Self-Assembly of the Second Transmembrane Domain of hCtr1 in

Article pubs.acs.org/JPCB

Self-Assembly of the Second Transmembrane Domain of hCtr1 in Micelles and Interaction with Silver Ion Zhe Dong,† Yunrui Wang,† Chunyu Wang,† Haoran Xu,‡ Liping Guan,† Zhengqiang Li,‡ and Fei Li*,† †

State Key Laboratory of Supramolecular Structure and Materials, Jilin University, 2699 Qianjin Avenue, Changchun 130012, PR China ‡ Key Laboratory for Molecular Enzymology & Engineering, The Ministry of Education, Jilin University, 2699 Qianjin Avenue, Changchun 130012, PR China S Supporting Information *

ABSTRACT: Human copper transporter 1 (hCtr1) transports copper and silver by a homotrimer. The protein contains three transmembrane domains in which the second transmembrane domain (TMD2) is a key component lining the central pore of the trimer. The MXXXM motif in the Cterminal end of TMD2 plays a significant role in the function of hCtr1. In this study, we characterized the structure and assembly of isolated TMD2 of hCtr1 in sodium dodecyl sulfate (SDS) micelles and the interaction of the micelle-bound peptide with silver ion using nuclear magnetic resonance, circular dichroism, isothermal titration calorimetry and electrophoresis techniques. We detected the formation of a trimer of the isolated hCtr1-TMD2 in SDS micelles and the binding of the trimer to Ag(I) by a chemical stoichiometry of 3:2 of peptide:Ag(I). We showed that either an intensive pretreatment of the TMD2 peptide by 1,1,1,3,3,3-hexafluoro-2-propanol solvent or a conversion from methionine to leucine in the MXXXM motif changes the aggregation structure of the peptide and decreases the binding affinity by 1 order of magnitude. Our results suggest that the intrinsic interaction of the second transmembrane domain itself may be closely associated with the formation of hCtr1 pore in cellular membranes, and two methionine residues in the MXXXM motif may be important for TMD2 both in the trimeric assembly and in a higher-affinity binding to Ag(I).

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N-terminal domain and an intracellular C-terminal domain.25,26 The protein forms a homotrimeric structure in phospholipid membranes and the transmembrane domains constitute a coneshaped pore passing through the lipid bilayer with a narrow extracellular entrance and a wider export on the cytosolic side.27−30 A generally accepted hypothesis for the transport mechanism of copper by hCtr1 is that the ion is captured and concentrated by the extracellular N-terminal metal-binding domain prior to entry into the transmembrane pathway, and then transferred as cuprous ion through the pore formed by the TMDs to a C-terminal metal-binding domain, where the ion is released and escorted to specific compartments by metallochaperones.29,31 The transport of silver by hCtr1 is believed to be performed by a similar mechanism.16 Each of the three domains of hCtr1 plays an important role for the ion transference across the membrane. The studies on the short peptides have shown that the N-terminal domain of hCtr1 can bind Cu(I)/Ag(I) by the coordination of Met32,33 and His,21,31 and the C-terminus can bind the ions by the His-Cys-His motif (hCtr1 188−190).34 The deletion or substitution of these

INTRODUCTION Silver has been widely used for a long time as an antimicrobial agent in the treatment of burns and infections caused by external trauma and chronic ulcers since it was originally used by humans in the Neolithic age.1,2 Silver and silver complexes also exhibit potential applications in the antitumor activity,3−5 particularly in the therapies of cisplatin resistance cancers.6−8 Compared with heavy metals such as arsenic, mercury, or lead, silver turned out to be less toxic to human cells. Nevertheless, exposure to silver for a long time could cause some adverse effects on immune functions,9−12 cellular detoxification systems,13,14 and central nervous systems.15 The toxicities of silver and silver complexes might be associated with the absorption and accumulation of silver in the mammalian cells and tissues. However, detailed mechanism for the absorption and accumulation of silver in cells is poorly understood. Human copper transporter 1 (hCtr1) is a high-affinity copper influx transporter in the mammalian cells. The protein is also believed to transport Ag(I).16−18 The regulations of hCtr1-mediated copper uptake by Zn(II) and Cd(II) as well as cisplatin17,19−23 are also identified. The transport of Ag(I) by hCtr1 may be linked with the toxicity of the ion.24 Human Ctr1 contains 190 amino acids, which are divided into three transmembrane domains (TMDs), an extracellular © 2015 American Chemical Society

