Copper(II)-Binding Induces a Unique Polyproline Type II Helical

Article pubs.acs.org/IC

Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX

Copper(II)-Binding Induces a Unique Polyproline Type II Helical Structure within the Ion-Binding Segment in the Intrinsically Disordered F‑Domain of Ecdysteroid Receptor from Aedes aegypti Magdalena Rowińska-Ż yrek,*,†,∥ Anna Wiȩch,‡,∥ Joanna Wa̧tły,† Robert Wieczorek,† Danuta Witkowska,§ Andrzej Ożyhar,‡ and Marek Orłowski*,‡ †

Faculty of Chemistry, University of Wrocław, 50-383 Wrocław, Poland Department of Biochemistry, Faculty of Chemistry, Wrocław University of Science and Technology, 50-370 Wrocław, Poland § Public Higher Medical Professional School in Opole, Katowicka 68, 45-060 Opole, Poland

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‡

S Supporting Information *

ABSTRACT: Reproduction of the dominant vector of Zika and dengue diseases, Aedes aegypti mosquito, is controlled by an active heterodimer complex composed of the 20-hydroxyecdysone receptor (EcR) and ultraspiracle protein. Although A. aegypti EcR shares the structural and functional organization with other nuclear receptors, its C-terminus has an additional long F domain (AaFEcR). Recently, we showed that the full length AaFEcR is intrinsically disordered with the ability to specifically bind divalent metal ions. Here, we describe the details of the exhaustive structural and thermodynamic properties of Zn2+- and Cu2+-complexes with the AaFEcR domain, based on peptide models of its two putative metal binding sites (AcHGPHPHPHG-NH2 and Ac-QQLTPNQQQHQQQHSQLQQVHANGS-NH2). Unexpectedly, only in the presence of increasing concentrations of Cu2+ ions, the Ac-HGPHPHPHGNH2 peptide gained a metal ion-induced poly-L-proline type II helical structure, which is unique for members of the family of nuclear receptors.

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INTRODUCTION A majority of the nuclear receptors (NRs) family members are ligand-dependent transcription factors that regulate gene expression by interacting with specific DNA sequences. Most of NRs exhibit common architecture and structural organization. A typical NR consists of a variable A/B region (Nterminal domain, NTD) responsible for transactivation, a DNA binding domain (DBD), a hinge region, and a ligand binding domain (LBD).1 Several NRs also contain a variable F domain at their C-terminus. For many years, research has been focused on the biochemical, functional, and structural characteristics of two globular domains: DBD and LBD.2 Therefore, they are the best known and characterized regions of the NRs, while knowledge about the unique F domain is rather poor. At the Cterminus of ecdysteroid receptor (EcR) from the Aedes aegypti mosquito, the main vector of the world’s most devastating human diseases, dengue, chikungunya, Zika, and yellow fever,3−5 there is the F domain (AaFEcR). Recently, we showed that AaFEcR exhibits characteristics of intrinsically disordered regions (IDRs) and intrinsically disordered proteins (IDPs).6 Our data also showed its ability to specifically bind Zn2+ and Cu2+ ions.6 The lack of significant secondary structure changes in the full-length AaFEcR in the presence of Zn2+ and Cu2+ raises the question whether metal ion binding could induce local structural changes. Therefore, we decided to work on two model peptides (Ac-HGPHPHPHG-NH2 and Ac-QQLTPNQQQHQQQHSQLQQVHANGS-NH2) (Figure © XXXX American Chemical Society

1) with the sequences that occur in AaFEcR and study the details of their interactions with metal ions. Like other NRs, EcR from A. aegypti exhibits a modular structure composed of the NTD, DBD, the hinge region, the LBD, and an additional F domain at its C-terminus.7 The F domain of A. aegypti EcR (AaFEcR) is intrinsically disordered.6 T w o p e p t i d e s (A c - H G P H P H P H G - N H 2 a n d A c QQLTPNQQQHQQQHSQLQQVHANGS-NH2) with the sequences that occur in AaFEcR (residues 617−625 and residues 626−650, respectively) were synthesized in order to investigate in detail their interactions with metal ions described in this paper. Residue numbers correspond to the sequence of EcRB from (UniProt ID: P49880/GenBank: AAA87394.1). Sequences of the two peptides used in this study are marked with arrows. AaFEcR possesses a specific proline-rich (P-rich) sequence motif, HGPHPHPHG (residues 617−625), resembling the one present in His(H)-Pro(P)-rich glycoproteins (HPRGs),8 which is responsible for Zn2+ and Cu2+ binding. Following this motif, there is a long glutamine-rich (Q-rich) segment (residues 626−650). Q-rich domains are highly abundant in eukaryotic transcription factors, where they can act as functional modulators of gene expression, providing the specific platform for interactions with co-activators or coReceived: June 19, 2019

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DOI: 10.1021/acs.inorgchem.9b01826 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry

Figure 1. Schematic representation of the structure of A. aegypti EcR showing sequences of the two peptides used in the studies.

repressors.9 Interestingly, highly variable and intrinsically disordered NTDs of many nuclear hormone receptors harbor the transactivation functions (AF-1) and Q-rich regions within their sequences.10−12 Here, we show the stoichiometry, structural, and thermodynamic properties of complexes consisting of AcHGPHPHPHG-NH2 or Ac-QQLTPNQQQHQQQHSQLQQVHANGS-NH2 with Zn2+ or Cu2+. Furthermore, our experimental data combined with density functional theory (DFT) calculations enabled us to map the histidine residues responsible for metal ion coordination. Although Zn2+ binding did not affect the content of the secondary structures in each of the peptides, the effect of interaction between Cu2+ and AcHGPHPHPHG-NH2 was unexpected. For the very first time, we report the Cu2+ binding-induced formation of a poly-Lproline helix II (PPII), which is very unique for members of the family of NRs. PPII helices play an important structural role in folded and unfolded proteins. The relevance of these findings is discussed.

