎛ ∂ΔG b ⎞ ΔVb = ⎜ ⎟ ⎝ ∂p ⎠T

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MATERIALS AND METHODS Protein Preparation. The N-terminal domains of the αand β-isoforms of Hsp90 protein (abbreviated as Hsp90αN and Hsp90βN, respectively) used in FPSA and densitometry experiments were expressed and purified as previously described.22,28 The 15N isotope-labeled Hsp90αN (Hsp90αN*) was expressed by using the Escherichia coli strain BL21(DE) grown in M9 minimal medium with 15N isotope-labeled ammonium chloride (1 mM of M2 trace elements, 1 mM magnesium sulfate, 42 mM sodium hydrogen phosphate, 22 mM potassium dihydrogen phosphate, 1 g of ammonium chloride, 4 g of glucose, and pH 7.2). The selected colony from the agar plate was inoculated into 25 mL of Luria−Bertani medium. Overnight culture was spun down at 5000g for 20 min and suspended in 1 L of M9 medium. The cells were grown up to OD600 = 0.7; the protein expression was induced with 0.4 mM IPTG and grown for 5 h at 37 °C. The expressed Hsp90αN* was purified using the same procedure used for unlabeled protein. Ligands. The chemical structures of the Hsp90 ligands used in this study are shown in Figure 1. Compounds AZ1, AZ2, and AZ3 are weak millimolar binders, and radicicol is a naturally occurring strong subnanomolar binder of Hsp90.21,26 A group of ligands, 5-aryl-4-(5-substituted-2,4-dihydroxyphenyl)-1,2,3thiadiazole compounds (hereafter abbreviated as ICPD), have been described previously21 and have a submillimolar (ICPD9) to nanomolar (ICPD47, ICPD62) range of Hsp90 binding affinities.21,22 FPSA Experiment and Data Analysis. In a usual FPSA experiment, the protein solution contained 10 μM Hsp90αN, 0.4 M guanidine hydrochloride (GndHCl), 1% dimethyl sulfoxide (DMSO), 50 mM NaCl, 100 μM 1,8-anilinonaphthalene sulfonate (ANS), and 10 mM Bis−Tris buffer, pH 7.0. All aqueous solutions were prepared in Milli-Q purified water, and ligand solutions were prepared in DMSO.

(1)

Thus, in general, to determine the volumetric properties of a protein−ligand system, one has to determine ΔGb as a function of pressure. The application of this general equation in particular cases using different experimental techniques will be described in the following section. The focus of this article is the determination and comparison of the changes in the volume of heat shock protein 90 (Hsp90) binding of several compounds having affinities in the millimolar to subnanomolar range. Hsp90 is a chaperone protein participating in a wide range of physiological processes. It stabilizes proteins against heat stress and assists the proper folding of other proteins. Hsp90 inhibitors are considered and studied as potential drugs against cancer,18 neurodegenerative disorders,19 infections,20 and other diseases. The thermodynamic properties of Hsp90 protein binding with numerous inhibitors have been previously described in detail using isothermal titration calorimetry, fluorescent thermal shift assay (FTSA), and other techniques.21−25 In this article, we show the volumes of compound binding to Hsp90 determined by three independent experimental techniques: fluorescent pressure shift assay (FPSA), vibrating tube densitometry, and high-pressure NMR. Densitometry is considered a direct technique used to measure volumetric properties of proteins, whereas the other two techniques are qualified as indirect methods, as they exploit conformational changes of proteins to obtain the volumetric information. Experimental data that were obtained by densitometry and NMR techniques support our previous findings26,27 that tightbinding ligands have a higher impact on the volume of Hsp90αN protein. 9904


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±1.0 × 10−6 g cm−3. All densitometric experiments were conducted at 25 °C. The aqueous Hsp90αN and Hsp90βN protein solutions used in the densitometry experiments were the same as those used in the FPSA experiments. The partial molar volume of a protein, V0, in the presence of a ligand can be calculated using the equation