Received: April 19, 2015 Revised: June 8, 2015 Published: June 10, 2015 8302

DOI: 10.1021/acs.jpcb.5b03744 J. Phys. Chem. B 2015, 119, 8302−8312

Article

The Journal of Physical Chemistry B

with Lys does not affect their native characteristics of insertion in membrane mimetic environments and fold as an α-helical structure, and even native oligomeric states.48 The peptides were purified by high performance liquid chromatography and mass spectroscopy, and the purity of the peptides was greater than 95%. Deuterated sodium dodecyl sulfate SDS-d25 (98%) and D2O (99.8%) were purchased from Cambridge Isotope Laboratories, Inc. (Cambridge, MA). The detergent SDS and the organic solvent HFIP were obtained from Sigma-Aldrich (St. Louis, MO) and Acros Organics (Morris Plains, NJ), respectively. Silver nitrate was purchased from Beijing Dingguo Changsheng Biotechnology Co, Ltd. (Beijing, China). All reagents and solvents were of analytical grade and utilized without further purification. Sample Preparation. Peptide (0.08 mg for the CD experiments, 0.10 mg for the ITC and SDS-PAGE experiments, or 1.77 mg for the NMR experiments) was weighted and solubilized in 200 μL HFIP. The solution was vortexed for 3−5 s and then sonicated using a bath-type sonicator for 30 min. Detergent SDS (3.46 mg for the CD experiments, 1.73 mg for the ITC and SDS-PAGE experiments), or SDS-d25 (18.80 mg for the NMR experiments) was weighted and solubilized in 1 mL deionized water separately. The HFIP solution of the peptide was added into SDS (or SDS-d25) aqueous solution. The mixture was vortexed for 3−5 s and sonicated for 30 min. The resulting solution was further diluted with 1.6 mL deionized water and lyophilized overnight. We also prepared the samples in which the peptides were treated with HFIP twice in order to disaggregate the hydrophobic peptides further. Briefly, the peptide was solubilized in 200 μL HFIP. After 1 h sonication the solution was lyophilized overnight. The resulting powder was resolubilized in 200 μL HFIP and the procedure of sample preparation for the peptide incorporated with SDS micelles just described above was repeated. The names of the samples containing the peptides treated with HFIP once and twice were discriminated by adding the number 1 (TMD2-1, M20L-1, M24L-1, and M20LM24L-1) and the number 2 (TMD2-2, M20L-2, M24L-2, and M20LM24L-2), respectively. The resulting dry powder was hydrated either using deionized water for the CD, ITC, and SDS-PAGE experiments or H2O/D2O mixture (9/1 in volume) for the NMR experiments. The final samples of 20 μM peptide in 10 mM SDS detergent solution (CD), 50 μM peptide in 10 mM SDS detergent solution (ITC and SDS-PAGE) and 1 mM peptide in 120 mM SDS-d25 detergent solution (NMR) were prepared. The pH values were adjusted with 0.1 mM NaOH or 0.1 mM HNO3 to 6 for all the solution samples. A stock solution of 200 mM AgNO3 in deionized water was prepared for the titration experiments. In all titration experiments, the addition of the maximum amount of AgNO3 solution led to an increase in pH by no more than 0.2. The effects of such small pH variations on the results of the experiments with regard to the peptides studied were insignificant. Far-UV Circular Dichroism Experiments. The far-UV CD experiments were performed on a PMS-450 spectropolarimeter (Biologic, France) at room temperature. A quartz cuvette with a path-length of 0.5 mm was used. The instrument was sufficiently purged with 99.9% nitrogen before starting the measurements. All CD spectra were collected in a wavelength range from 190 to 260 nm at a scan speed of 0.1 nm/s. Three spectra were accumulated and averaged and the reference spectrum of the respective medium was subtracted. The intensity was expressed as molar ellipticity (θ) in a unit of

sulfur-containing residues (Met and Cys) and iminazolecontaining residues (His) in the terminal domains reduces or eliminates the transport function of the protein.22,31,32,34−40 Of the three transmembrane domains, the helical bundle of the second transmembrane domain (TMD2) constitutes the inner wall of the pore and is directly related to the transport function of the protein.41 A MXXXM motif in TMD2 150−154 has been proved to be very important for the copper and silver transport into cells.16,39,41−45 The first and the third transmembrane domains are believed to be involved in the tight packing of helices. Though some results are obtained, the mechanism by which hCtr1 transports Ag(I)/Cu(I) cross cellular membranes remains largely unknown. Two possible transport modes are proposed: (1) the changes in the electrostatic field along the pore drive ion movement through the pore;46 (2) a series of ionic exchange reactions between distinct binding sites induce the conformational changes, which provides a driving force for the movement of the ion through the pore.28 To understand the mechanism of hCtr1 transporting Ag(I)/Cu(I), a knowledge of the trimeric structure at the atomic level is needed. However, such a structure is not available until now. S. J. Opella et al. expressed hCtr1 45−190 and some mutants and acquired the 15N/1H-HSQC NMR spectra of the proteins in DPC micelles, but they did not obtain high resolution structures of these proteins likely because of poorly resolved NMR spectra.35 We previously studied the structures and assemblies of the peptides corresponding to three transmembrane domains of hCtr1 in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) aqueous solution using 1H NMR spectroscopy.47 Our results demonstrated that TMD1 and TMD2 peptides form a continuous αhelix, while TMD3 peptide forms a noncontinuous α-helix in which a flexible segment including the GXXXG motif separates the helix into two parts. The TMD2 peptide has more propensity for self-association than other two TMD peptides. TMD2 is trimeric, whereas TMD1 and TMD3 are dimeric. Because TMD2 is a key element lining the central pore of hCtr1 trimer, this result gives a hint at a possibility of getting insight into hCtr1 pore structure and transport property by studying TMD2 trimer. In light of the significance of TMD2 in the construction of hCtr1 pore and the trafficking of Ag(I)/Cu(I) by hCtr1, we studied the structure and assembly of isolated hCtr1−TMD2 in sodium dodecyl sulfate (SDS) micelles, which were used as a membrane-mimics, as well as the binding of the peptide to Ag(I) using nuclear magnetic resonance (NMR), circular dichroism (CD), isothermal titration calorimetry (ITC) and electrophoresis (SDS-PAGE) techniques. We also undertook a similar study using the variant forms of the peptide in which either one or two methionines were converted to leucines to get insight into the roles of the Met residues in the peptide assembly and binding to Ag(I).

