Báo cáo khoa học: An NMR study of the interaction between the human copper(I) chaperone and the second and fifth metal-binding domains of the Menkes protein

An NMR Study of the Interaction Between the Human Copper(I) Chaperone and the Second and Fifth Metal-Binding Domains of the Menkes Protein

Tác giả: Lucia Banci, Ivano Bertini, Simone Ciofi-Baffoni, Christos T Chasapis, Nick Hadjiliadis, and Antonio Rosato

Lĩnh vực: Hóa sinh, Sinh học phân tử, Vật lý sinh học

Nội dung tài liệu: Nghiên cứu này sử dụng phương pháp cộng hưởng từ hạt nhân (NMR) dị nhân để khảo sát sự tương tác giữa protein chaperone đồng (I) ở người (HAH1) và hai miền gắn kim loại của protein Menkes (ATP7A), cụ thể là miền thứ hai (MNK2) và miền thứ năm (MNK5). Nghiên cứu đã thực hiện các phép đo chuẩn độ trong dung dịch với sự hiện diện của đồng (I). Kết quả cho thấy các đặc tính chuyển giao đồng (I) của MNK2 và MNK5 tương tự nhau và khác biệt đáng kể so với hệ thống tương đồng ở nấm men. Không có sự hình thành phức hợp ổn định giữa các miền MNK và HAH1. Phản ứng chuyển giao đồng (I) diễn ra chậm trên thang thời gian dịch chuyển hóa học NMR, với trạng thái cân bằng nghiêng về phía hình thành phức hợp đồng (I)-MNK2/MNK5. Nghiên cứu cũng báo cáo cấu trúc dung dịch của cả dạng apo và dạng có đồng (I) của MNK5. Các kết quả được thảo luận trong sự so sánh với dữ liệu có sẵn trong tài liệu cho tương tác giữa HAH1 và các đối tác của nó từ các kỹ thuật phổ khác.

Mục lục chi tiết:

• An NMR study of the interaction between the human copper(I) chaperone and the second and fifth metal-binding domains of the Menkes protein
• Keywords: copper(I); metal homeostasis; metallochaperone; protein-protein interaction
• Correspondence
• (Received 30 September 2004, revised 30 November 2004, accepted 13 December 2004)
• doi:10.1111/j.1742-4658.2004.04526.x
• The interaction between the human copper(I) chaperone, HAH1, and one of its two physiological partners, the Menkes disease protein (ATP7A), was investigated in solution using heteronuclear NMR. The study was carried out through titrations involving HAH1 and either the second or the fifth soluble domains of ATP7A (MNK2 and MNK5, respectively), in the presence of copper(I). The copper-transfer properties of MNK2 and MNK5 are similar, and differ significantly from those previously observed for the yeast homologous system. In particular, no stable adduct is formed between either of the MNK domains and HAH1. The copper(I) transfer reaction is slow on the time scale of the NMR chemical shift, and the equilibrium is significantly shifted towards the formation of copper(I)-MNK2/MNK5. The solution structures of both apo- and copper(I)-MNK5, which were not available, are also reported. The results are discussed in comparison with the data available in the literature for the interaction between HAH1 and its partners from other spectroscopic techniques.
• Copper, an essential trace metal, is utilized as a cofactor in a variety of redox and hydrolytic proteins, which, in eukaryotes, are found in various cellular locations [1]. However, the amount of copper is presumably strictly controlled and a complex machinery of proteins that bind the metal ion strictly controls the uptake, transport, sequestration and efflux of copper in vivo [2-4]. In particular, so-called metallochaperones deliver copper to specific intracellular targets, acting like enzymes to lower the activation barrier for copper transfer to their specific partners [5]. A fast kinetics of metal transfer may circumvent the significant thermodynamic overcapacity for copper chelation of cytoplasm components [6].