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Tunemix mixture. Data were processed by application of the Compass DataAnalysis 4.0 program (BrukerDaltonic). Potentiometric Measurements. Stability constants for proton and metal complexes were calculated from titration curves registered over the pH range of 2−11 at T = 298 K, in a mixed DMSO-water (30:70, v/v) solution of 4 mM HClO4 and ionic strength 0.1 mol· dm−3 (NaClO4), using a total volume of 3 mL. Potentiometric titrations were performed with a Metrohm 905 Titrando pH-meter system provided with a Mettler-Toledo InLabMicro, glass-body, micro combination pH electrode, and a dosing system 800 Dosino equipped with a 2 mL micro buret. High-purity grade argon was gently blown over the test solution in order to maintain an inert atmosphere. A constant-speed magnetic stirring was applied throughout. Solutions were titrated with 0.1 mol·dm−3 carbonatefree NaOH. The electrode was daily calibrated for hydrogen ion concentration by titrating HClO4 with alkaline solution under the same experimental conditions as above. The ligand concentration was about 5 × 10−4 mol·dm−3, and the metal-to-ligand ratio was 1:1. The standard potential and the slope of the electrode couple were computed by means of the Glee program.13 The purities and the exact concentrations of the ligand solutions were determined by the Gran method.14 The HYPERQUAD 200615 program was employed for the overall (β) and step (K) stability constant calculations, referring to the following equilibrium:

MATERIALS AND METHODS

Ac-HGPHPHPHG-NH2 (a fragment of the cDNA (residues 617− 625)) and Ac-QQLTPNQQQHQQQHSQLQQVHANGS-NH2 (a fragment of the cDNA (residues 626−650)) peptides (98% purity) (chosen motifs and their localization in AaFEcR are presented in Figure 1) were purchased from KareBio and used without further purification. Residue numbering corresponds to the sequence of EcRB from A. aegypti7 (UniProt ID: P49880/GenBank: AAA87394.1). Cu2+ and Zn2+ perchlorides were extra-pure products (Sigma-Aldrich). The concentrations of stock solutions of these salts were determined by inductively coupled plasma mass spectrometry. The carbonate-free stock solution of NaOH (0.1 M) was purchased from Sigma-Aldrich and then potentiometrically standardized with potassium hydrogen phthalate as a primary standard. All of the sample solutions were prepared with freshly doubly distilled water. Mass Spectrometric Measurements. High-resolution mass spectra were obtained on a BrukerMicrOTOF-Q spectrometer (BrukerDaltonik, Bremen, Germany) equipped with an Apollo II electrospray ionization source with an ion funnel. Measurements were carried out in the range of positive values of mass-to-charge ratio (m/ z) from 150 to 2500. The instrumental parameters were as follows: scan range, m/z 400−2500; dry gas, nitrogen; temperature, 170 °C; capillary voltage, 4500 V; ion energy, 5 eV. The capillary voltage was optimized to the highest signal-to-noise ratio. Small changes in voltage (±500 V) did not significantly affect the optimized spectra. The Cu2+ and Zn2+ complexes (metal:ligand stoichiometry of 1:1, [ligand]tot = 5 × 10−4 M) were prepared in a 1:1 MeOH/H2O mixture at pH 7. The samples were infused at a flow rate of 3 μL/min. Before each experiment, the instrument was calibrated externally with the

pM + qL + rH V M pLqHr (charges omitted; p is 0 in the case of ligand protonation; r can be negative). Reported Ka values are instead the acid dissociation constants of the corresponding species. The computed standard deviations (referring to random errors only) were given by the program itself and are shown in parentheses as uncertainties on the last significant figure. Hydrolysis constants for Zn2+ and Cu2+ ions were taken from the literature.16,17 The distribution and competition diagrams were computed using the HYSS program.18 Spectroscopic Studies. The absorption spectra in the UV−vis region were recorded on a Varian Cary 300 Biospectrophotometer. Circular dichroism (CD) spectra over the 250−800 nm range were recorded on a Jasco J 1500 spectropolarimeter in a 1 cm path length. Direct CD measurements (Θ) were converted to mean residue molar ellipticity (Δε) using the Jasco Spectra Manager. UV−vis and CD spectroscopic studies were carried out in 1 cm path length quartz cells at 298 K; solution concentrations were similar to those employed in the potentiometric experiments. Electron paramagnetic resonance (EPR) spectra were recorded in liquid nitrogen on a Bruker ELEXSYS E500 CW-EPR spectrometer at X-band frequency (9.5 GHz) and equipped with an ER 036TM NMR teslameter and an E41 FC frequency counter. The ligands were prepared in an aqueous solution of HClO4 at I = 0.1 M (NaClO4). The concentration of Cu2+ was 1 × 10−3 M, and the M:L molar ratio was 1:1.1. Ethylene glycol (25%) was used as a cryoprotectant for EPR measurements. The EPR parameters were analyzed by computer simulation of the experimental B


Article

Inorganic Chemistry

Table 1. Thermodynamic Parameters for Ac-HGPHPHPHG-NH2 and Its Cu2+ and Zn2+ Complexes at 298 K in DMSO/Water (30:70) Solutiona UV−vis species Ligand HL H2L H3L H4L Cu2+ Complex CuH2L

CuHL

log β 7.34(1) 13.82(1) 19.98(1) 25.45(1)

pKa

Λ (nm)

CD

ε (M

−1

Λ (nm)

cm )

EPR −1

Δε(M

−1

cm )

A∥

g∥

g⊥

120.6

2.42

2.08

182

2.25

2.05

182

2.25

2.05

184

2.25

2.04

186.2

2.25

2.04

7.34 6.48 6.16 5.47

19.99(5)

613

58.40

15.64(1)