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EXPERIMENTAL SECTION Materials. All peptides, including a peptide from hCtr1 132−157 with a sequence of KHLLQTVLHIIQVVISYFLMLIFMTYNKK (assigned as to TMD2) and three Met-to-Leu mutants of TMD2 (assigned as to M20L, M24L, M20LM24L), were synthesized by GL Biochem. Ltd. (Shanghai, China). The first Lys residue at the N-terminal end and the last two Lys residues at the C-terminal end in these peptides were not included in the hCtr1 protein. They were added in these peptides to facilitate the purification of the samples. Previous studies have demonstrated that tagging hydrophobic peptides 8303


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The Journal of Physical Chemistry B deg cm2 dmol−1. The secondary structure contents were estimated by the CDPro software package using the program CONTIN/LL. A reference set of SMP56 including 56 proteins was used in the secondary structure analysis. SDS-PAGE Experiments. A Tricine buffer system with 0.5% SDS in the gel was employed.49 The samples of 50 μM peptide in 10 mM SDS aqueous solution were diluted with 4 × SDS loading buffer containing the tracking dye Serva Blue G, and boiled for 5 min before loading on to a 16.5%polyacryalamide/Tricine/SDS gel. The electrophoresis was performed at 4 °C using a voltage of 30 V before the sample entered the separation gel and 130 V afterward. After gel electrophoresis, the gel was placed in a solution containing 0.5% gluteraldehyde and 30% ethanol for 20 min and stained with Coomassie Brilliant Blue stain. The protein standards with ultra low molecular weight (3.3−20.1 kDa) were used. NMR Experiments and Structural Calculation. All NMR spectra were performed at 25 °C on a Bruker Avance 600 spectrometer equipped with a cryoprobe. Sodium 2,2-dimethyl2-silapentane-5-sulfonate (DSS) was used as an internal standard. The TOCSY (total correlation spectroscopy) and NOESY (nuclear Overhauser effect spectroscopy) spectra were recorded with a mixing time of 100 ms. The WATERGATE technique was used in both experiments for water signal suppression and the MLEV-17 pulse sequence was used in the TOCSY experiments. The 2 K data points in F2 dimension and 512 increments in F1 dimension were collected. Standard Bruker software (TOPSPIN 3.1) was used to process the 2D NMR spectra and software Sparky was used for the resonance assignments. The tertiary structures of the peptides were calculated by program CYANA.50 The cross-peak intensities in the NOESY spectra were transformed to the upper limits of the distance restraints using the CALIBA program. These distance restraints were subjected to a systematic analysis of the local conformation around the Cα atom of each residue, including the dihedral angles ϕ, ψ, χ1, and χ2, using the macro GRIDSEARCH. Torsion angle dynamic calculations were performed by the macro ANNEAL. The calculations were started from 200 initial conformers with random torsion angle values and the structures with violations no more than 0.2 Å for distance restraints and 5.0° for angle restraints were accepted. The 20 structures with the lowest target functions were assessed by software PROCHECK-NMR51 and visualized using program MOLMOL.52 The NMR restraints used in calculations and structural statistics were listed in Table S1, Supporting Information. Isothermal Titration Calorimetry. The ITC experiments were performed on a MicroCal ITC200 instrument at 25 °C. The reference cell was filled with deionized water and the sample cell was filled with the solution of 50 μM peptide incorporated with 10 mM SDS at pH 6. The syringe cell was injected with 1 mM AgNO3 solution containing 10 mM SDS at pH 6. Aliquots of titrant were successively injected into the sample cell at an interval of 240 s and a total of 27 injections were performed. Control was performed with SDS micellar solution to measure the dilution heat of the titrant and was subtracted from the sample data. The ITC data were analyzed using the Origin 7.0 software package. The parameters including the enthalpy change (ΔH), binding constant (Ka), and number of binding site (n) were obtained by a nonlinear least-squares fitting of the experimental data using the one-site binding model. The Gibbs free energy

(ΔG) and the entropy change (ΔS) were calculated from the equations: ΔG = −RT ln K

(1)

ΔG = ΔH − T ΔS

(2)

where T is the absolute temperature and R the gas constant.

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RESULTS AND DISCUSSION Structure and Assembly of TMD2 in SDS Micelles. The secondary structure and assembly of TMD2 alone in SDS micelles were characterized first by CD spectra (Figure 1). One

Figure 1. CD spectra of 20 μM TMD2 in 10 mM SDS micelles at pH 6 in the absence and presence of 50 μM AgNO3 at room temperature.

positive absorbance at 194 nm and two negative absorbances at 208 and 222 nm were observed in the spectra of TMD2-1 and TMD2-2, suggesting the formation of an α-helix structure in both cases. However, there was an evident difference in the absorbance intensity of the two minima. Whereas the absorbance at 222 nm was more negative than that at 208 nm in the spectrum of TMD2-1, the intensities at the two minima were very close in the spectrum of TMD2-2. The ratio of the molar ellipticities at 222 and 208 nm ([θ]222/[θ]208) has been used as a criterion in several proteins to evaluate the presence of coiled-coil helices.53−55 The value is 0.83 for noncoiled-coil helices, while it is 1.03 for two-stranded coiled coils. In our case, the ratio was about 1.23 and 1.02 for TMD21 and TMD2-2, respectively, implying that TMD2 is prone to aggregation in SDS micelles by coiled-coil interaction and TMD2-1 is more aggregated than TMD2-2. The pulse field gradient (PFG) NMR method was also used to monitor the self-assembly of the TMD2 peptide in SDS-d25 micelles by the measurement of diffusion coefficient. The SDSd25 micelles incorporated with TMD2-1 showed a diffusion rate distinctly slower than that of the micelles incorporated with TMD2-2 (Figure 2). The diffusion coefficients of 5.85 × 10−12 m2 s−1 and 9.63 × 10−12 m2 s−1 were obtained for the complexes of the micelles with TMD2-1 and TMD2-2, respectively. The PFG data further suggest that TMD2-1 is self-assembly at a higher degree than TMD2-2 in the micelles. The self-assembly states of the micelle-bound peptide treated with HFIP once and twice were further tested by the SDSPAGE gel electrophoresis (Figure 3). It indicated that TMD2-1 in SDS micelles was trimeric predominantly (the molecular weight of TMD2 monomer is 3.5 kDa), while TMD2-2 was a mixture of monomer and dimer. 8304