• One of the pathways of copper transfer present in humans involves HAH1 (also known as Atoxl), a small soluble metallochaperone [7,8], which is capable of delivering copper(I) both to the Menkes and the Wilson disease proteins (ATP7A and ATP7B, respectively; EC 3.6.3.4) [2-4]. The latter two proteins are membrane-bound P-type ATPases that translocate copper in the trans-Golgi network or across the plasma membrane [2-4], depending on environmental conditions [9]. In fact, both proteins experience copper-regulated trafficking between the Golgi and plasma membranes [9]. ATP7A and ATP7B have a long N-terminal cytosolic tail containing six putative metal-binding domains. Homologues of HAH1 and
• Abbreviations
• HSQC, heteronuclear single quantum coherence; MNK2, second metal binding domain of the human Menkes protein (ATP7A); MNK5, fifth metal binding domain of the human Menkes protein (ATP7A); RMSD, root mean square deviation.
• FEBS Journal 272 (2005) 865-871 © 2005 FEBS
• Interaction between HAH1 and ATP7A
• L. Banci et al.
• ATP7A/ATP7B are found in a large number of prokaryotic and eukaryotic organisms. The number of metal-binding domains in ATP7A/ATP7B homologues is variable, ranging from one to six, with proteins from higher eukaryotic organisms, e.g. mammals, having a higher number of such domains than prokaryotic (typically one or two) or yeast (two) homologues [10,11]. The reasons why higher organisms have as many as six metal-binding domains are still unclear. Available studies on ATP7A or ATP7B trying to address this matter indicate some functional differentiation between the first four (counting from the N-terminus) and the last two domains, and suggest that the last two domains are sufficient for function [12-14]. In addition, the mechanism of copper(I) transfer from HAH1 to either human ATPase is not completely elucidated. In this respect, it is noteworthy that homology modelling of the ATP7A metal-binding domains shows significant variations among the various domains in the electrostatic surface implicated in partner recognition, potentially making it possible for them to interact with one another [11].
• At present, high-resolution data mapping the regions of interaction between an HAH1 homologue and a soluble metal-binding domain from an ATPase are available only for the yeast [15] and the Bacillus subtilis [16] systems. The data obtained on the yeast proteins have been used to determine a three-dimensional structure for the protein adduct [17]. Even though the sequence similarity between yeast Atx1 and HAH1, as well as between the domains of yeast Ccc2 and human ATP7A/ATP7B, is remarkable, there are several well-documented structural differences that warrant direct investigation of the human proteins. In particular, human HAH1 has been shown to bind copper(I) in a linear bidentate fashion [18,19], whereas in Atxl the copper(I) ion is tricoordinate [20], with two ligands provided by the protein and a third by a reductant molecule recruited from the solution. Also the extent of structural variation upon copper(I) binding observed in Atx1 is different and significantly larger than for HAH1 [19]. The electrostatic potential at the surface of Atx1 and HAH1 is quite similar, but that of the metal binding domains of Ccc2 is somewhat different from ATP7A/ATP7B [11]. In addition, although the two metal-binding domains of Ccc2 are very similar as far as electrostatic features are concerned, the six domains of ATP7A/ATP7B differ widely in this same respect, even showing charge reversals. There seems also to be some differentiation among the ATP7A domains with respect to the structural and dynamic effects of copper(I) binding [21].
• In this study we investigated using high-resolution NMR the interaction between HAH1 and two different soluble domains of ATP7A: the second (MNK2 hereafter) and the fifth (MNK5 hereafter). The solution structure of both the apo- and copper(I)-form of MNK2 was already available [21]. No NMR assignment or structural data were instead available for MNK5, which has been expressed in Escherichia coli, and structurally characterized by NMR in this study. Particular interest in the study of the interaction between HAH1 and MNK2 is due to the recent proposition that the second soluble domain of ATP7B, which has a pl quite close to that of MNK2, is the first entry point for delivery of copper(I) ions by HAH1 to the ATPase [22].
