Nramp2-exo-loop1相关肽与金属离子的相互作用
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摘要
天然抗性相关巨噬细胞蛋白2(Nramp2)是一个拥有重要生物活性功能的膜蛋白,广泛存在于人类、哺乳动物、细菌等生物体内,能够传输生命活动中所需要的二价金属阳离子。在Nramp2的十二个预测跨膜区中,第一跨膜区C端和与之相连的环区(exo-loop1)可能在该蛋白捕获金属离子方面起重要作用。我们采用核磁共振方法研究了Nramp2-exo-loop1片段及基于这个肽片段的系列突变体在水溶液和胶束溶液中与金属离子Pb~(2+)、Cd~(2+)、Zn~(2+)、Ca~(2+)、Ba~(2+)、Mn~(2+)、Cu~(2+)、Co~(2+)、Ni~(2+)、Fe~(2+)、Cr~(3+)、Fe~(3+)的相互作用,包括结合位点、金属离子的选择性、各配位残基对配合物稳定性的贡献、关键残基突变对相互作用的影响等,并采用分子动力学模拟方法计算了肽段/铅离子配合物的结构。结果发现Nramp2-exo-loop1区域能够选择性地结合二价金属阳离子(至少包括Mn~(2+)、Co~(2+)、Ni~(2+)、Fe~(2+)、Cu~(2+)、Pb~(2+)),而不与三价金属阳离子如Cr~(3+)和Fe~(3+)结合。顺磁性二价金属离子的NMR实验结果和肽段/铅离子配合物的分子动力学计算都表明,在与二价金属离子的结合中该肽段的残基Asp1、Glu6和Asp8的侧链羧基氧原子参与配位,残基Asn4通过骨架羰基氧原子也参与配位。其中Glu6对配合物的稳定性起关键的作用,E6A突变将导致配合物完全解离,Asp8和Asp1的作用次之,但仍很重要,两者突变成Ala后将导致配合物大部分解离。Asn4在配位中起辅助作用,对配合物的稳定性影响最小。由此可以推断,exo-loop1区域可能对Nramp2传输体识别和捕获二价金属阳离子起重要作用。我们的发现可能对揭示Nramp2整体膜蛋白的离子传输机理有重要的意义。
Natural resistance-associated macrophage protein 2 (Nramp2) is a vital membrane protein which contains important biological activities and transfers divalent metal ions in life activity of human, mammal and bacteria. In human body,Nramp2 is crucially important for the balance of metal ions. The overabundance or deficiency in body leads to sorts of diseases or even death, such as Parkinson's disease and Iron-deficiency anemia. It is of great importance to find out the mechanism of Nramp2 in the prevention and cure of these diseases. Nramp2 belongs to Nramp protein family. Presently, the knowledge of the metal-iron-transfer of Nramp is mainly from the electrophysiological researches of the Nramp2 and Smflp in oocyte, while there are few reports on the researches of the interactions of Nramp with metal ions and transport mechanism at the level of atom.
It has been previously demonstrated that Nramp2 is able to transport a variety of divalent metal cations including Fe~(2+), Mn~(2+), Zn~(2+), Co~(2+), Cd~(2+), Cu~(2+), Ni~(2+) and Pb~(2+) by a proton-coupled mechanism. Among the 12 predicted Nramp transmembrane domains, certain residues from the C-terminal end of the first transmembrane domain and its connected external loop (exo-loop1) might be essential for transport activity, responsible for capture of divalent metal cations and playing a crucial role in proton/metal ion coupling. Therefore, understanding the roles of these key residues of exo-loop1 in metal ion transport is very significant for uncovering the function of Nramp2.
In this paper, we study the interactions of this wildtype peptide and its various mutants with metal ions, including binding sites, dissociation constants, specificity of the peptide for metal ion and effects of key residues, in water and micelle environments (SDS and DPC) usisng NMR methods. On the basis of NMR results, we apply molecular dynamics simulation to calculate the structure of complex of the peptide with Pb~(2+) ion.