The FPSA method has been described previously.26,27,29,30 Here, we provide a brief summary of this technique. Pressureinduced protein denaturation experiments were performed using an ISS PC1 photon-counting spectrofluorimeter equipped with a high-pressure cell connected to a hydrostatic pump. A pressure of 380 MPa was attained by gradual increments of 20 MPa while maintaining a constant temperature of 25 °C using a circulating water bath. The system was allowed to equilibrate for at least 2 min after each pressure increment before recording the fluorescence signal of intrinsic tryptophan residues (observed by exciting at 295 nm and recording the emission at 332 nm) or extrinsic probe ANS (observed by exciting at 350 nm and recording the emission at 480 nm). Volumetric properties of the studied protein−ligand systems were determined as described previously.26 FPSA equations were derived exploiting the reaction scheme, which describes the equilibrium between native (N) and unfolded (U) protein states in the presence of a ligand (L) Dissociation

Unfolding

Binding

Folding

[NL] HoooooooooooI [N] + [L] HooooooooI [U] + [L]

V0 =

Pt

stoichiometric ratio of protein−ligand association, the partial protein volume at an arbitrary value of r, V0(r), can be described in terms of the partial molar volume of protein in the absence of a ligand, V(0), and the change in protein volume associated with the ligand binding, ΔVb

(2)

V 0(r ) = V 0(0) + αΔVb

α= (3)

ΔGx = ΔG0_x + ΔVx(pm − p0 ) −

Δβx 2

(4)

Here, R is the universal molar gas constant and T is the absolute temperature. Indices U and b stand for the changes in Gibbs energy, ΔGx, related to protein unfolding and protein− ligand binding, respectively. ΔG0 and ΔV denote the changes in Gibbs energy and volume at the reference pressure p0, respectively. The parameter Δβ is defined as −

( ) ∂ΔV ∂p

and

T

referred to as the differential partial molar compressibility (note that another common term is the compressibility factor). It is related to the isothermal compressibility, ΔβT, by the following

( )

equation: ΔβT = −V −1

∂ΔV ∂p

= V −1Δβ . The values of pm

T

were determined by fitting the protein unfolding profiles as a function of pressure f (p) = fN +

fU − fN 1 + exp(ΔG U /RT )

K ⎞ 1⎛ ⎜1 + r + d ⎟ − 2⎝ Pt ⎠

17

2 K ⎞ 1⎛ ⎜1 + r + d ⎟ − r 4⎝ Pt ⎠

(8)

Here, Kd is the dissociation constant of the protein−ligand complex. The determination of protein binding volume could be simplified in the case of tight-binding ligands, because the fraction of ligand-bound protein at r > 1 in eq 8 is approximately equal to one. High-Pressure NMR Data Analysis. The protein solution for NMR studies contained 0.25 mM Hsp90αN*, 9 mM Bis− Tris, 45 mM NaCl, 4% DMSO, 0−2 mM ligand, 0.5 mM 4,4dimethyl-4-silapentane-1-sulfonic acid, 0.04% sodium azide, and 10% D2O, and the pH of the solution was 7. The two-dimensional (2D) 1H−15N heteronuclear single quantum coherence (HSQC) spectra were recorded at a high hydrostatic pressure using a 600 MHz Bruker Avance III spectrometer connected to a high-pressure generator (Daedalus Innovations LLC, Philadelphia, PA).33 A zirconium tube was filled with 0.35 mL of protein solution and overlaid with silicon oil. The chemical shift differences of each amino acid residue in the protein backbone between bound and free states were calculated as

(pm − p0 )2 ;

x = U, b

(7)

where α is the fraction of ligand-bound protein

⎛ Pt L t = (exp( −ΔG U /RT ) − 1)⎜ ⎝ 2 exp( −ΔG U /RT ) ⎞ 1 ⎟ exp( −ΔG b /RT ) ⎠

(6)

where M and C are the molecular mass and molar concentration of the protein, respectively, while d0 and d are the densities of the solvent and protein solution, respectively. In our experiment, the “solvent” is the ligand solution of constant concentration Lt. During the experiments, different volumes of the protein stock solution were added to reach various concentrations Pt. The partial molar volume of the protein is then measured at different values of r = Lt . For the one-to-one

where [NL] is the equilibrium concentration of the ligandbound protein. The main system of equations relating the concentration of the added ligand, Lt, total protein concentration, Pt, and the midpoint of unfolding transition, pm, is

