Study on structure and assembly of the third transmembrane domain of Slc11a1.

The structure and self-assembly of the peptide corresponding to the third transmembrane domain (TMD3) of Slc11a1 and its E139A mutant are studied in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) aqueous solution by NMR and CD experiments. Slc11a1 is an integral membrane protein with 12 putative TMDs and functions as a pH-coupled divalent metal cation transporter. Glu139 of Slc11a1 is highly conserved within predicted TMD3 of the Slc11 protein family and function-associated. Here, we provide the first direct experimental evidence for the structural features of two 24-residue peptides corresponding to TMD3 of Slc11a1 and its E139A mutant in 60% HFIP-d(2) aqueous solution using CD and NMR spectroscopies. Our study shows that the membrane-spanning peptide folds as a typical amphipathic alpha-helix structure from Ile5 to Met20 with hydrophilic residues Glu12 (Glu139 in Slc11a1) and Asp19 lying on the same side of the helix. The substitution of Glu139 by an alanine residue has little effect on the structure of the peptide, but increases hydrophobicity and facilitates self-assembly of the peptide. Although the wildtype peptide is monomeric in HFIP aqueous solution, the E139A mutant forms a dimer. The increase in hydrophobicity of the membrane-spanning peptide and/or change in the interactions between transmembrane segments induced by E139A mutation may affect the metal ion transport of the protein.