Firstly, we study the interactions of the wildtype peptide and its mutants with diamagnetic metal ions Cd~(2+), Zn~(2+), Ca~(2+), Ba~(2+)and Pb~(2+). The addition of Pb~(2+) in WT aqueous solution induces remarkable upfield or downfield shifts of the protons from the residues Asp1, Ile5, Glu6, Ser7 and Asp8, indicating that the WT peptide binds to Pb~(2+) by the residues in the N-terminal and central regions of the peptide, while the residues in the C-terminal part do not participate in coordination. The substitution of Ala for either Glu6 or Asp8 (E6A or D8A) leads to disappearance of Pb~(2+) effects on the proton chemical shifts of entire peptide; the separate mutations of D1A, G3A, N4A and D1A/N4A only eliminate the effects of Pb~(2+) on the resonances of the N-terminal residues; The effect of Pb~(2+) on the proton resonances of Q10A mutant is similar to that of Pb~(2+) on the proton resonances of WT peptide. The results from these mutants support the conclusion that the residues fron the N-terminal and central regions are involved in coordination with Pb~(2+). The studies of the interactions of Q10D and D8A/Q10D mutants with Pb~(2+) reveal that these mutaions mainly affect the coordination of the C-terminal residue Asp10. The Asp10 in the mutant Q10D is involved in binding to Pb~(2+), changing the binding mode. The dissociation constant of WT/Pb~(2+) in water is estimated to be ca. 10-4 M by titration experiment, indicating a weak binding interaction. The interaction of the wildtype peptide with the metal ion is dependent on pH value, weaker at pH 4 and stronger at pH 5.5 or above. In DPC micelles, the WT peptide also forms chelate to Pb~(2+) with coordination similar to that in water but weaker affinity, while the interaction of the peptide with Pb~(2+) in SDS micelles is rather weak. The the interactions of the peptide with Cd~(2+), Zn~(2+), Ca~(2+) and Ba~(2+) are unobservable in both water and SDS micelles. We can not find resonance shifting of protons of any residues even if larger amounts of metal ions are added. Based on the NMR data, we obtain the structure of WT/Pb~(2+) complex by molecular dynamics simulation and find that six carboxyl oxygen atoms from the side-chains of Asp1, Glu6 and Asp8 and one carbonyl oxygen atom from the backbone of Asn4 are involved in the binding.
Then, we analyze the interactions of the WT peptide and its various mutants with paramagnetic metal ions including Mn~(2+), Cu~(2+), Co~(2+), Ni~(2+), Fe~(2+), Cr~(3+), Fe~(3+) in water and micelle media, trying to obtaine the information of binding sites, contribution of each binding residue to stabilizing the chelate and specificity of the peptide to metal ion. The addition of Co~(2+) in WT aqueous solution induces downfield shifting of the proton resonances related to the residues Asn4, Glu6 and Asp8 and selective broadening even disappearing of these resonance signals. Although the N4A mutation reduces the effects of Co~(2+) on the resonance shifting and signal intensities of Glu6 and Asp8, the evident changes are still observed. However, selective broadening and shifting of certain residues induced by Co~(2+) ion are eliminated by any of the mutations of D1A, E6A and D8A. These results imply that Asp1, Glu6 and Asp8 bind to Co~(2+) ion by the carboxyl oxygen atoms of their side-chains, which can be dissociated by the substitution of Ala. Moreover, the three residues nearly play equally important role in coordination. The substitution of any of these residues by Ala will break the binding of entire peptide with Co~(2+) ion. The backbone atoms of Asn4 may be involved in the binding and subsidiary for stability of complex. In tht mutant D8A/Q10D with double site substitutions, the Asp10 in the C-terminal region can partially replace lost Asp8 to participate coordination with Co~(2+) ion and the interaction of D8A/Q10D with Co~(2+) is slightly stronger than that of D8A with the metal ion. The results obtained by the peptide/Mn~(2+) systems demonstrate that Asp1, Glu6 and Asp8 are also coordinated by carboxyl oxygen atoms of their side-chains and play important role for stability of peptide/Mn~(2+) complex. Among the three binding residues, an Ala substitution for any residues in Glu6 and Asp8 will lead to dissociation of peptide/Mn~(2+) complex. The substitution of Asp1 by Ala decreases the interaction of peptide with the metal ion. The residue Asn4 may bind to Mn~(2+) through backbone atoms, the N4A mutation has less effect on the coordination of peptide with Mn~(2+) than the mutaions of other three binding residues. Furthermore, we find that the residue Asp8 is irreplaceable in the binding to Mn~(2+) by comparing the interactions of D8A and D8A/Q10D mutants with Mn~(2+), indicative of a highly specific coordination of Asp8 to Mn~(2+). The four residues Asp1, Asn4, Glu6 and Asp8 are also involed in the association with Cu~(2+). Among them, the E6A mutation weakens the binding affinity, while the D1A, N4A and D8A mutaions have less influence on the binding affinity, suggesting that Glu6 is a key residue for stabilizing the binding to Cu~(2+). The coordinations of WT peptide with Fe~(2+) and Ni~(2+) are similar to the coordinations of the peptide with the paramagnetic divalent metal ions mentioned above. The interactions of WT peptide with Mn~(2+) and Cu~(2+) ions in SDS and DPC micelles are also similar to those obtained in water. It is noteworthy that WT peptide can not bind to trivalent metal ions such as Cr~(3+) and Fe~(3+), indicating that the peptide has a specific binding to the divalent metal cations.