4.35

611

63.79

CuL

8.97(3)

6.67

597

72.07

CuH−1L

0.79(6)

8.18

583

83.83

CuH−2L

−8.17(4)

8.96

578

93.35

CuH−3L

−19.19(4)

11.02

565

111.74

17.81(3) 6.87(1) −0.94(4) −8.84(2)

7.81 7.90

Zn2+Complex ZnH2L ZnL ZnH−1L ZnH−2L

−1

624.8 526.5 472.7 301.4 524.5 302.2 509.2 302.4 562.1 494.7 300.3 621.5 537.1 473 329.9 620.3 561.7 482.7 309.3

0.50 0.04 0.17 −0.22 −0.02 −0.28 0.03 −0.25 0.44 0.04 −0.15 0.03 0.49 0.09 0.45 0.22 0.54 0.06 0.42

[Cu2+] 0.001 M; Cu2+ to ligand ratio of 1:1. Standard deviation on the last significant figure is given in parentheses. L = Ac-HGPHPHPHG-NH2.

a

spectra using WIN-EPR SIMFONIA software, version 1.2 (Bruker). The pH was adjusted with appropriate amounts of HCl and NaOH solutions. CD spectra for Ac-HGPHPHPHG-NH 2 and AcQQLTPNQQQHQQQHSQLQQVHANGS-NH2 peptides, which indicated the structure of the studied species, were recorded with a JASCO J-715 CD spectropolarimeter (Japan), and the temperature of 293 K in the cell was maintained by a Peltier Type Temperature Control System (JASCO, Japan). Peptides were dissolved in H2OmQ (pH 7.5). For every sample, far-UV spectra were recorded from 260 nm up to the wavelength when the voltage on the photomultiplier was lower than 600 mV, namely up to 190 nm for Ac-HGPHPHPHGNH2 and up to 202 nm for Ac-QQLTPNQQQHQQQHSQLQQVHANGS-NH2 (except from unfolding experiment in the presence of guanidinium chloride). Every sample contained a peptide at a concentration equal to 100 μM. Using a 2 mm path length cell, data were collected in a 5-fold repetition at a scanning speed of 50 nm/ min. For every sample, a corresponding baseline (spectrum recorded for H2OmQ and derivatives containing appropriate amount of Zn2+, Cu2+ ions, TFE, or GdmCl) was subtracted. Spectra were smoothed with Savitzky−Golay filter19 and converted to molar residual ellipticity units.20 DFT Calculations. Computational methods of theoretical chemistry have been used to predict structure and properties of organic and inorganic compounds.21−23 The molecular orbital studies on 1:1 complexes of Ac-QQLTPNQQQHQQQHSQLQQVHANGSNH2 and Ac-HGPHPHPHG-NH2 capped peptides with Cu2+ and Zn2+ cations have been done on the DFT level of theory. The starting structures of the peptides for DFT calculations were generated on the basis of the amino acid sequence after 175 ps for Ac-

QQLTPNQQQHQQQHSQLQQVHANGS-NH2 and 75 ps AcHGPHPHPHG-NH2 simulation at 300 K, without cutoffs using BIO+ implementation of CHARMM force field. Gaussian 09 E.0124 suite of programs using the ωB97X-D25 long-range corrected hybrid density functional with damped atom−atom dispersion corrections with 6-31G basis set was used. All presented structures were fully optimized.

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RESULTS Coordination of Cu2+ and Zn2+ at Various pH Values. Structural and thermodynamic properties of Zn2+- and Cu2+Ac-HGPHPHPHG-NH 2 and Ac-QQLTPNQQQHQQQHSQLQQVHANGS-NH2 complexes were studied by mass spectrometry, potentiometric measurements, and a variety of spectroscopic techniques: UV−vis, CD, and EPR spectroscopy. Mass spectrometry showed the binding stoichiometry, potentiometry allowed to calculate the complex stability constants, and spectroscopic measurements gave hints about Cu2+ binding sites and coordination geometry. Ac-HGPHPHPHG-NH 2 Complexes. All four AcHGPHPHPHG-NH2 protonation constants come from the imidazole rings of His residues (log K values in the range of 5.47−7.34, Table 1) and are similar to values found in similar systems.26 Electrospray ionization mass spectrometry (ESI-MS) confirmed the purity of the studied ligand and showed the metal C


Article

Inorganic Chemistry Table 2. Thermodynamic Parameters for Ac-QQLTPNQQQHQQQHSQLQQVHANGS-NH2 and Its Cu2+ and Zn2+ Complexes at 298 K in DMSO/Water (30:70) Solutiona UV−vis species

CD −1

log β

pKa

7.39(1) 14.41(1) 20.97(1)

7.39 7.02 6.56

15.66(1) 10.23(3) −4.55(4)

5.43

736 635 602

22.70 34.10 57.02

−13.47(4)

8.92

518

78.99

13.43 8,04(1) 0.99(4) −6.96(2)

5.39 7.05 7.95

Λ (nm)

ε (M

−1

cm )

Λ (nm)

EPR −1

Δε(M

−1

cm )