+

d − d0 M − d0 Cd0

Δδ = (5)

where f N and f U denote the experimental fluorescence yields for the native and unfolded protein states, respectively. The binding constants shown in Table 3 were determined previously by FTSA or isothermal titration calorimetry (ITC).22,31,32 Densitometry Experiment and Data Analysis. Densities of solutions were measured using a vibrating tube densitometer DMA 5000M (Anton Paar, Graz, Austria) with a precision of

1

H (δ bound

−

1

H 2 δfree )

⎛ γ15N + ⎜⎜ 1 ⎝ γH

⎞2 15 15 N ⎟⎟ (δ bound − δfreeN)2 ⎠ (9)

1

where δ H and δ

15

N

are the chemical shifts of amide hydrogen 1

15

and nitrogen and γ H and γ N are the gyromagnetic ratios of hydrogen and nitrogen, respectively. The dissociation constant of Hsp90αN* interaction with a ligand was determined from the chemical shift change dependence of the total ligand concentration in the protein solution34 9905


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The Journal of Physical Chemistry B Δδ = Δδmax

(L t + Pt + Kd) −

(L t + Pt + Kd)2 − 4L tPt 2Pt (10)

where Δδmax is the maximum chemical shift difference between the fully ligand-saturated and ligand-free protein states and Δδ is the change in the observed chemical shift. In the case of slow chemical exchange of nuclei between ligand-free and ligandbound protein states, the values of Kd were determined using the equation Kd =

[P](L t − [PL]) [PL]

(11)

where [P] and [PL] are the concentrations of the ligand-free and ligand-bound proteins, respectively. The values of [P] and [PL] were estimated from the peak intensity of a particular residue in the 1H−15N HSQC spectrum. All recorded NMR spectra were analyzed using the CcpNmr software.35

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RESULTS The FPSA technique was used to determine the protein−ligand binding volume by measuring protein denaturation profiles (i.e., the dependence of fluorescence yield on pressure) at various concentrations of the added ligand. Typical unfolding profiles of Hsp90αN are shown in Figure 2a. The measurement of ANS fluorescence in the absence of a protein demonstrated that its quantum yield changed by 2.5% upon increase of pressure from 0 to 380 MPa. This effect was negligible, and thus no correction was necessary for the fluorescence yield curves. Protein unfolding parameters and, subsequently, the melting pressure, pm, were determined by fitting the unfolding profiles using model eq 5. The increase in ligand concentration stabilizes Hsp90αN protein and shifts the value of pm toward a higher pressure. The melting pressure dependence on added ligand concentration (referred to as a dosing curve) encodes the change in protein volume associated with ligand binding. The plotted shifts in melting pressure, Δpm, of four Hsp90αN− ligand systems are shown in Figure 2b. Δpm is defined as the difference between protein melting pressures in the presence and absence of a ligand. Equation 3 was used to fit experimental data points of dosing curves and to determine volumetric parameters associated with ligand binding to Hsp90αN. Correlations between fitting parameters could sometimes result in a high uncertainty of the determined parameters. This general problem was partially overcome with an assumption that the change in Δβb weakly depends on the chemical structure of a ligand, which means that a common (global) fitting parameter Δβb can be applied for all data sets in Figure 2b. The determined value of Δβb was 0.015 ± 0.002 cm3 mol−1 bar−1. The standard state Gibbs energies and the binding volumes are shown in Table 1. The fitted parameters of binding are very sensitive to the given values of unfolding parameters, thus it was necessary to determine the unfolding parameters with the highest possible accuracy. Seven independent pressureinduced unfolding profiles of unliganded Hsp90αN were collected and the common set of unfolding parameters, that is, ΔG0_U = 4.0 ± 0.3 kJ mol−1, ΔVU = −14 ± 3 cm3 mol−1, and ΔβU = 0.019 ± 0.002 cm3 mol−1 bar−1, was determined by fitting to eq 5. FPSA experiments with other above-mentioned ligands, namely, AZ1, AZ3, ICPD47, and radicicol, have been published

Figure 2. (a) Unfolding profiles of Hsp90αN protein at various concentrations of added ligand AZ2. (b) Shift in Hsp90αN melting pressure as a function of ligand concentration (dosing curves). Lines in (a) and (b) are fits to eqs 5 and 3, respectively. All tested ligands stabilized the Hsp90αN protein against denaturation by pressure by up-shifting the melting pressure in a dose-dependent manner.