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Figure 2. PFG-NMR experimental results of 1 mM peptides TMD2-1 (■), TMD2-2 (□), M20L-1 (▲), M24L-1 (▽), and M20LM24L-1 (×) in 120 mM SDS-d25 micelles at pH 6, recorded at 25 °C.

Figure 4. Hα-HN region of the NOESY spectra and NOE connectivities along with the CSI data for 1 mM TMD2-1 in 120 mM SDS-d25 micelles at pH 6 before (A and B) and after (C and D) the addition of 2 mM AgNO3 recorded at 25 °C. The threshold levels of the spectra were set to the values at which the signal/noise ratios were good enough. A few cross-peaks with intensities lower than the thresholds were not displayed in the partial spectra.

Figure 3. SDS-PAGE data of 50 μM peptides TMD2-1 (band A) and TMD2-2 (band B) in 10 mM SDS detergent at pH 6.

In order to obtain the spatial structure of TMD2 in trimer, we recorded two-dimensional TOCSY and NOESY spectra of TMD2-1 in SDS-d25 micelles. Although TMD2-1 was selfassociated predominantly as a trimer in the micelles as predicted by the SDS-PAGE experiment, the NOESY spectrum of the peptide was quite well dispersed. The tertiary structure of TMD2-1 in SDS-d25 micelles was determined by a combining analysis of TOCSY and NOESY spectra as well as the dynamic calculation based on a geometrical optimization. In the NOESY spectrum, a series of NOE cross-peaks of Hα(i)/HN(i+3) and Hα(i)/Hβ(i+3) in the region of His2-Lys28 were assigned (Figure 4, parts A and B). No cross-peaks from intermolecular interactions were identified in the NOESY spectrum. In addition, the CSI (chemical shift index from the Hα resonances56) mode with a series of −1 from Leu4 to the Cterminus of the peptide was obtained (Figure 4B). Both the NOE connectivity and CSI mode predicted a highly helical structure for the TMD2-1 peptide in the micelles, which was well consistent with the result of the CD measurement. An αhelix spanning from Leu4 to Tyr26 was well-defined for the structure of TMD2-1 in SDS micelles by the calculation (Figure 5A). Binding of the Micelle-Bound TMD2 with Ag(I). In the presence of AgNO3, the proton chemical shifts of the residues in the C-terminal half of the peptide (Tyr17-Lys28) were affected significantly. In addition, the chemical shifts of the residues Leu8, Gln12 and Val13 were also changed considerably. Other residues in the N-terminal half of the peptide were almost not affected (Table 1 and Table S2). This indicates that the peptide binds Ag(I) mainly by the residues in the C-terminal part. Although the chemical shifts of the peptide were significantly affected by the addition of Ag(I), the mode of CSI obtained from the chemical shifts of Hα was not changed (Figure 4D), compared with the CSI mode obtained in the

Figure 5. Ensemble of 20 structures with the lowest target functions presented as backbone atoms and a ribbon representation of the mean structure for 1 mM TMD2-1 in 120 mM SDS-d25 micelles at pH 6 in the absence (A) and presence (B) of 2 mM Ag(I).

absence of Ag(I) (Figure 4B). Similar to the case in the absence of Ag(I), the medium-range NOE connectivities including Hα(i)/HN(i+3), Hα(i)/HN(i+4) and Hα(i)/Hβ(i+3) in the region of Leu3-Asn27 were also obtained in the presence of Ag(I) (Figure 4C and 4D). The structural calculation based on the NOE restraints assumed a well-defined α-helix in the same region (Figure 5B). The structure of the peptide was almost not affected by the presence of Ag(I). The same conclusion was also obtained by the CD data. As shown in Figure 1, the introduction of Ag(I) in the micellar solution of TMD2-1 did not cause a considerable change in the CD spectrum of the peptide. Of the residues with larger chemical shift changes, Met20 and Met24 displayed dramatic downfield shifts in the HN, Hγ (−CH2−S−) and Hε (−S−CH3) resonances (Table 1 and 8305


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Table 1. Changes in the Proton Chemical Shifts of 1 mM TMD2-1 in 120 mM SDS-d25 Micelles Induced by the Addition of 2 mM Ag(I) at pH 6, 25°Ca

a

number

residue

ΔδHN

ΔδHα

ΔδHβ

ΔδHγ

ΔδHδ

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

K H L L Q T V L H I I Q V V I S Y F L M L I F M T Y N K K

0.001 0.003 −0.001 0 0.008 −0.009 −0.004 0.006 0.003 −0.009 −0.012 0.009 0.04 −0.003 −0.001 0.006 0.057 −0.093 0.018 0.144 0.163 −0.083 0.002 0.277 −0.06 −0.076 −0.037 −0.044 −0.008

