Structures And Topologies Of Nramp1-TM4 Related Peptides In Membrane-mimetic Environments

Natural resistance-associated macrophage protein 1 (Nramp1) is an important integral membrane protein which is involved in defense against intracellular pathogens such as Leishmania donovan, Salmonella typhimurium and Mycobacterium bovis. As a proton-coupled divalent metal cation transporter, Nramp1 can transport essential metal ions for life including Fe2+ and Mn2+. Nramp1 belongs to Nramp family which is highly conserved from bacteria to human. For human, NRAMP1 is associated with the diseases such as tuberculosis, leprosy and rheumatoid arthritis, etc. Understanding the functional mechanism of Nramp1 may be helpful to develop new therapeutic methods for these diseases. A basic work to reveal the mechanism of Nramp1 is getting structural information of the protein. Nramp1 contains 12 putative transmembrane domains (TM). However, there has been no report on the tertiary structure and topology of Nramp1 up to now due to the intrinsic difficulty in study of membrane protein.Recently, model peptides corresponding to isolated transmembrane domains have proved to be very useful in providing qualitative structural information and in guiding complete structure determination for integral proteins. Disease-causing mutation in Nramp1 occurring at glycine 169 (G169D) located within TM4 suggests functional importance of TM4. In this thesis, we studied the structures, assemblies and topologies of the peptides, including the TM4 of the wild-type Nramp1 and its G169D mutant, and the effects of G169D mutation in various membrane-mimetic environments, such as sodium dodecyl sulfate (SDS), 2,2,2-trifluoroethanol (TFE) and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) aqueous solution, using NMR and CD spectroscopies.First, the three-dimentional structures of the wild-type peptides in model membranes were studied using CD, NMR and molecular dynamic simulation. The peptides adopt dominatedα-helical conformations in various membrane-mimetic environments. The C-terminus of theα-helical fragment in SDS micelles is regulated by pH both in length and orientation. There is a smaller extension ofα-helix to the C-terminus at alkalescent environment than at acid environments. Moreover, the folding of the C-terminus is bent towards to the orientation perpendicular to the helix backbone in SDS at pH 7.4. Second, the topology of the wild-type peptide was explored by paramagnetic probes, 16-DSA and Mn2+, and pH titration experiments. The results showed that the wild-type peptide is completely embedded in SDS micelles with the N- and C-terminus located at the inner surface of the micelles and the helical segment positioned in the hydrophobic core of SDS micelles. Third, the aggregation state of the wild-type peptides was measured based on DOSY experiments, CD spectra, and pH titration experiments. The wild-type peptides can self-associate in various mimetic-membranes and may form tetramer or pentamer in which the structure of monomer is similar with each other. In model membranes, the hydrophilic and polar residues of helices may locate in the inside of assembly for the easy entry of metal ions into the membrane, while the outside of assembly consists of the hydrophobic residues of helices in favor of stability of the peptide in membrane. The accessibility of Mn2+ ions to Asp14 located in the interior of SDS micelles was observed. The interaction of the residue with Mn2+ strengthens with increase of pH, but weakens with decrease of pH. The Hill coefficient larger than 1 indicates that the proton binding of Asp14 embedded in the center of micelles occurs in a positive cooperative manner.In order to further understand the function of TM4 in Nramp1 and the pathogenic mechanism of the G169D mutation, we also characterized the G169D peptides in model membranes using the same methods as those used in the study of the wild-type peptides and compared the results of the G169D peptides with those of the wild-type peptides in detail. In the same model membranes, the structures and topologies of the G169D peptides are similar to those of the wild-type peptides except for a small difference in the local conformation near mutation site. The diffusion coefficients of the G169D peptides are identical to those of the wild-type peptides, suggesting that the G169D peptides can also self-associate in model membranes and the aggregation number is the same as that of the wild-type peptides. Based on the results of dilution and pH titration experiments, it was found that the cooperative manner in proton binding is changed from the wild-type peptide to the G169D mutant, a negative cooperativity for the residue Asp14 of the G169D mutant vs. a positive cooperativity for Asp14 of the wild-type peptide, indicating that the assembly mode of the peptide is changed due to G169D mutation. In HFIP aqueous solution, the intermolecular interaction in assemblies was found in the C-terminal region of helical segment for both peptides, a little stronger for the wild-type peptide than for the G169D mutant, further supporting the conclusion that the G169D mutation varies the aggregation mode of the peptide. Though the G169D mutant in SDS micelles can interact with Mn2+, the interaction is reduced or partially prevented compared with the wild-type peptide. These results indicate that the positive cooperativity in proton binding for the wild-type peptide may facilitate the cotransport of metal ions and protons, while the opposite cooperative manner in proton binding for the G169D mutant may disfavor the cotransport of metal ions and protons. The difference in the assembly modes and cooperativities in proton binding for the wild-type and G169D peptides may lead to the difference in their behaviors of ion-transport. This study may be meaningful for understanding the relationship between structure and function of integral membrane protein Nramp1.