A∥

g∥

g⊥

118.0 167 160

2.41 2.29 2.23

2.08 2.06 2.05

157

2.23

2.05

Ligand

HL H2L H3L Cu2+ Complex CuHL CuL CuH−2L

CuH−3L

Zn2+ Complex ZnHL ZnL ZnH−1L ZnH−2L

− − 623.3 481 354.7 267.7 624.8 489.3 320.1 270.4

− − 4.41 −8.91 −4.66 82.05 24.10 −36.06 23.32 76.68

[Cu2+] 0.001 M; Cu2+ to ligand ratio of 1:1. Standard deviation on the last significant figure is given in parentheses. L = AcQQLTPNQQQHQQQHSQLQQVHANGS-NH2. a

binding stoichiometry of its Zn2+ and Cu2+ complexes at pH 7.4 (Figure S1). Only equimolar complexes were detected under the studied conditions, with m/z values of 537.7 and 538.2 which correspond to [CuL]2+ and [ZnL]2+ complex species, respectively (Figure S1A,B). Isotopic distributions of the metal complexes fit perfectly with the simulated ones (Figure S2A,B). The first Cu2+-Ac-HGPHPHPHG-NH2 complex, CuH2L, starts to form above pH 3.5 and coexists in solution with the major CuHL species, which reachs a maximum at pH around 5 (Figure S3A). The log K value of this deprotonation step (4.35, Table 1) is much lower than the one detected in the free ligand (6.48) and most likely corresponds to the binding of an imidazole nitrogen to the Cu2+ ion, resulting in a [3Nim] coordination mode of CuHL, which is suggested by a UV−vis band above 600 nm (Figure S4A) and by the increase of the A∥ value to 182. The CuL complex dominates in solution from pH 6 to pH 9, and its metal donor binding set does not change with respect to the previous CuHL species; the log K value of 6.67 (Table 1) comes from the deprotonation of the fourth His imidazole that is not involved in metal binding, which is evidenced by the lack of spectroscopic changes up to pH 9. The uniqueness of this complex comes from the fact that, unlike any other Cu2+ species, it cannot form typical square planar complexes that involve the deprotonation of subsequent amide nitrogens; in the discussed sequence, each histidine is preceded by a proline, which, formally speaking, is an imino and not amino acid and does not contain an amide nitrogen. In Ac-HGPHPHPHG-NH2, apart from the four available imidazole nitrogens (from His1, His4, His6 or His8), Cu2+ can only bind to one of the amides of amino acids located in the direction of the C-terminus of the coordinating histidines (Gly2 or Gly9). What are the binding modes of CuH−1L, CuH−2L, and CuH−3L then? Most likely, CuH−1L and CuH−2L (with log K values that lead to their formation

equal 8.18 and 8.95, respectively (Table 1) have the same [3Nim] Cu2+ coordination mode, and the stepwise deprotonations correspond to water molecules bound apically to the complex core; the UV−vis spectroscopic parameters do not undergo any significant change, and CD does not show any sign of d−d transitions, typical for Cu2+ square planar complexes (Figure S5A). Such d−d bands (at around 550 and 620 nm) appear above pH 10 (together with a strong blue shift in UV−vis (Figure S4A) and change in EPR parameters (Table 1), which suggests the involvement of one amide nitrogen in the binding,27 leading to a [3Nim, 1N−] type of binding. The Ac-HGPHPHPHG-NH2 peptide forms four complex species with Zn2+ ions, with the first minor one, ZnH2L, reaching a maximum concentration at pH 5.5 (Figure S3B). In this complex, most likely two imidazole nitrogen atoms are bound to the central Zn2+. The next observed species, ZnL, reachs a maximum at pH 6.5 and, due to the lack of spectroscopic data available for d10 metal, it can only be speculated that 3 imidazoles are involved in binding, based only on the decrease of pKa for Zn2+ complex in comparison to the free ligand. In this case, the speculation is even more sophisticated, because of the simultaneous loss of two protons (with an average log K value equal 5.47, the exact value of the pKa of the corresponding imidazole in the free ligand (Table 1)). Keeping in mind that Zn2+ is normally unable to displace amide protons, the complex binding mode should consist of imidazole nitrogens and water molecule oxygens; most likely, the pKa values of 7.81 and 7.90 (Table 1), corresponding to ZnH−1L and ZnH−2L species, respectively, are associated with the deprotonation of two water molecules bound to the central Zn2+ ion. Ac-QQLTPNQQQHQQQHSQLQQVHANGS-NH2 Complexes. All three Ac-QQLTPNQQQHQQQHSQLQQVHANGS-NH2 protonation constants come from histidine D


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Inorganic Chemistry

Figure 2. Competition plot for (A) Cu2+, Ac-HGPHPHPHG-NH2, and Ac-QQLTPNQQQHQQQHSQLQQVHANG-NH2. (B) Zn2+, AcHGPHPHPHG-NH2, and Ac-QQLTPNQQQHQQQHSQLQQVHANG-NH2.

and, most importantly, CD d−d bands at ca. 500 and 650 nm, typical for square planar, amide involving complexes, start to appear (Figure S5B, Table 2). The last deprotonation with a log K = 8.92, which leads to the CuH−3L species, corresponds to the binding of the third amide and results in a [Nim, 3N−] binding mode (as evidenced by a further UV−vis blue shift and an increase of the previously mentioned CD d−d bands (Figures S4B and S5B, Table 2). Zn2+-Ac-QQLTPNQQQHQQQHSQLQQVHANGS-NH2 species forms a ZnHL complex at a pH of around 5, and a ZnL complex with a maximum at a pH of around 6.5. The log K of 5.39, significantly lower than the corresponding 6.56 detected in the free ligand, suggests a change of the coordination mode from [2Nim] to [3Nim] (Table 2, Figure S3D). Further two deprotonations (log K 7.05 and 7.95) lead to ZnH−1L and ZnH −2 L species and most likely correspond to the deprotonation of water molecules bound to the Zn2+ ion (Table 2, Figure S3D). What is the difference in stability between the metal complexes of the two studied AaFEcR fragments? As for the Cu2+ complexes, below pH 7, where only imidazoles take part

imidazoles and lie within the range 6.56−7.39 (Table 2). The purity of the ligand and the equimolar stoichiometry of its Cu2+ and Zn2+ complexes at pH 7.4 were confirmed by ESI-MS (Figure S1) (m/z values of 1498.7 and 1499.2 correspond to [CuL]2+ and [ZnL]2+ complexes, respectively) (Figure S1C,D); isotopic distributions of the metal complexes perfectly correspond to the simulated ones (Figure S2C,D). Cu2+-Ac-QQLTPNQQQHQQQHSQLQQVHANGS-NH2 complexes start to form at pH around 4 (Figure S3C), with the CuHL species, in which two imidazoles are involved in binding, as evidenced by the UV−vis band at 680 nm (Figure S4B, Table 2).28 This maximum undergoes a blue shift to 620 nm at pH 7, which, together with the log K value of 5.43 (much lower with respect to the corresponding log K of 6.56) and with the increase of the A∥ value to 167, proves the involvement of the third imidazole in the coordination of Cu2+ in the CuL species (Figure S4B, Table 2). A drastic change in the binding mode occurs at pH 8, where two amide nitrogens start to participate in binding (CuH−2L, Figure S3C), most likely displacing one of the previously bound imidazoles; the UV−vis band shifts to around 520 nm (Figure S4B, Table 2), E