−0.002 −0.001 −0.002 −0.004 −0.005 −0.004 0 −0.002 −0.007 −0.006 −0.005 −0.019 −0.003 −0.003 0 −0.016 −0.063 −0.025 0.037 −0.003 −0.12 −0.017 −0.073 −0.033 0.015 −0.025 −0.018 −0.013 −0.003

−0.002 0.004 −0.003 −0.001 0.003 −0.008 0 0.018 −0.004 0.001 −0.001 −0.02 −0.028 −0.012 −0.002 0 0.054 −0.023 0.055 0.041 0.044 −0.004 −0.027 0.101 −0.008 −0.014 −0.054 0.004 −0.014

−0.005

0.005 −0.003 0.002 −0.002

−0.009 −0.01 −0.001 −0.005 0.002 −0.027 −0.005 0.003 −0.019 −0.020 −0.013 −0.002

−0.024 0.323 −0.06 0.025

ΔδHε −0.005

−0.005

−0.015 −0.005 −0.004 −0.004 −0.07/−0.035

−0.002 −0.081 0.011 −0.001

−0.002

−0.02 0.008 −0.019

0.238 0.007

−0.017 −0.009

−0.018 −0.066 −0.003 −0.009

−0.007 0.001 −0.001

The data larger than 0.02 ppm are in italics.

Figure S1), suggesting that Met20 and Met24 are involved in the binding to Ag(I) by the coordination of thioether in the side-chains. Other chemical groups in the C-terminal part of the TMD2 peptide, such as the aromatic ring in Tyr17 side-chain, the acylamino groups in Gln12 and Asn27 side-chains, the nitrogen atom in the backbone of Leu21, likely also contributed to the binding of the peptide to Ag(I). The binding of the peptide to Ag(I) may be accompanied by the regulation of the helical bundle mostly in the topology and intermolecular interaction, rather than in the folding structure of the peptide. The changes in the chemical shifts of Leu8, Val13 and some residues in the C-terminal part may be attributed to the variations in the interactions between peptide molecules. The stoichiometry of TMD2-1 binding to Ag(I) was estimated by the NMR titration experiments. The dependences of the chemical shifts of Hγ in Met20 and Met24 upon the molar ratios of Ag(I) to peptide were obtained (Figure 6). A transition of chemical shift occurred at a Ag(I)/peptide molar ratio of ∼0.7 was observed in the titration curve, indicating that the peptide binds Ag(I) with a stoichiometry of ∼3:2. The binding of TMD2-1 with Ag(I) was further determined by the ITC experiments. The isotherm of TMD2-1 titrated with AgNO3 showed an exothermic process (Figure 7A). The leastsquares fitting of the isotherm resulted in a n value of 0.8, corresponding to a stoichiometry of ∼3:2 for the binding of TMD2-1 to Ag(I). This is in agreement with the result of the NMR titration experiment. A model of three peptides binding two Ag(I) ions could explain the stoichiometric result quite

Figure 6. Hγ chemical shift changes of Met20 (■) and Met24 (□) of 1 mM TMD2-1 in 120 mM SDS-d25 micelles with the titration of AgNO3 at pH 6, 25 °C.

well. In this model, three peptide molecules in the trimer are self-associated as a parallel head-to-head pattern and bind two Ag(I) by three Met20 and three Met24, likely with the help of other coordinating residues, such as Gln12, Tyr17, Leu21, and Asn27. With respect to the peptide aggregation mode, we could not exclude the existence of the oligomers as head-to-tail pattern definitely. However, we did not observe any cross-peaks from the long distance interactions between the N- and Cterminal residues in the NOESY spectra. This suggests that the antiparallel head-to-tail aggregation state, if it is existent, would 8306


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Figure 7. ITC profiles of 50 μM peptides TMD2-1 (A) and TMD2-2 (B) in 10 mM SDS micelles at pH 6 titrated with AgNO3.

Table 2. ITC Data of the 50 μM Peptides Titrated with AgNO3 in 10 mM SDS Micellar Solution at pH 6, 25 °C peptide

n

Ka (M−1)

TMD2-1 M20L-1 M24L-1 M20LM24L-1 TMD2-2 M20L-2 M24L-2 M20LM24L-2

± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ±

0.80 2.61 2.19 1.23 2.95 2.48 1.89 1.49

0.01 0.01 0.06 0.05 0.01 0.02 0.01 0.06

(2.13 (3.25 (5.26 (5.17 (2.68 (4.52 (3.46 (5.93

0.33) 0.21) 1.99) 2.27) 0.17) 0.39) 0.21) 3.12)