Table 1. Ligand-Binding-Induced Changes in Standard State Gibbs Energy, Volume, and Differential Partial Molar Compressibility of Hsp90αN Protein Determined by FPSA ΔG0_b/kJ mol−1 ICPD91 ICPD9 AZ2 ICPD1

−23 −21 −23 −24

± ± ± ±

1 1 1 1

ΔVb/cm3 mol−1 −1 −2 −9 −9

± ± ± ±

5 5 6 6

Δβb/cm3 mol−1 bar−1 0.015 0.015 0.015 0.015

± ± ± ±

0.002 0.002 0.002 0.002

previously.26,27 A summary of the determined binding volumes is given in Table 3. Densitometry experiments were performed using Hsp90αN and Hsp90βN proteins and four ligands: radicicol, ICPD47, ICPD62, and AZ3. Most of the experiments were performed at a 50 μM ligand concentration, whereas for radicicol, the ligand concentration was 20 μM. To eliminate the possible concentration-based effects on the observed data, several experiments were repeated using both lower and higher concentrations of the ligand, but no statistically significant differences in the measured values were observed. 9906


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Figure 3. (a) Raw densitometry data of the Hsp90αN protein solution at different ligand concentrations (lines serve only as a guide to the eye). (b) Calculated fraction of ligand-bound Hsp90αN protein at a fixed ligand concentration Lt = 50 μM. (c) Partial molar volumes of Hsp90αN at various ligand-to-protein ratios calculated using eq 6. (d) Comparison of Hsp90αN (solid symbols) and Hsp90βN (open symbols) partial molar volumes at various ligand-to-protein ratios using ICPD47 (red) and radicicol (black). The lines in parts (c) and (d) are fits to eq 7.

volumes were determined by fitting partial protein volumes at various r using eq 7 with fitting parameters being V0(0) and ΔVb. Dissociation constants, Kds, determined by FTSA and/or ITC methods were taken from Zubrienė et al.28 and Kazlauskas et al.22 and were kept fixed during the parameter minimization procedure. Densitometry experiments were used to investigate the differences in binding volume between the α- and β-isoforms of Hsp90 N-terminal domains (Figure 3d). Although the partial molar volumes of these two proteins are different, the comparison of ligand-induced changes in volume does not show statistically significant differences for all tested ligands. The summarized densitometry data of ligand-induced volume changes of both Hsp90αN and Hsp90βN proteins are given in Table 2. NMR spectroscopy was used to study the interaction of Hsp90αN* with two ligands (AZ3 and ICPD9) at elevated pressures to determine the protein−ligand binding volume. AZ3 and ICPD9 bind Hsp90αN with Kd in the mM range, which allowed us to study this interaction by NMR spectroscopy. The titration of Hsp90αN* with AZ3 and ICPD9 showed the shifts of a number of peaks in the 1H−15N-HSQC spectrum (Figure 4). The largest changes in the chemical shift were observed for Val92, Gly95, Ile96, Gly97, and other amino acids

Raw densitometry data of Hsp90αN interaction with the ligands at various values of ligand-to-protein molar ratios, r, are shown in Figure 3a. Before titration, the partial molar volumes of Hsp90αN and Hsp90βN in ligand-free protein solutions (r = 0) were determined to be 21 494 cm3 mol−1 (0.7280 cm3 g−1) and 21 146 cm3 mol−1 (0.7281 cm3 g−1), respectively. These values support the previous findings36−39 that partial specific volumes for the majority of globular proteins in aqueous solutions fall within a narrow range between 0.70 and 0.75 cm3/1 g of protein. The calculated fractions of ligand-bound Hsp90αN protein, α, in the tested range of ligand-to-protein molar ratios are shown in Figure 3b. All tight-binding ligands demonstrate indistinguishable α dependence on r, thus only the ICPD47 case is shown in Figure 3b. For weak-binding ligands (such as AZ3), the saturation regime is achieved at higher concentrations, which is important to properly account for the change in protein binding volume. The measured solution densities were used to calculate the partial molar volumes, V0, using eq 6, which are shown in Figure 3c. Note that, in a strict manner, V0 is the apparent molar protein volume, but at low concentrations (which is the case in our experiments), the partial molar volume is very close to the apparent molar volume. The ligand−Hsp90 binding 9907