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Inorganic Chemistry in the coordination, the complex stabilities are quite comparable. A drastic change is observed at basic pH, at which, in the case of Ac-QQLTPNQQQHQQQHSQLQQVHANGS-NH2, amide nitrogens begin to take part in binding; at pH 10, in a hypothetical situation, in which equimolar amounts of the ligands and Cu2+ are mixed (Figure 2A), almost 100% of the available metal would be bound to Ac-QQLTPNQQQHQQQHSQLQQVHANGSNH2 (as already discussed, Ac-HGPHPHPHG-NH2 has only one amide available for binding). Previously calculated stability constants are applied to a theoretical situation, in which equimolar amounts of Me2+, Acand AcHGPHPHPHG-NH2, QQLTPNQQQHQQQHSQLQQVHANG-NH2 are present. The case of Zn2+ complexes is equally interesting. Since this metal is not likely to displace amide protons, it can be assumed that the binding donors are the same in both complexes (up to 3 imidazole nitrogens). In an analogous hypothetical situation, in which equimolar amounts of both ligands and Zn2+ were mixed, throughout the studied pH range, no more than 20% of available Zn2+ is bound to the shorter Ac-HGPHPHPHG-NH2 peptide (Figure 2B). This can most likely be explained by the additional stabilizing effect of the hydrogen bonds formed by the numerous glutamines in Ac-QQLTPNQQQHQQQHSQLQQVHANGS-NH2, which protect the metal core from hydrolysis.29 Structural Changes Induced by Cu2+ and Zn2+ at Physiological pH. To summarize the previous section, at around physiological pH (pH 7.4), Ac-HGPHPHPHG-NH2 binds both Cu2+ and Zn2+ ions via a set of 3 histidine imidazoles. What is the influence of the addition of metals on the structure of the peptide? To start with, surprisingly, the CD spectrum for the native Ac-HGPHPHPHG-NH2 peptide (Figure S6) is similar to α-helical structures: two negative minima at ∼208 nm and ∼230 nm are present. This peptide is likely to undergo both induced folding (Figure S7) and unfolding (Figure S6). An increase of θ value at ∼208 nm and decrease at ∼230 nm upon increasing concentration of TFE is observed. Secondary structures present in Ac-HGPHPHPHGNH2 were readily available for the denaturating agent. Even low concentrations of GdmCl caused an increase of the θ value at ∼220 nm (Figure S6). A really interesting phenomenon can be observed on the spectra recorded in the presence of Cu2+: Increasing concentration of Cu2+ strongly enhances the formation of a positive maximum at ∼222 nm with a simultaneous blue shift of the negative band around 200 nm. (Figure 3A). This indicates that upon Cu2+ ion binding, the Ac-HGPHPHPHGNH2 peptide gains a PPII helical structure, which is a lefthanded helix that often occurs in collagen triple-helix structures, but can also be found in peptides and globular proteins.30,31 This structure is formed already in the presence of 1 Cu2+ equivalent, and no significant changes in the far UV CD spectra are observed in the presence of 2.5 metal equivalents. Surprisingly, although the coordination mode of Cu2+- AcHGPHPHPHG-NH2 and Zn2+-Ac-HGPHPHPHG-NH2 complexes are the same (a [3Nim] binding), increasing the concentration of Zn2+ ions did not induce any particular changes in the secondary structure content of the AcHGPHPHPHG-NH2 peptide (Figure 3B). How can this phenomenon be explained? DFT calculations helped us to find the answer and also revealed structural details

Figure 3. Far-UV CD spectra of Ac-HGPHPHPHG-NH2 recorded in the presence of different molar ratios of (A) Cu2+ and (B) Zn2+ ions.

of the complexes, showing that at physiological pH, both Cu2+ (Figure 4B) and Zn2+ (Figure 4A) complexes with Ac-

Figure 4. Structures of the Ac-HGPHPHPHG-NH2 complexes obtained with DFT calculations. From left 3Nim with Zn2+ and Cu2+. The blue tubes follow backbones, red tube indicates polyproline II helix fragment.

HGPHPHPHG-NH2 involve the same 3 imidazole nitrogens in metal binding: ones from His4, His6, and His8 (corresponding to His620, His622, and His624 in AaEcR (UniProt ID: P49880/GenBank: AAA87394.1), and both are stabilized by a moderate number of hydrogen bonds due to the presence of proline in the sequence (four hydrogen bonds in F