× × × × × × × ×

ΔH (kcal mol−1) −12.21 −12.67 −12.18 −4.97 −10.55 −19.67 −14.98 −6.39

6

10 105 105 105 105 105 105 105

± ± ± ± ± ± ± ±

0.16 0.1 0.46 0.27 0.07 0.16 0.11 0.37

ΔG (kcal mol−1) −8.6 −7.49 −7.80 −7.76 −7.40 −7.71 −7.55 −7.87

± ± ± ± ± ± ± ±

0.08 0.04 0.19 0.22 0.04 0.05 0.04 0.25

TΔS (kcal mol−1) −3.61 −5.18 −4.38 2.79 −3.15 −11.96 −7.43 1.48

± ± ± ± ± ± ± ±

0.24 0.14 0.65 0.49 0.11 0.21 0.15 0.62

∼3:1. The intensive treatment by HFIP resulted in a dissociation of the peptide oligomer from a trimer to a dimer or a mixture of dimer and monomer (Figure 3). In contrast to the trimeric construction in which six Met residues provide two binding sites accommodating two Ag(I) simultaneously, the dimeric aggregate or the mixture of dimer and monomer binds more Ag(I) with a lower binding affinity and a poor binding cooperativity. This suggests that the aggregate pattern of peptide is significant for the binding affinity and the trimerization of the peptide is necessary for a higher affinity binding to Ag(I). Structures and Assemblies of the M-to-L Mutants in SDS Micelles. In order to get insight into the role of Met residues in the trimerization of the TMD2 peptide, we studied the structures and assemblies of the mutants M20L, M24L, and M20LM24L in SDS micelles using CD, PFG-NMR, and SDSPAGE methods. We used Leu as a substituent of Met in the study because Leu is the closest native amino acid to Met.57 The CD spectra of the micelle-bound mutants treated with HFIP once demonstrated two negative absorbances at 208 and 222 nm and one positive absorbance at 194 nm (Figure 8), characteristic of an α-helix structure. The molar ellipticities at 208 and 222 nm in each of the CD spectra were approximately equal ([θ]222/[θ]208 ≈ 1). The ratios were similar to that of TMD2-2, but smaller than that of TMD2-1. Further treatment with HFIP did not give rise to a considerable change in the CD

have a very small proportion compared with that of the headto-head aggregation state, which makes the long distance crosspeaks too weak to be observed. Additionally, if a peptide monomer adopts an antiparallel orientation to other two monomers in the trimer with quite a proportion, the binding of the peptide molecules to Ag(I) by Met20 and Met24 in their Cterminal parts should induce considerable changes in the chemical shifts of the N-terminal part residues. In fact, we only observed the effects of Ag(I) on the chemical shifts of the Cterminal part residues, but not observed the changes in the chemical shifts of the N-terminal part residues. These results indicate that the mode of all-parallel molecular arrangement is reasonable for the TMD2-1 trimeric structure. The binding affinity and thermodynamic parameters were also obtained by the ITC curve fitting and the calculations using eq 1 and 2 (Table 2). The thermodynamic data showed that the binding of TMD2-1 to Ag(I) is driven by the enthalpy change. The hydrogen bond and van der Waals force may play important roles in the formation of the relatively stable coordination structure of the peptide trimer. Interestingly, the ITC isotherm of TMD2-2 was dramatically different from that of TMD2-1 (Figure 7B). The binding affinity of TMD2-2 was smaller approximately by 1 order of magnitude than that of TMD2-1 (the Ka was 2.1 × 106 M−1 for TMD2-1 vs 2.7 × 105 M−1 for TMD2-2), and the molar ratio of Ag(I)/peptide for the binding was increased from ∼2:3 to 8307


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Figure 9. SDS-PAGE data of 50 μM peptides TMD2-1 (band A), M20L-1 (band B), M24L-1 (band C) and M20LM24L-1 (band D) in 10 mM SDS detergent at pH 6.

The one-dimensional 1H NMR spectra of the three mutants in SDS-d25 micelles were also acquired. The signals in the spectra of M20L-1 and M24L-1 were fairly dispersed, while the signals of M20LM24L-1 were poorly dispersed and severely broadened. Therefore, we only measured the two-dimensional NMR spectra of M20L-1 and M24L-1 in SDS-d25 micelles. However, the cross-peaks in the 2D NOESY spectra of the two mutants were broad and weak, which prevented us from unambiguous assignment for all signals. Although the NOESY spectra of M20L-1 and M24L-1 in SDS-d25 micelles were relatively poor, two sets of signals for some residues (such as Gln5, Thr6, Ile15, Phe18, Tyr26, and Asn27) were observed (Figure 10). Either the coexistence of parallel and antiparallel structure of the aggregates in the micelles or an unsymmetric aggregation of the peptide molecules in a dimer or trimer may result in a spreading resonance and reducing signal intensity. In contrast, TMD2-1 formed a predominant and symmetric trimer in the micelles, which allowed us to obtain a NMR spectrum with a relatively high quality. The larger steric effect of Leu than Met may cause the changes in the aggregation behavior of the mutants. Interactions of the M-to-L Mutants in SDS Micelles to Ag(I). The interactions of the mutants with Ag(I) were also investigated using CD, NMR and ITC techniques. As shown in Figure 8, the presence of Ag(I) had little effect on the CD spectra of the mutants, indicating that the helical structures of the mutants are less affected by the presence of Ag(I). However, in the NMR spectra of the mutants titrated by Ag(I), dramatic downfield shifts of the Hγ and Hε in Met side-chains (in the resonance region of 2.0−3.0 ppm) were observed at the molar ratios of Ag(I)/peptide of 2.5−3.0 and 2.0−3.0 for M20L-1 and M24L-1, respectively (Figure S2 and S3). This suggests that the unsubstituted Met in the mutants is still involved in the coordination with Ag(I). Other residues that displayed the resonance changes in the presence of Ag(I) were difficult to be identified for M20L-1 due to broadening and weakening of the resonances. For M24L-1, evident downfield shifts of the HN protons of His9, Gln12, Val13, Tyr17, Leu19, Leu21, and Ile22, the side-chain protons of Leu8, the Hε protons on the aromatic ring of Tyr17 and the acylamide protons in the side-chain of Asn27 were observed in the presence of 4.0 equiv of Ag(I). The changes in the chemical shifts of these residues may be associated partly with direct Ag(I) binding by some of them and partly with the regulation in the intermolecular interactions induced by the binding. For the double M-to-L substitute, the one-dimensional 1H NMR spectra looked like similar in the entire resonance region in the absence and presence of Ag(I). The details in the resonance differences were not identified because of severe broadening of the resonance lines.

