Interactions Of Nramp2-exo-loop1 Related Peptides With Metal Ions

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 Fe²⁺, Mn²⁺, Zn²⁺, Co²⁺, Cd²⁺, Cu²⁺, Ni²⁺ and Pb²⁺ 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 Pb²⁺ ion. Firstly, we study the interactions of the wildtype peptide and its mutants with diamagnetic metal ions Cd²⁺, Zn²⁺, Ca²⁺, Ba²⁺and Pb²⁺. The addition of Pb²⁺ 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 Pb²⁺ 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 Pb²⁺ effects on the proton chemical shifts of entire peptide; the separate mutations of D1A, G3A, N4A and D1A/N4A only eliminate the effects of Pb²⁺ on the resonances of the N-terminal residues; The effect of Pb²⁺ on the proton resonances of Q10A mutant is similar to that of Pb²⁺ 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 Pb²⁺. The studies of the interactions of Q10D and D8A/Q10D mutants with Pb²⁺ 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 Pb²⁺, changing the binding mode. The dissociation constant of WT/Pb²⁺ 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 Pb²⁺ with coordination similar to that in water but weaker affinity, while the interaction of the peptide with Pb²⁺ in SDS micelles is rather weak. The the interactions of the peptide with Cd²⁺, Zn²⁺, Ca²⁺ and Ba²⁺ 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/Pb²⁺ 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 Mn²⁺, Cu²⁺, Co²⁺, Ni²⁺, Fe²⁺, Cr³⁺, Fe³⁺ 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 Co²⁺ 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 Co²⁺ 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 Co²⁺ ion are eliminated by any of the mutations of D1A, E6A and D8A. These results imply that Asp1, Glu6 and Asp8 bind to Co²⁺ 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 Co²⁺ 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 Co²⁺ ion and the interaction of D8A/Q10D with Co²⁺ is slightly stronger than that of D8A with the metal ion. The results obtained by the peptide/Mn²⁺ 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/Mn²⁺ complex. Among the three binding residues, an Ala substitution for any residues in Glu6 and Asp8 will lead to dissociation of peptide/Mn²⁺ complex. The substitution of Asp1 by Ala decreases the interaction of peptide with the metal ion. The residue Asn4 may bind to Mn²⁺ through backbone atoms, the N4A mutation has less effect on the coordination of peptide with Mn²⁺ than the mutaions of other three binding residues. Furthermore, we find that the residue Asp8 is irreplaceable in the binding to Mn²⁺ by comparing the interactions of D8A and D8A/Q10D mutants with Mn²⁺, indicative of a highly specific coordination of Asp8 to Mn²⁺. The four residues Asp1, Asn4, Glu6 and Asp8 are also involed in the association with Cu²⁺. 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 Cu²⁺. The coordinations of WT peptide with Fe²⁺ and Ni²⁺ are similar to the coordinations of the peptide with the paramagnetic divalent metal ions mentioned above. The interactions of WT peptide with Mn²⁺ and Cu²⁺ 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 Cr³⁺ and Fe³⁺, 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 Mn²⁺, Co²⁺, Ni²⁺, Fe²⁺, Cu²⁺ and Pb²⁺) 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.