Domain Features of the Peripheral Stalk Subunit H of the Methanogenic A1AO ATP Synthase and the NMR Solution Structure of H1-47

Abstract

A series of truncated forms of subunit H were generated to establish the domain features of that protein. Circular dichroism analysis demonstrated that H is divided at least into a C-terminal coiled-coil domain within residues 54–104, and an N-terminal domain formed by adjacent α-helices. With a cysteine at the C-terminus of each of the truncated proteins (H1-47, H1-54, H1-59, H1-61, H1-67, H1-69, H1-71, H1-78, H1-80, H1-91, and H47-105), the residues involved in formation of the coiled-coil interface were determined. Proteins H1-54, H1-61, H1-69, and H1-80 showed strong cross-link formation, which was weaker in H1-47, H1-59, H1-71, and H1-91. A shift in disulfide formation between cysteins at positions 71 and 80 reflected an interruption in the periodicity of hydrophobic residues in the region 71AEKILEETEKE81. To understand how the N-terminal domain of H is formed, we determined for the first time, to our knowledge, the solution NMR structure of H1-47, which revealed an α-helix between residues 15–42 and a flexible N-terminal stretch. The α-helix includes a kink that would bring the two helices of the C-terminus into the coiled-coil arrangement. H1-47 revealed a strip of alanines involved in dimerization, which were tested by exchange to single cysteines in subunit H mutants.