• Results
• NMR spectra assignment and structural calculations
• Backbone assignments for MNK5 were obtained using standard strategies based on triple resonance experiments [23]. In 15N-heteronuclear single quantum coherence (HSQC) spectra the resonances of the backbone amide moieties of residues 13-17 were not detectable nor were those of the residues in the C-terminal tag. As in the case of MNK2, where only two residues escaped detection [21], the lack of signals from residues in the metal-binding loop is likely to originate from conformational exchange processes. Variations in the chemical shifts between apo- and copper(I)-MNK5 are observed for residues close (in sequence) to the binding loop, as reported previously for similar systems [21,24,25], and, to a small extent, for residue 65. NMR assignments have been deposited in the BMRB.
• One thousand two hundred and twenty-seven and 1121 meaningful upper distance limits were used for structure calculations of apo-MNK5 and copper(I)-MNK5, respectively. In addition, 37 θ and 37 ψ torsion angles were constrained in each protein form. The structures obtained and the constraints used for calculations have been deposited in the PDB (codes 1Y3K and 1Y3J). The final (after REM refinement) apo-MNK5 and copper(I)–MNK5 families have an average total target function of ≈ 0.30 Ų (CYANA units), and an average backbone root mean square deviation (RMSD) values (over residues 2–73) of ≈ 0.70 Å; the all heavy atoms RMSD value instead was instead ≈ 1.20 Å.
• Figure 1 shows a comparison of the structures of apo-MNK5 and copper(I)–MNK5, highlighting the metal site structure in the latter. Both structures adopt
• the ferredoxin-like βαββαβ fold. The RMSD between the backbone atoms for the mean structures of the two families of conformers, excluding the metal-binding loop region and the poorly defined C-terminal tail is ≈ 1.1 Å.
• Interaction between MNK2 and HAH1
• To investigate the interaction of MNK2 with HAH1, we titrated 15N-enriched copper(I)–MNK2 with unlabelled apo-HAH1, and followed the process via 1H-15N HSQC spectra. No variation in the chemical shifts of the amide signals in copper(I)-MNK2 could be observed at any stage of the titration. Instead, the intensities of signals decreased with increasing HAH1 concentration. Concomitantly, signals corresponding to apo-MNK2 appeared and increased in intensities along the titration (Fig. 2). No additional signals from a possible (transiently populated) intermediate could be detected at any point of the titration.
• The above data thus indicate that an adduct between MNK2 and HAH1 does not form at detectable concentration, even if an interaction between the two proteins does occur, resulting in copper(I) transfer. The latter process is slow on the chemical shift time scale, setting an upper limit for the equilibration rate of ≈ 102-103 s⁻¹ (determined by the smallest chemical shift difference between apo-MNK2 and copper(I)-MNK2 that can be detected, i.e. ≈ 0.1 p.p.m). The profiles of signal intensity as a function of the MNK2/HAH1 molar ratio can be fitted with an equilibrium constant for the transfer of copper(I) from HAH1 to MNK2 between 5.0 and 10 (Fig. 3). The relatively high spread of the data in Fig. 3 is due to the fact that during the titration some broadening of the signals occurs, to a different extent at different HAH1/MNK2 ratios. This contributes to scattering the values of the signal integrals.
• Interaction between MNK5 and HAH1
• The interaction of MNK5 and HAH1 was studied by titrating 15N-enriched apo–MNK5 into 15N-enriched copper(I)-HAH1. As observed for MNK2, there is no detectable formation of a protein/protein adduct, and the copper(I) transfer equilibrium is slow on the chemical shift time scales. Already at the first addition of apo-MNK5 (MNK5/HAH1 ratio ≈ 1 : 5), signals due to copper(I)-MNK5 appeared, with an intensity significantly higher than those of apo-MNK5. Only after an excess of apo-MNK5 with respect to copper(I)-HAH1 is reached, was a steady increase of the intensities of apo-MNK5 signals observed, although the signals of copper(I)–MNK5 did not increase significantly. These data are consistent with the copper(I) transfer process favouring the formation of copper(I)-MNK5. The titration data can be fit to an equilibrium constant similar to that observed in the case of HAH1. In parallel, the intensity of the signals of copper(I)–HAH1 in the HSQC spectra decreased steadily along all the titration, and apo(I)-HAH1 was formed.