Based on the results above, one can infer that exo-loop1 may play a important role for Nramp2 selectively capturing divalent metal cations (at least Mn~(2+), Co~(2+), Ni~(2+), Fe~(2+), Cu~(2+) and Pb~(2+)) before transport. The flexible region of Nramp2 at the surface of membrane may initially recognize divalent metal cations specifically, and then transfer them into channel by proton/metal ion coupling mechanism. Our finding may have a significant implication for uncovering the transport mechanism of integral Nramp proteins.
Natural resistance-associated macrophage protein 2 (Nramp2) is a vital membrane protein which contains important biological activities and transfers divalent metal ions in life activity of human, mammal and bacteria. In human body,Nramp2 is crucially important for the balance of metal ions. The overabundance or deficiency in body leads to sorts of diseases or even death, such as Parkinson's disease and Iron-deficiency anemia. It is of great importance to find out the mechanism of Nramp2 in the prevention and cure of these diseases. Nramp2 belongs to Nramp protein family. Presently, the knowledge of the metal-iron-transfer of Nramp is mainly from the electrophysiological researches of the Nramp2 and Smflp in oocyte, while there are few reports on the researches of the interactions of Nramp with metal ions and transport mechanism at the level of atom.
It has been previously demonstrated that Nramp2 is able to transport a variety of divalent metal cations including Fe~(2+), Mn~(2+), Zn~(2+), Co~(2+), Cd~(2+), Cu~(2+), Ni~(2+) and Pb~(2+) by a proton-coupled mechanism. Among the 12 predicted Nramp transmembrane domains, certain residues from the C-terminal end of the first transmembrane domain and its connected external loop (exo-loop1) might be essential for transport activity, responsible for capture of divalent metal cations and playing a crucial role in proton/metal ion coupling. Therefore, understanding the roles of these key residues of exo-loop1 in metal ion transport is very significant for uncovering the function of Nramp2.
In this paper, we study the interactions of this wildtype peptide and its various mutants with metal ions, including binding sites, dissociation constants, specificity of the peptide for metal ion and effects of key residues, in water and micelle environments (SDS and DPC) usisng NMR methods. On the basis of NMR results, we apply molecular dynamics simulation to calculate the structure of complex of the peptide with Pb~(2+) ion.
Firstly, we study the interactions of the wildtype peptide and its mutants with diamagnetic metal ions Cd~(2+), Zn~(2+), Ca~(2+), Ba~(2+)and Pb~(2+). The addition of Pb~(2+) in WT aqueous solution induces remarkable upfield or downfield shifts of the protons from the residues Asp1, Ile5, Glu6, Ser7 and Asp8, indicating that the WT peptide binds to Pb~(2+) by the residues in the N-terminal and central regions of the peptide, while the residues in the C-terminal part do not participate in coordination. The substitution of Ala for either Glu6 or Asp8 (E6A or D8A) leads to disappearance of Pb~(2+) effects on the proton chemical shifts of entire peptide; the separate mutations of D1A, G3A, N4A and D1A/N4A only eliminate the effects of Pb~(2+) on the resonances of the N-terminal residues; The effect of Pb~(2+) on the proton resonances of Q10A mutant is similar to that of Pb~(2+) on the proton resonances of WT peptide. The results from these mutants support the conclusion that the residues fron the N-terminal and central regions are involved in coordination with Pb~(2+). The studies of the interactions of Q10D and D8A/Q10D mutants with Pb~(2+) reveal that these mutaions mainly affect the coordination of the C-terminal residue Asp10. The Asp10 in the mutant Q10D is involved in binding to Pb~(2+), changing the binding mode. The dissociation constant of WT/Pb~(2+) in water is estimated to be ca. 10-4 M by titration experiment, indicating a weak binding interaction. The interaction of the wildtype peptide with the metal ion is dependent on pH value, weaker at pH 4 and stronger at pH 5.5 or above. In DPC micelles, the WT peptide also forms chelate to Pb~(2+) with coordination similar to that in water but weaker affinity, while the interaction of the peptide with Pb~(2+) in SDS micelles is rather weak. The the interactions of the peptide with Cd~(2+), Zn~(2+), Ca~(2+) and Ba~(2+) are unobservable in both water and SDS micelles. We can not find resonance shifting of protons of any residues even if larger amounts of metal ions are added. Based on the NMR data, we obtain the structure of WT/Pb~(2+) complex by molecular dynamics simulation and find that six carboxyl oxygen atoms from the side-chains of Asp1, Glu6 and Asp8 and one carbonyl oxygen atom from the backbone of Asn4 are involved in the binding.