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The Journal of Physical Chemistry B Table 2. Ligand-Binding Volumes, ΔVb, of Hsp90αN and Hsp90βN Proteins Determined by Fitting Densitometry Data with eq 7a ligand AZ3 ICPD47 ICPD62 radicicol a

Hsp90αN

Hsp90βN

−10 −49 −50 −124

n.d. −46 ± 4 −48 ± 4 −119 ± 8

± ± ± ±

3 5 4 7

The largest chemical shift changes induced by the AZ3 ligand were observed on Val92, Gly95, and Gly97 amino acid residues. The binding seems to occur in a slow chemical exchange regime as both ligand-free and ligand-bound states of Hsp90αN were observed. Gly95 and Gly97 residues could not be used to determine Kd, as their peaks were observed only as the Hsp90αN*−AZ3 complex or ligand-free Hsp90αN* in the spectrum. However, in the case of Val92, two peaks were observed in the same spectrum: one corresponding to the Hsp90αN*−AZ3 complex and the other corresponding to a ligand-free state of Hsp90αN*. The concentrations of a ligand-free Hsp90αN* protein, [P], and Hsp90αN*−ligand complex, [PL], were determined by integrating the Val92 peak intensity of the ligand-free and ligand-bound states in the spectrum. Using eq 11, Kd was determined to be 60 μM at 5 MPa and 20 μM at 120 MPa, which resulted in ΔVb = −9 ± 4 cm3 mol−1 and Δβb = 0.023 ± 0.009 cm3 mol−1 bar−1. Although lower in magnitude, a concentration-dependent chemical shift change was observed for Val136. The Kd values at different pressures were determined using eq 10. The analysis of Hsp90αN*−AZ3 Kd dependence on pressure yielded a similar value of binding volume ΔVb = −8 ± 4 cm3 mol−1 and Δβb = −0.0014 ± 0.006 cm3 mol−1 bar−1.

All values are given in cm3 mol−1.

for AZ3 and ICPD9, as shown in Figure 5. Gly97 is located within the ATP binding site of Hsp90αN*. The intensity of the Gly97 peak disappeared with an addition of ICPD9 due to the intermediate exchange rate of the nucleus. Therefore, this residue was excluded from the data analysis. ICPD9 binding to Hsp90αN* affects the chemical shift of Val92 and other amino acids in a fast chemical exchange regime, which allows us to gradually follow the chemical shift change in the 1H−15N-HSQC spectrum during the titration. The dissociation constant, Kd, at a particular pressure was determined by global analysis of all amino acids showing statistically significant shifts using a model described by eq 10. Global fit analysis of the shift dependence on concentration yielded Kd = 0.25 mM at 5 MPa pressure (Figure 6a). Kd gradually increased (affinity weakened) with an increased pressure (shown in Figure 6b). The dependence could be fit linearly (Δβb = 0, yielding ΔVb = 14 ± 1 cm3 mol−1) or by including a nonlinear pressure term yielding ΔVb = 24 ± 6 cm3 mol−1 and Δβb = 0.014 ± 0.004 cm3 mol−1 bar−1. The latter value matched the FPSA measurements (Δβb = 0.015 cm3 mol−1 bar−1). Interestingly, it seems that the volumetric parameters of binding determined from the Kd dependence on pressure that were obtained from Val92 analysis alone were sufficiently precise and did not require a global analysis of all other studied amino acid chemical shift changes with ligand concentration.

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DISCUSSION In this study, we compared the ligand-binding volumes of Hsp90αN protein, obtained using three different experimental techniques. Both direct and indirect approaches were in good agreement when determining the protein−ligand binding volumes. The exploited methods have not enabled the dissection of the binding volumes into separate contributions of, for example, solvent-mediated interactions or thermal fluctuations; thus, all conclusions drawn are based on the fact that the determined ligand-induced changes in volume should be classified as “observed”.