• Discussion
• As expected, in solution MNK5 adopts the classical βαββαβ ferredoxin fold regardless of the presence of the metal ion. As observed for other proteins of this class [26,27], in copper(I)-MNK5 the copper ion is close to the protein surface and solvent exposed. Chemical shift variations observed between apo-MNK5 and copper(I)-MNK5 indicate that perturbations due to copper(I) binding affect mainly the Cys-containing loop (loop 1). Indeed, the comparison of the two structures highlights that this is the region where structural rearrangement occurs upon metal binding, while the remainder of the polypeptide chain does not experience significant conformational changes (Fig. 1). For the two copper(I)-binding cysteines, it is difficult to appreciate the extent of conformational rearrangement as their conformation in the two families is not very precisely defined. Overall, the behaviour of MNK5 upon copper(I) binding is similar to what observed for MNK2 [21].
• The behaviour observed for the interaction of HAH1 with MNK2 and MNK5 is somewhat different from that observed for the yeast homologues [15], and from that observed for Bacillus subtilis CopZ and CopA [16]. In the latter two systems an adduct is formed in fast (with respect to the time scale of NMR chemical shifts) equilibrium with the two separate proteins. This was evident from the fact that in a mixture of two partners in the presence of only one equivalent
• Fig. 1. Comparison of the solution structures of apo-MNK5 (left) and copper(I)-MNK5 (right). The side chains of Cys14 and Cys17 are shown as sticks; the copper(I) ion is shown as a sphere. This figure was prepared with MOLMOL [31].
• the ferredoxin-like βαββαβ fold. The RMSD between the backbone atoms for the mean structures of the two families of conformers, excluding the metal-binding loop region and the poorly defined C-terminal tail is ≈ 1.1 Å.
• Interaction between MNK2 and HAH1
• To investigate the interaction of MNK2 with HAH1, we titrated 15N-enriched copper(I)–MNK2 with unlabelled apo-HAH1, and followed the process via 1H-15N HSQC spectra. No variation in the chemical shifts of the amide signals in copper(I)-MNK2 could be observed at any stage of the titration. Instead, the intensities of signals decreased with increasing HAH1 concentration. Concomitantly, signals corresponding to apo-MNK2 appeared and increased in intensities along the titration (Fig. 2). No additional signals from a possible (transiently populated) intermediate could be detected at any point of the titration.
• The above data thus indicate that an adduct between MNK2 and HAH1 does not form at detectable concentration, even if an interaction between the two proteins does occur, resulting in copper(I) transfer. The latter process is slow on the chemical shift time scale, setting an upper limit for the equilibration rate of ≈ 102-103 s⁻¹ (determined by the smallest chemical shift difference between apo-MNK2 and copper(I)-MNK2 that can be detected, i.e. ≈ 0.1 p.p.m). The profiles of signal intensity as a function of the MNK2/HAH1 molar ratio can be fitted with an equilibrium constant for the transfer of copper(I) from HAH1 to MNK2 between 5.0 and 10 (Fig. 3). The relatively high spread of the data in Fig. 3 is due to the fact that during the titration some broadening of the signals occurs, to a different extent at different HAH1/MNK2 ratios. This contributes to scattering the values of the signal integrals.
• Interaction between MNK5 and HAH1
• The interaction of MNK5 and HAH1 was studied by titrating 15N-enriched apo–MNK5 into 15N-enriched copper(I)-HAH1. As observed for MNK2, there is no detectable formation of a protein/protein adduct, and the copper(I) transfer equilibrium is slow on the chemical shift time scales. Already at the first addition of apo-MNK5