Figure 8. CD spectra of 20 μM peptides M20L (A), M24L (B), and M20LM24L (C) in 10 mM SDS micelles at pH 6 in the absence and presence of 50 μM AgNO3.

spectra of the mutants (Figure 8). All the CD results indicate that the M-to-L conversions mainly affect the interactions between peptide molecules rather than the folding structure of a peptide molecule. The PFG-NMR experiments showed that all the diffusion coefficients of the SDS-d25 micelles incorporated with M20L-1, M24L-1 and M20LM24L-1 were 8.8 × 10−12 m2 s−1, a value very close to that of TMD2-2/SDS-d25, but distinctly larger than that of TMD2-1/SDS-d25 (Figure 2). The SDS-PAGE results showed that M20L-1 and M24L-1 most likely aggregate as a dimer or a mixture of dimer and monomer in the micelles, while M20LM24L-1 likely aggregates as a trimer (Figure 9). The fact that more aggregated M20LM24L-1 has a diffusion coefficient equal to that of less aggregated M20L-1 and M24L-1 suggests that M20LM24L-1 may be packed more tightly in SDS-d25 micelles by certain aggregate structures with intermolecular interactions stronger than M20L-1 and M24L-1. 8308


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Figure 10. Hα−HN region of the NOESY spectra of 1 mM M20L-1 (A) and M24L-1 (B) in 120 mM SDS-d25 micelles at pH 6 recorded at 25 °C. The threshold levels of the spectra were set to the values at which the signal/noise ratios were good enough. Some cross-peaks with intensities lower than the thresholds were not displayed in the partial spectra. The two sets of cross-peaks including the cross-peaks from the acylamino protons in the side chains of Q5 and N27 (shown in the inserted figures) were also assigned.

TMD2 was confirmed to locate at the inner of the hCtr1 trimer and line the pore of the protein.28 Therefore, the study on the structure and oligomerization of isolated TMD2 peptide in SDS micelles may provide an insight into the pore formation of hCtr1 trimer. Interestingly, the trimer formation of hCtr1− TMD2 peptide in SDS micelles was observed by proper HFIP pretreatment to the synthesized peptide sample in the present study. The oligomerization of isolated hCtr1−TMD2 as a trimer was also observed in the previous study of three hCtr1− TMD peptides in 40%HFIP aqueous solution.47 This indicates that the intrinsic coiled-coil interactions between TMD2 segments may play an important role for the formation of hCtr1 trimer in cellular membranes. In particular, the reconstructed trimeric structure by the isolated TMD2 in SDS micelles displayed a capacity of binding Ag(I) with a stoichiometry of 3:2 of peptide:Ag(I). The residues Met24 and Met20 (corresponding to Met154 and Met150 in hCtr1) were evidenced to participate directly in the binding, likely by two Met triads in the TMD2 trimer. This result is in agreement with the binding mode predicted for hCtr1 trimer well. This suggests that the oligomeric structure of the trimer reconstructed by the isolated TMD2 in SDS micelles may be comparable to some extent with that of TMD2 in hCtr1 trimer. Previous study demonstrated that His139 is a crucial residue in the copper transport. Conversion of His139 to Arg decreases the affinity for Cu(I) and increases the transport rate dramatically due to introducing repulsive interaction between pore and cation.44 Tyr147 in hCtr1−TMD2 was also shown to play a role in hCtr1 activity. The substitution of Tyr147 by Phe or Ala results in a decrease in the rate of copper transport, although the effect is smaller than that of Met substitutions.44 In addition, Gln142 in hCtr1−TMD2 is believed to be an attractive site for transient binding of Cu(I).29 Our results in this study showed that the polar residues Asn27, Tyr17 and Gln12 (Asn157, Tyr147 and Gln142 in hCtr1) may provide a subsidiary coordination in the binding of two methionine triads to Ag(I). In contrast, the coordination of the polar residues in the N-terminal part of the peptide, such as His9, Gln5 and His2 (corresponding to His139, Gln135 and His132 in hCtr1) were not observed based on the NMR experiments. One possible explanation is that the binding affinity of HX2QX3H motif for