Figure 4. 1H−15N HSQC spectra of Hsp90αN* with (blue) and without (red) the ICPD9 ligand. Emphasized amino acids are used to illustrate calculations of protein−ligand binding volume from ligand-induced chemical shift changes. 9908


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Figure 5. Chemical shift changes of the selected amino acids of Hsp90αN*, which showed the highest chemical shift change or the nonlinear behavior after the addition of 1.2 mM of AZ3 (solid bars) and ICPD9 (open bars) ligands. Several amino acids experiencing the strongest AZ3induced shifts are shown in the Hsp90αN crystal structure (PDB ID: 1UYL).

Figure 6. Interaction of ICPD9 with Hsp90αN* by NMR. (a) An example of Kd calculation from the chemical shift change of several amino acids. Curves were obtained by global fitting (the same Kd for all curves) to eq 10. (b) Increase in ΔGb at increasing pressure yields a positive ΔVb = 24 ± 6 cm3 mol−1 with nonzero Δβb = 0.014 ± 0.004 cm3 mol−1 bar−1. If we fit the data with Δβb = 0, then ΔVb = 14 ± 1 cm3 mol−1.

tendencies become more clear, showing that nanomolar binders induce considerably larger binding volumes. Experimental densitometry data support the previous findings26,27 that tight-binding ligands induce more negative binding volumes than weak binders (see Figure 7 and Table 3). Although all tested weakly binding ligands induce detectable shifts in the protein melting pressure as shown in Figure 2b, small ΔVb absolute values of, for example, ICPD9 and ICPD91 compounds indicate that volumetric rearrangements are mostly governed by the changes in compressibility associated with both protein unfolding and protein−ligand binding. Differential partial molar compressibilities that appear in eq 4 are difficult to determine experimentally. Thus, these parameters are often calculated from nondirect experiments and precise data analysis

The obtained values of binding volumes by FPSA and densitometry were negative and spanned 2 orders of magnitude depending on the interacting ligand. Negative binding volumes suggest that the protein−ligand complex together with its hydration shell occupies less volume than the separate constituents with their hydration shells. If we consider only weak-binding ligands in the Kd range of 10−2−10−5 M (shown in Figure 7), the values of ΔVb appear to be scattered and do not seem to correlate with the binding affinity. The only reasonable conclusion that could be drawn after taking into account relatively large errors of binding volumes and dissociation constants in this range is that weak-binding ligands induce small negative or even positive change in Hsp90αN volume. However, on the larger scale of binding affinities, the 9909


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values, describing experimental data with roughly the same sum of least squares. Dosing curves (Figure 2b) were fitted with an assumption that the change in compressibility weakly depends on the chemical structure of the ligand. On one hand, this assumption limits the ability to account for the differences in Δβb, which might be related to different ligand structures. However, on the other hand, this assumption allowed exploitation of the global parameter fitting technique, which considerably reduced the correlations and improved the accuracy of the fitted experimental data. Without this assumption, the benefits of acquiring ligand structure related details would be buried under statistical errors arising from parameter correlation. Densitometry experiments revealed that for tight-binding ligands, the binding volume model could be simplified by dividing into two regions: (1) at r < 1, the apparent binding volume linearly decreases with an increase in relative ligand concentration and (2) the saturation regime at r > 1, when the apparent binding volume is not affected anymore by a relative increase in ligand concentration. The simple difference between experimental values of V0 at saturation and ligand-free states provides a good estimate of ΔVb. However, such an approach could lead to significant errors in the determination of ΔVb in the case of weakly binding ligands. There were no statistically significant differences between the α- and β-isoforms of Hsp90 protein in terms of volumetric properties (FPSA data of Hsp90βN are not shown). This could be expected because there is only one amino acid difference (Ala−Ser) in the ligand-binding pocket.28 All three techniques used here are suitable to determine the binding volumes for different ranges of protein−ligand binding affinities. FPSA and densitometry cover rather similar and a much broader interval of protein−ligand affinities than NMR, with Kds ranging from several millimoles to subnanomoles. Although even extremely tight binders should not be a limiting factor to determine the ligand−protein binding volume by the densitometry technique, it could become an obstacle when using FPSA because tight-binding ligands usually stabilize proteins against pressure denaturation and it becomes very difficult to obtain proper unfolding profiles. This is the reason why the ΔVb value for radicicol was determined with a relatively large error using FPSA. It was not possible to determine the ligand−protein binding volume for tight-binding ligands using high-pressure NMR. This limitation arises from the sensitivity of the NMR spectrometer, as the protein concentration in the tube and the dissociation constant should be of the same order. The