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Inorganic Chemistry Cu 2 + -Ac-HGPHPHPHG-NH 2 and five in Zn 2 + -AcHGPHPHPHG-NH2). Theoretical calculations are in perfect agreement with our experimental findings and confirm the presence of a polyproline helix in the Cu2+-Ac-HGPHPHPHG-NH2 complex (but not in the Zn2+-Ac-HGPHPHPHG-NH2 one). The ideal polyL-proline II helix is expected to display a φ angle with value of −75° and ψ dihedral angle of 160°. The ideal values of these angles may vary depending on the computational approach used. We have calculated the Ac-P10-NH2 ideal PPII helix (Figure S10) at the level of theory presented here. The typical calculated dihedrals in the middle of the PPII helix are −72° and 163°. As expected, due to the moderate size of the ligand and the metal-peptide bonding, only a fragment of the Cu2+Ac-HGPHPHPHG-NH2 complex shows the typical structure of a PPII helix (Figure 4B), with values of φ and ψ dihedrals equal to −74.7° and 167.7°. The presence of the PPII helix is not detected neither in the free ligand nor in its Zn2+ complex (Figure 4A). Its formation is initiated only by the coordination of Cu2+ ions. The Ac-QQLTPNQQQHQQQHSQLQQVHANGS-NH2 peptide CD spectrum resembles one registered for peptides with random coil characteristics: one negative minimum of ∼200 nm.32,33 The structure is susceptible to induced folding in the presence of TFE (Figure S8). Samples containing 40% (or more) TFE were typical for helical peptides spectrum with decreasing values of θ at ∼208 nm and ∼222 nm. Some residual secondary structures are, however, present and were unfolded by the increasing concentration of GdmCl (Figure S9). At physiological pH, Ac-QQLTPNQQQHQQQHSQLQQVHANGS-NH2 binds Cu2+ via a set of three imidazoles (as in the case of the previously discussed peptide). Does the addition of Cu2+ also alter the previously unstructured peptide? We hypothesized, based on the high content of glutamines which are likely to form numerous hydrogen bonds, that they may help to obtain a given structure and/or to additionally stabilize the formed complex.27 Here, spectra registered for samples with the molar ratio of Cu2+:AcQQLTPNQQQHQQQHSQLQQVHANGS-NH2 equal to 1.0 and 2.5 demonstrated only a slight decrease in θ value in the 215−225 nm range (Figure 5A), showing that no pronounced structural changes were observed upon the addition of Cu2+. In the case of the 3 imidazole-bound Zn2+ complexes, no changes in the CD spectra were detected in the presence of increasing Zn2+ concentrations (Figure 5B). Similarly, no spectacular structural changes in prothymosin-α, an intrinsically disordered protein, in the presence of Zn2+ were observed in ref 34. DFT calculations found two 3N complexes built by AcQQLTPNQQQHQQQHSQLQQVHANGS-NH2 and Zn2+ and Cu2+ cations (Figure 6A,B, respectively) and one 2N connected complex with Cu2+ (Figure 6C). The 3N complexes with both investigated cations engage all 3 imidazole rings form the sequence. The histidines build imidazole-based docking sockets located outside of the folded peptide (see Figure 6A,B). The metal−nitrogen strong bonds display values slightly bigger than 2 Å, which are values typical for a 3N type of imidazole connections (Table S1). Blue Tubes Follow Backbones. The two imidazole connected complex has only been found for the Cu2+ complex, where imidazole rings from His14 and His21 (His639 and His646 in AaEcR, respectively) are engaged in binding. The

Figure 5. Far-UV CD spectra of AcQQLTPNQQQHQQQHSQLQQVHANG-NH2 recorded in the presence of different molar ratio of (A) Cu2+ and (B) Zn2+ ions.

Figure 6. Structures of the AcQQLTPNQQQHQQQHSQLQQVHANGS-NH2 complexes. (A) 3N complex with Zn2+, (B) 3N with Cu2+, and (C) 2N with Cu2+.

metal−ligand bond lengths are slightly shorter than in 3N type complexes, ∼1.9 Å, due to high flexibility of the side chains. Noteworthy, one supporting interaction between the oxygen form carbonyl group and the copper cation with length 2.236 Å has been found. The 2N type complex with Cu2+ (Figure 6C) forms the shortest metal−peptide bond (1.854 Å) among all complexes as well as the shortest average metal−peptide bond. All Ac-QQLTPNQQQHQQQHSQLQQVHANGS-NH2 complexes display a rich network of up to 20 hydrogen bonds. However, well-defined regular fragments based on 3− 10 and 4−13 helical pattern fragments have not been found. G


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Inorganic Chemistry

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NH 2 species are more stable than the Zn 2 + -AcHGPHPHPHG-NH2 ones due to the stabilizing effect of the hydrogen bonds in Ac-QQLTPNQQQHQQQHSQLQQVHANGS-NH2, which protect the metal core from hydrolysis. The glutamine-rich fragment seems to be the primary region of choice for metal binding, however, from the structural point of view, the role of the proline-rich fragment is of particular importance. Results of CD measurements in the presence of varying concentrations of GdmCl showed that both peptides possess some residual secondary structure (Figures S7 and S9). To verify the structure-forming potential of both peptides, TFE, a reagent often used for probing structural potential which promotes α-helical structure formation,51 even in IDPs,6,47 was used. The recorded spectra revealed the increase in the content of helical structure which suggested the strong structureforming potential encoded in the sequences of both peptides (Figures S6 and S8). The most interesting finding was observed for the Cu2+-Ac-HGPHPHPHG-NH2 interaction CD analysis: Increasing concentrations of Cu2+ enhanced the formation of a positive maximum at ∼222 nm. This indicates that upon binding Cu2+ ions, the Ac-HGPHPHPHG-NH2 peptide gained a unique poly-L-Pro helical structure (Figure 3A). The significance of CD data in the detection of PPII helices has been well documented in the literature.52,53 Several papers underline the fact that one has to be careful about an univocal assignment of a PPII conformation based on CD data only54 and point to DFT calculations as an additional method to show the formation of this structure.53 Indeed, DFT calculations confirmed the formation of a PPII -like helix also in the case of the Cu2+-Ac-HGPHPHPHG-NH2 complex studied in this work (Figure 4B). In general, the PPII-like structure can also be observed for polypeptides that are not P-rich or even do not contain any proline residues in the sequence.55 Interestingly, the addition of Cu2+ may change the structure of the coordinating peptide, leading to a PPII-like conformation.30 Recently, we reported the first evidence of a Cu2+-induced formation of a PPII structure in a sequence that does not contain any proline residues.56 The discussed PPII has emerged clearly as a structural class not only of fibrillar proteins but also of the folded and unfolded proteins.31 The PPII helix is an extended (3.1 Å per residue compared to 1.5 Å in the α-helix), left-handed helix defined by the φ, ψ torsional angle cluster with the distribution maximum at −75° and 145°.31 The PPII helix is far more flexible than the typical α-helix and β-sheet structures. It is the dominant conformation in the structure of the Pro-rich regions in proteins.57 PPII helices play a crucial role in protein−protein interactions58,59 and in interactions between proteins and nucleic acids60 and references in.31 Beside its important structural role in both folded and unfolded proteins, PPII helices are thought to be directly involved or mediate a wide range of molecular functions including signaling, transcription, cell motility, immune response, elasticity of elastomeric proteins, and possibly conformational transitions in amyloid proteins.31 It is believed that many of the possible PPII functions are yet undiscovered. What is significantly interesting in the case of the Ac-HGPHPHPHG-NH2 peptide is that the PPII structure is clearly induced by the binding of Cu2+. It suggests that A. aegypti EcR may use this segment in the F