Then, we analyze the interactions of the WT peptide and its various mutants with paramagnetic metal ions including Mn~(2+), Cu~(2+), Co~(2+), Ni~(2+), Fe~(2+), Cr~(3+), Fe~(3+) in water and micelle media, trying to obtaine the information of binding sites, contribution of each binding residue to stabilizing the chelate and specificity of the peptide to metal ion. The addition of Co~(2+) in WT aqueous solution induces downfield shifting of the proton resonances related to the residues Asn4, Glu6 and Asp8 and selective broadening even disappearing of these resonance signals. Although the N4A mutation reduces the effects of Co~(2+) on the resonance shifting and signal intensities of Glu6 and Asp8, the evident changes are still observed. However, selective broadening and shifting of certain residues induced by Co~(2+) ion are eliminated by any of the mutations of D1A, E6A and D8A. These results imply that Asp1, Glu6 and Asp8 bind to Co~(2+) ion by the carboxyl oxygen atoms of their side-chains, which can be dissociated by the substitution of Ala. Moreover, the three residues nearly play equally important role in coordination. The substitution of any of these residues by Ala will break the binding of entire peptide with Co~(2+) ion. The backbone atoms of Asn4 may be involved in the binding and subsidiary for stability of complex. In tht mutant D8A/Q10D with double site substitutions, the Asp10 in the C-terminal region can partially replace lost Asp8 to participate coordination with Co~(2+) ion and the interaction of D8A/Q10D with Co~(2+) is slightly stronger than that of D8A with the metal ion. The results obtained by the peptide/Mn~(2+) systems demonstrate that Asp1, Glu6 and Asp8 are also coordinated by carboxyl oxygen atoms of their side-chains and play important role for stability of peptide/Mn~(2+) complex. Among the three binding residues, an Ala substitution for any residues in Glu6 and Asp8 will lead to dissociation of peptide/Mn~(2+) complex. The substitution of Asp1 by Ala decreases the interaction of peptide with the metal ion. The residue Asn4 may bind to Mn~(2+) through backbone atoms, the N4A mutation has less effect on the coordination of peptide with Mn~(2+) than the mutaions of other three binding residues. Furthermore, we find that the residue Asp8 is irreplaceable in the binding to Mn~(2+) by comparing the interactions of D8A and D8A/Q10D mutants with Mn~(2+), indicative of a highly specific coordination of Asp8 to Mn~(2+). The four residues Asp1, Asn4, Glu6 and Asp8 are also involed in the association with Cu~(2+). Among them, the E6A mutation weakens the binding affinity, while the D1A, N4A and D8A mutaions have less influence on the binding affinity, suggesting that Glu6 is a key residue for stabilizing the binding to Cu~(2+). The coordinations of WT peptide with Fe~(2+) and Ni~(2+) are similar to the coordinations of the peptide with the paramagnetic divalent metal ions mentioned above. The interactions of WT peptide with Mn~(2+) and Cu~(2+) ions in SDS and DPC micelles are also similar to those obtained in water. It is noteworthy that WT peptide can not bind to trivalent metal ions such as Cr~(3+) and Fe~(3+), indicating that the peptide has a specific binding to the divalent metal cations.
Based on the results above, one can infer that exo-loop1 may play a important role for Nramp2 selectively capturing divalent metal cations (at least Mn~(2+), Co~(2+), Ni~(2+), Fe~(2+), Cu~(2+) and Pb~(2+)) before transport. The flexible region of Nramp2 at the surface of membrane may initially recognize divalent metal cations specifically, and then transfer them into channel by proton/metal ion coupling mechanism. Our finding may have a significant implication for uncovering the transport mechanism of integral Nramp proteins.
引文
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