Figure 7. ΔVb of Hsp90αN−ligand interaction plotted against binding affinities, which were determined by FTSA. The standard errors of measured volumes are shown in Table 3. The dashed line serves only as a guide to the eye.

could yield a good estimate about the sign and magnitude of differential partial molar compressibilities. Our analysis involved the global parameter fitting technique (i.e., usage of the same fitting parameter for several experimental data sets) and fitting of experimental data with ΔβU varied versus set to zero. The latter resulted in negative and higher (by absolute value) volumes of unfolding, but the unfolding profiles were fit with a considerably lower accuracy than that on using nonzero compressibility parameters. The determined values of ΔβU and Δβb, which are related to Hsp90αN unfolding and binding to the various ligands, fell near the range of the corresponding compressibility parameters reported in the literature.3 For example, the adiabatic compressibility of tri-N-acetylglucosamine binding to lysozyme was 0.0022 ± 0.0004 cm3 mol−1 bar−1,3,17 whereas that of cAMP binding to the cAMP-binding domain was 0.0034 ± 0.0009 cm3 mol−1 bar−1.40 These numbers are 4−6-fold lower than our value of 0.015 ± 0.002 cm3 mol−1 bar−1. The difference could be due to the significantly different binding affinities or even experimental conditions. The potential source of error in equations with two or more fitting parameters (e.g., eq 3 or 5) is the correlation between these parameters, which could lead to many different sets of

Table 3. Ligand-Binding Volumes of Hsp90αN Using FPSA, Densitometry, and NMR Experimental Techniquesa ICPD91 ICPD9 AZ3 AZ2 ICPD1 AZ1 ICPD47 ICPD62 radicicol

ΔVb (FPSA)

ΔVb (densitometry)

ΔVb (NMR)

−1 ± 5 −2 ± 5 −7 ± 626 −9 ± 6 −9 ± 6 −21 ± 1126 −40 ± 1427 n.d. −170 ± 6027

n.d. n.d. −10 ± 3 n.d. n.d. n.d. −49 ± 5 −50 ± 4 −124 ± 7

n.d. 24 ± 6 −9 ± 4 n.d. n.d. n.d. n.d. n.d. n.d.

Kd 3.3 2.0 9.1 1.0 2.5 2.0 5.0 2.0 2.0

× × × × × × × × ×

10−5 M 10−5 M 10−5 M26 10−3 M 10−5 M 10−3 M26 10−9 M22 10−9 M22 10−10 M28

All values of binding volumes are given in cm3 mol−1. Dissociation constants of Hsp90αN interaction with the ligands were determined by FTSA and/or ITC at 25 °C.

a

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The Journal of Physical Chemistry B minimum concentration of the protein to record a 2D 1H−15N HSQC spectrum in 2−4 h should be about 0.1 mM. The twofold reduction in protein concentration would require a 4 times longer experiment to obtain the same signal-to-noise ratio in the spectrum. Protein solutions of nanomolar concentration have to be used to measure the Kd dependence on pressure of, for example, ICPD47 or ICPD62 interaction with Hsp90αN. Such small concentrations would require unacceptably long times to register a single NMR spectrum. Protein−ligand systems with millimolar Kds could be determined by all three techniques. However, the relative errors of the determined binding volumes were greater than those obtained for systems having intermediate binding affinities.

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CONCLUSIONS

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AUTHOR INFORMATION

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Hsp90 protein−ligand binding volumes determined by FPSA, densitometry, and NMR were similar. The binding volumes were increasingly negative with increasing binding affinity.

Corresponding Author

*E-mail: [email protected]. Phone: +370 52 239408. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was funded by a grant (No. MIP-004/2014) from the Research Council of Lithuania. HP-NMR work (CBS, France) was supported by the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-0.

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