DISCUSSION The unique F domain located at the C-terminus of some NRs is one of the least evolutionary conserved regions in members of these ligand-dependent transcription factors. This short Cterminal region is not clearly defined and is absent in many members of the NR family and therefore is often considered as a part of the LBD.35 The residual F domains of mammalian NRs are involved in modulating NRs activities by affecting transcriptional activation, dimerization, and interactions with co-activators or co-repressors and by influencing ligand binding conformations within LBDs.36 Some of the F domains of NRs were previously described as crucial for maintaining their function. Bianchetti and co-workers37 showed that the F domain of the glucocorticoid receptor-α ligand binding domain (GRα) forms a steric obstacle to the well-known canonical LBD dimer assembly. In the case of the F domain of the estrogen receptor-α ligand binding domain (ERα), it was shown that this region of ERα plays a significant role in a ligand-mediated transactivation in the species-specific mode.38 Recently, we showed that the isolated recombinant fulllength F domain of EcR from A. aegypti (AaFEcR) exhibits characteristics of IDPs with residual secondary structures and exhibits the ability to bind metal ions such as Cu2+ and Zn2+.6 Our extensive studies revealed that both metal ions did not induce any statistically relevant changes in the secondary structure content of AaFEcR. Thus, we suggested that the AaFEcR interactions with Zn2+ and Cu2+ do not lead to a significant change in secondary structure, but result in protein compaction.6 At this point, we asked ourselves: Maybe the metal ions induce some local structural changes in the area of their interactions with the F domain, and we are not able to detect them having the intrinsically disordered full-length AFEcR. It is experimentally proved that IDPs or IDRs can adopt distinct structures by interacting with particular protein partners or by binding other ligands.39−41 In contrast to the highly evolutionarily conserved DBDs and LBDs of NRs, the isolated NTDs of, for example, GR,42 ER,43 AR,44 ecdysteroid receptor (Drosophila melanogaster EcR),45 HR38,46 and ultraspiracle protein (A. aegypti Usp)47 are disordered in solution. Binding the right protein partner may induce the folding of NTDs, as it was shown for, for example, GR-AF1, ERα, PR, and AR, and changes in the secondary structure content often limited to the fragment of NTDs possessing the transactivation function (AF1).43,48−50 Therefore, we decided to focus on the core sequence of AaFEcR, rich in the putative metal ion binding motifs, and to work on two model peptides (Ac-HGPHPHPHG-NH2 and Ac-QQLTPNQQQHQQQHSQLQQVHANGS-NH2), which encompass wild-type AaFEcR residues 617−625 and 626−650, respectively (Figure 1). At physiological pH, the glutamine-rich region forms more stable complexes with both Zn2+ and Cu2+ than the prolinerich one. In the case of Cu2+ complexes, the difference can be explained by participation of amide nitrogens in metal binding and the formation of stable, square planar five and sixmembered chelate rings in the case of Cu 2+ -AcQQLTPNQQQHQQQHSQLQQVHANGS-NH 2 (AcHGPHPHPHG-NH2 has only one amide available for binding). Although both complexes have the same [3Nim] binding donors, Zn2+-Ac-QQLTPNQQQHQQQHSQLQQVHANGSH


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Inorganic Chemistry domain as the Cu 2+ -dependent platform for multiple interactions with, for example, modulatory proteins or specific ligands controlling gene expression. Interestingly, increasing concentration of Zn2+ ions did not induce any particular changes in the secondary structure content of the AcHGPHPHPHG-NH 2 and Ac-QQLTPNQQQHQQQHSQLQQVHANGS-NH2 peptides (Figures 3B and 5B). Only minor structural changes in the AcQQLTPNQQQHQQQHSQLQQVHANGS-NH 2 peptide can be observed in the presence of Cu2+ (Figure 5A). In summary, both peptides are able to bind Zn2+ and Cu2+ ions, but only the Cu2+ ions trigger the dramatic structural changes. It may indicate that the ion-binding-dependent functions of the AaFEcR lie in the core segment of the domain with the sequence corresponding to the peptide sequences (617−650). The 3D structures of the AaFEcR alone or in the complexes with metal ions are not determined yet.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work was supported by the National Science Centre (UMO-2017/26/E/ST5/00364) and by a statutory activity subsidy from the Polish Ministry of Science and Higher Education for the Faculty of Chemistry of Wrocław University of Science and Technology. We are grateful to Michał Szumiński for painting the Aedes aegypti mosquito for the table of contents graphic.

