Structural Study Of The Third And Sixth Transmembrane Domains Of Solute Carrier Family 11

The Slc11 family defines a family of membrane proteins highly conserved throughout evolution. Slc11a1 (also known as Nramp1) and Slc11a2 (also know as Nramp2, DMT1, DCT1) are two members of this family. Slc11a1 is expressed exclusively in late endosomal/lysosomal compartment of macrophages and polymorphonuclear leukocytes. It is crucial for normal functioning of cells. Defective Slc11a1 causes susceptibility to infection by several intracellular pathogens including Mycobacterium, Leishmania, and Salmonella and is implicated in a number of diseases known as autoimmune or inflammatory diseases. Slc11a2 is ubiquitously expressed. It shares 64% amino acid sequence identity and 78% similarity with Slc11a1 and has a broad substrate range including Fe2+, Zn2+, Co2+, Mn2+, Cu2+, Cd2+, Ni2+ and Pb2+, the driving force for the metal-ion transport is proton gradient. Slc11a2 has been postulated to play important roles in intestinal iron absorption, erythroid iron utilization, hepatic iron accumulation, placental iron transfer, and other processes. Bacterial Slc11 homologs MntH is a proton-dependent manganese transporter. It has demonstrated that the Slc11 family possesses a common topological TM organization, including 10-12 transmembrane domains (TMD), a glycosylated extracellular loop, and several phosphorylation sites. Research on the structures of Slc11 proteins is expected to be helpful to understand the functions of the proteins at a molecular level and may provide important clues for uncovering nosogenesis of intracellular pathogens infections and ion imbalance.In the transmembrane domains of Slc11 proteins, TMD3 and TMD6 were found to be important for specific aspects of transport. The mutation from a Glu to an Ala (E154A) within the putative transmembrane domain 3 of Slc11a2 caused complete loss of function. Similar results were also found in MntH, the conservative mutations of Glu102 (analogous to Glu154 in Slc11a2 and Glu139 in Slc11a1) resulted in a complete loss of transport activity. The conservative mutations of D109E, D109N, E112D, and E112Q in TMD3 of MntH showed partial loss of transport function. Two highly conserved histidine residues located in the sixth transmembrane domain (TMD6) of Slc11 homologs were also found to play an important role in proton-dependent metal ion transport of the proteins. Both smf1/smf2 complementation results in yeast and transport data in mammalian cells demonstrated that this histidine pair are mutation sensitive, with Ala substitution at either or both sites causing deficiency of function and shifting the pH required to achieve maximal transport to a more acidic value, suggesting that the two histidines in TMD6 may have a unique and crucial role in pH regulation of metal ion transport by Slc11 proteins. In this work, we investigated the structures, self-assembly and topologies of Slc11a1-TMD3 peptide and its E139A mutant, Slc11a2-TMD6 peptide and its H267A and H272A mutant in HFIP aqueous solution, TFE aqueous solution and SDS micelles by CD and NMR spectra.The study reveals that TMD3 of Slc11a1 adopts predominantly anα-helical structure in both 60% HFIP-d₂ aqueous solution and SDS micelles. The helix length of the peptide in SDS-d₂₅ micelles at acidic pH conditions is similar to that in HFIP-d₂ aqueous solution, while the helix has a shorter extension to the C-terminus at alkalescent pH valueThe peptide exists as a monomer in HFIP aqueous solution, but severely assembles in SDS micelles at acidic pH values. The association is destabilized when the pH of solution is close to neutral or higher or by addition a small amount of HFIP to the solution. The structure of the E139A mutant is very similar to that of the WT peptide in 60% HFIP-d2 aqueous solution. However, the E139A mutation makes the peptide more hydrophobic and more tendentious to aggregate than the wildtype peptide. The pH dependence of the aggregate in the WT peptide is largely eliminated by the mutation. The topologies of the two peptides in SDS micelles mixed with a little amount of HFIP are similar in general: both peptides are inserted in SDS micelles with most residues from the beginning of the N-termini locating in the interior of the micelles and C-termini close to the surface of the micelles. However, a small but significant difference in the topology of the peptides is observed in the results of the spin label experiments. The resonances of the C-terminal residues of the E139A mutant are less broadened by Mn2+ ions than those of the WT peptide in the SDS micelles mixed with HFIP, whereas the resonances of the N-terminal residues (Arg4-Leu6) and C-terminal residues (Val23 and Ile24) are more affected by 16-DSA. This indicates that the N- and C- termini of the E139A mutant are embedded more deeply than those of the WT peptide. Our study indicates that the ionizable Glu139 is a crucial residue for assembly and topology of the transmembrane domain 3 of Slc11a1 in membranes, the increase in hydrophobicity of the membrane-spanning peptide and/or change in the location and orientation of TMD3 induced by E139A mutation may affect the metal ion transport of the protein.In this study, we verify in the first time that TMD6 of Slc11a2 adopts an"α-helix-extended segment-α-helix"structure in SDS micelles, His267 locates near the central part of the extended segment, while the His272 is involved in theα-helical folding. The structure of the wildtype peptide is evidently changed by the mutations of H267A and H272A. The H267A mutant forms an ordered structure consisting of anα-helix from the C-terminus to the central part and continuous turns in the residual part. The H272A mutation mainly induces unfolding of the short helix in the N-terminal side, while the short helix in the C-terminal side and unordered conformation in the central part remain. The pH value has little effect on theα-helical contents of the three peptides. All the three peptides are embedded in SDS micelles, and the H267A mutant is inserted more deeply due to increasing hydrophobicity in the central part of the peptide. We obtain similar results in TFE aqueous solution, we observe more helical content induced by H267A mutation making the central part of the peptide more rigid, and less helical content by H272A mutation making peptide structure less rigid, as compared with the wildtype peptide. DOSY experiments demonstrated that each of the three peptides assembles as a trimer in 30% TFE-d2 aqueous solution. In trimers, each monomer may be structurally heterogeneous for the wildtype peptide, but homogeneous for the H267A and H272A mutants. The intermolecular interactions decrease according to the order H267A>WT>H272A, obtained by dilution experiments.The particular"α-helix-extended segment-α-helix"structure of TMD6 would have important implication for the functions of the protein. Compared toα-helix, the flexible central segment could create a space for accommodation of charged metal ions in a tightly packedα-helical environment. In addition, the protonation at acidic pH or deprotonation at neutral pH of histidine imidazole protons may lead to breaking or building of compensatory electrostatic interactions and/or disruption or formation of salt bridges between adjacent residues, inducing overall conformational changes in response to pH through the bending and twisting of the associated helical elements, controlling the active and inactive state of the channel. The structure and the strength of the intermolecular interactions are changed due to H267A and H272A mutation, these changes may affect the metal ion transport of the protein.The findings in this article may be meaningful for understanding the structure and function of the entire membrane protein.