The ITC isotherms from the titration of Ag(I) in SDS micellar solution of the M-to-L mutants were measured (Figure 11) and the relevant data were obtained (Table 2). Contrary to the observation in the ITC experiments of the wild-type TMD2 peptide, the isotherms of the M-to-L mutants treated by HFIP once and twice were very similar. No significant differences were obtained in their ITC data. For each of the three mutants, the peptides treated with HFIP once and twice displayed similar Ka values. The Ka values were smaller than that of TMD2-1 by about 1 order of magnitude, but very close to that of TMD2-2 (Table 2). Notably, the TMD2 peptide variant with double M-to-L conversions was still able to bind Ag(I) with a binding affinity as large as the single M-to-L converted peptides. This implies that some polar residues in the TMD2 peptide may be capable to bind Ag(I) if these residues are exposed to Ag(I). The thermodynamic data showed that the binding processes were driven by the enthalpy changes in all the cases of the peptides containing Met residues, while the binding process was driven by an entropy change in the case of the peptide without Met residue. Thus, we can assume that the participation of Met in Ag(I) binding is unfavorable for the entropy, but favorable for the enthalpy. In contrast, M20LM24L provides polar residues to bind Ag(I), by which more water molecules are released from the hydrated polar groups, leading to the increase in the entropy. Biological Implications. Previous studies have demonstrated that hCtr1 transports copper in cells by trimerization and the binding of Cu(I) by two triad rings of Met in TMD2 is important for the function.29,46 The substitutions of two Met residues in the motif MXXXM in hCtr1-TMD2 by amino acids that are incapable of binding Cu either inactivated copper uptake almost completely or dramatically reduced copper uptake.41,44,58 Ctr1 has also been confirmed to be able to transport Ag(I) and contribute to the accumulation of Ag(I) in cells.16 The presence of excess Ag inhibits Cu uptake by Ctr1 at the plasma membrane.17 The replacements of both Met150 and Met154 in hCtr1-TMD2 by Leu fail to facilitate cellular accumulation of either Cu or Ag.16 These studies suggested that Ctr1 may transport Cu(I) and Ag(I) by a similar mechanism. 8309


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the tilting angles of helical axes and the axial rotations of helices as well as the conformational adjustment, the Met residues in the trimer could be separated apart and Ag(I) ions could be released to the His-Cys189-His sink at the wider dipolar intracellular exit through an intermediary process likely involving the transient binding of certain polar residues in the other half part of the helices, e.g., Gln142 and His139, to the ion. The substitution of either one of two Met residues in the TMD2 peptide by Leu resulted in a decrease in the interactions between peptide molecules and a dissociation of the peptide oligomer to a lower aggregate state, suggesting that both Met residues may be important for the formation of the peptide trimer. The aggregate states of these mutants, whether they were treated by HFIP once or twice, were more similar to that of TMD2-2 in SDS micelles rather than to that of TMD2-1. In addition, the mutants and TMD2-2 displayed similar binding affinities (smaller than that of TMD2-1 by 1 order of magnitude) and a common property of binding Ag(I) with an n value larger than 1. This indicates that the specific oligomeric structure is very significant for a higher affinity binding of the peptide with Ag(I). The interaction of M20LM24L with Ag(I) was observed, which may be associated with certain polar residues in the peptide, although they were not identified in this study due to broad NMR lines. Previous study demonstrated that the double mutation of M150I/M154I reduces the copper transport to about 30% of the rate of the wild-type protein. The authors concluded that the pair of Met in TMD2 of hCtr1 is important but not essential for the copper transport.44 Our results suggest that certain polar residues in TMD2 may contribute to a transient association with Ag(I) (or Cu(I)) and the association could occur with a low affinity in the absence of Met coordination.

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CONCLUSIONS

By the study of the hCtr1−TMD2 reconstructed in SDS micelles, it is revealed that the second transmembrane helix alone has an intrinsic propensity to form a trimer by the coiledcoil interaction and the trimer can bind Ag(I) by a chemical stoichiometry of 3:2 of peptide:Ag(I). The silver binding has little effect on the helical structure of TMD2, instead, affects the interactions between the helical bundles by changing the topologies of the molecules. Our results confirm that Met150 and Met154 participate in the binding to Ag(I) and play a predominant role not only in the high affinity binding to Ag(I), but likely also in the trimer formation of the TMD2 peptide. Either one-site or two-site substitution of Met by Leu changes the aggregate state of the peptide and decreases the binding affinity for Ag(I). Certain polar residues in the C-terminal part of the TMD2 peptide may also provide ligands in the binding. Our results indicate that a weak association between hCtr1− TMD2 and Ag(I) could occur even in the absence of Met residues, likely due to the coordination of some polar residues. Our results suggest that an intrinsic interaction of the second transmembrane domain itself may be significant for the formation of hCtr1 pore in cellular membranes, and the trimeric structure of the TMD2 may be crucial for a higheraffinity binding of MXXXM motif to Ag(I).

Figure 11. ITC profiles of 50 μM peptides M20L-1 (A), M20L-2 (B), M24L-1 (C), M24L-2 (D), M20LM24L-1 (E), and M20LM24L-2 (F) in 10 mM SDS micelles titrated with AgNO3 at pH 6.

Ag(I) is much smaller than the affinity of MX3M motif for Ag(I). Alternatively, the specific aggregate topology of the peptide molecules disenables the binding of the polar residues in the N-terminal part of TMD2 to Ag(I). Both cases imply that the binding of the TMD2 trimer to Ag(I) by Met residues at the extracellular side could not happen simultaneously with the binding by the polar residues at the intracellular side. If this is true in the ion transport process of hCtr1 protein, a change in the topologies of protein molecules and even a change in the protein structure are likely needed for the movement of the metal ions from the extracellular side to the intracellular side, by certain unknown driving force. Along with the changes in 8310


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ASSOCIATED CONTENT

S Supporting Information *

Chemical shifts and structural calculation data of TMD2-1 in the absence and presence of AgNO3 and 1H NMR spectra of three peptides in SDS-d25 micelles with the titration of AgNO3. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.5b03744.

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AUTHOR INFORMATION

Corresponding Author

*(F.L.) Fax: +86-431-85193421. Telephone: +86-43185168548. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the NSFC (20934002). REFERENCES

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Self-Assembly of the Second Transmembrane Domain of hCtr1 in

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