Implications of Threonine Hydrogen Bonding in the Glycophorin A Transmembrane Helix Dimer

Abstract

The transmembrane helix of glycophorin A contains a seven-residue motif, LIxxGVxxGVxxT, that mediates protein dimerization. Threonine is the only polar amino acid in this motif with the potential to stabilize the dimer through hydrogen-bonding interactions. Polarized Fourier transform infrared spectroscopy is used to establish a robust protocol for incorporating glycophorin A transmembrane peptides into membrane bilayers. Analysis of the dichroic ratio of the 1655-cm −1 amide I vibration indicates that peptides reconstituted by detergent dialysis have a transmembrane orientation with a helix crossing angle of <35°. Solid-state nuclear magnetic resonance spectroscopy is used to establish high resolution structural restraints on the conformation and packing of Thr-87 in the dimer interface. Rotational resonance measurement of a 2.9-Å distance between the γ-methyl and backbone carbonyl carbons of Thr-87 is consistent with a gauche− conformation for the χ1 torsion angle. Rotational-echo double-resonance measurements demonstrate close packing (4.0 ± 0.2 Å) of the Thr-87 γ-methyl group with the backbone nitrogen of Ile-88 across the dimer interface. The short interhelical distance places the β-hydroxyl of Thr-87 within hydrogen-bonding range of the backbone carbonyl of Val-84 on the opposing helix. These results refine the structure of the glycophorin A dimer in membrane bilayers and highlight the complementary role of small and polar residues in the tight association of transmembrane helices in membrane proteins.