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(1) Renaud, J. P.; Moras, D. Structural Studies on Nuclear Receptors. Cell. Mol. Life Sci. 2000, 57 (12), 1748−1769. (2) Nuclear Receptors: From Structure to the Clinic; McEwan, I. J., Kumar, R., Eds; Springer International Publishing: Cham, 2015; pp 1−14. (3) Guzman, M. G.; Halstead, S. B.; Artsob, H.; Buchy, P.; Farrar, J.; et al. Dengue: A Continuing Global Threat Europe PMC Funders Author Manuscripts. Nat. Rev. Microbiol. 2010, 8, S7−S16. (4) Barrett, A. D. T.; Higgs, S. Yellow Fever: A Disease That Has yet to Be Conquered. Annu. Rev. Entomol. 2007, 52, 209−229. (5) Marchette, N. J.; Garcia, R.; Rudnick, A. Isolation of Zika Virus from Aedes Aegypti Mosquitoes in Malaysia. Am. J. Trop. Med. Hyg. 1969, 18 (3), 411−415. (6) Wiȩch, A.; Rowińska-Ż yrek, M.; Wa̧tły, J.; Czarnota, A.; Hołubowicz, R.; Szewczuk, Z.; Ożyhar, A.; Orłowski, M. The Intrinsically Disordered C-Terminal F Domain of the Ecdysteroid Receptor from Aedes Aegypti Exhibits Metal Ion-Binding Ability. J. Steroid Biochem. Mol. Biol. 2019, 186, 42−55. (7) Cho, W. L.; Kapitskaya, M. Z.; Raikhel, A. S. Mosquito Ecdysteroid Receptor: Analysis of the CDNA and Expression during Vitellogenesis. Insect Biochem. Mol. Biol. 1995, 25 (1), 19−27. (8) Ranieri-Raggi, M.; Moir, A.; Raggi, A. The Role of HistidineProline-Rich Glycoprotein as Zinc Chaperone for Skeletal Muscle AMP Deaminase. Biomolecules 2014, 4 (2), 474−497. (9) Gemayel, R.; Chavali, S.; Pougach, K.; Legendre, M.; Zhu, B.; Boeynaems, S.; van der Zande, E.; Gevaert, K.; Rousseau, F.; Schymkowitz, J.; Madan Babu, M.; Verstrepen, K. J. Variable Glutamine-Rich Repeats Modulate Transcription Factor Activity. Mol. Cell 2015, 59 (4), 615−627. (10) Davies, P.; Watt, K.; Kelly, S. M.; Clark, C.; Price, N. C.; McEwan, I. J. Consequences of Poly-Glutamine Repeat Length for the Conformation and Folding of the Androgen Receptor AminoTerminal Domain. J. Mol. Endocrinol. 2008, 41 (5), 301−314. (11) Lavery, D. N.; McEwan, I. J. Structure and Function of Steroid Receptor AF1 Transactivation Domains: Induction of Active Conformations. Biochem. J. 2005, 391 (3), 449−464. (12) Kumar, R.; Litwack, G. Structural and Functional Relationships of the Steroid Hormone Receptors’ N-Terminal Transactivation Domain. Steroids 2009, 74 (12), 877−883. (13) Gans, P.; O’Sullivan, B. GLEE, a New Computer Program for Glass Electrode Calibration. Talanta 2000, 51 (1), 33−37. (14) Gran, G.; Dahlenborg, H.; Laurell, S.; Rottenberg, M. Determination of the Equivalent Point in Potentiometric Titrations. Acta Chem. Scand. 1950, 4, 559−577. (15) Gans, P.; Sabatini, A.; Vacca, A. SUPERQUAD: An Improved General Program for Computation of Formation Constants from Potentiometric Data. J. Chem. Soc., Dalton Trans. 1985, No. 6, 1195. (16) Petitt, L.; Powell, H. K. J. The IUPAC Stability Constants Database. Chem. Int. 2006, 28 (5), 14−15. (17) Arena, G.; Cali, R.; Rizzarelli, E.; Sammartano, S. Thermodynamic Study on the Formation of the Cupric Ion Hydrolytic Species. Thermochim. Acta 1976, 16 (3), 315−321. (18) Alderighi, L.; Gans, P.; Ienco, A.; Peters, D.; Sabatini, A.; Vacca, A. Hyperquad Simulation and Speciation (HySS): A Utility Program

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CONCLUSIONS The full-length F domain of EcR from A. aegypti (AaFEcR) exhibits characteristics of IDPs with residual secondary structures, and the coordination of Cu2+ and Zn2+ to this region does not induce any statistically relevant changes in its overall secondary structure, as it was published recently. Here, in this work, we focused on the detection of local structural changes in AaFEcR that may occur due to metal binding in two putative metal ion binding motifs, Ac-HGPHPHPHG-NH2 and Ac-QQLTPNQQQHQQQHSQLQQVHANGS-NH 2 (wild-type AaFEcR residues 617−625 and 626−650). At physiological pH, the glutamine-rich region forms more stable complexes with both Zn2+ and Cu2+ than the proline-rich one. In the case of Cu2+ complexes, the difference can be explained by participation of amide nitrogens, whereas in the case of Zn2+, the presence of glutamines in the longer peptide stabilizes the complex due to the hydrogen bonds that protect the metal core from hydrolysis. Although the glutamine-rich fragment seems to be the primary region of choice for metal binding, from the structural point of view, the role of the proline-rich fragment is of particular importance. The addition of Cu2+ to the proline-rich region changes the structure of the coordinating peptide, leading to a PPII helix-like conformation. This dramatic structural change sheds new light on the yet undiscovered ionbinding-dependent physiological functions of the AaFEcR domain.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.9b01826.

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REFERENCES

Figures S1−S10 (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Phone: +48713206286. ORCID

Magdalena Rowińska-Ż yrek: 0000-0002-0425-1128 Author Contributions ∥

These authors contributed equally to this work. I


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