Engineered tryptophan in the adenine-binding pocket of catalytic subunit A of A-ATP synthase demonstrates the importance of aromatic residues in adenine binding, forming a tool for steady-state and time-resolved fluorescence spectroscopy.

Abstract

A reporter tryptophan residue was individually introduced by site-directed mutagenesis into the adenine-binding pocket of the catalytic subunit A (F427W and F508W mutants) of the motor protein A(1)A(O) ATP synthase from Pyrococcus horikoshii OT3. The crystal structures of the F427W and F508W mutant proteins were determined to 2.5 and 2.6 Å resolution, respectively. The tryptophan substitution caused the fluorescence signal to increase by 28% (F427W) and 33% (F508W), with a shift from 333 nm in the wild-type protein to 339 nm in the mutant proteins. Tryptophan emission spectra showed binding of Mg-ATP to the F427W mutant with a K(d) of 8.5 µM. In contrast, no significant binding of nucleotide could be observed for the F508W mutant. A closer inspection of the crystal structure of the F427W mutant showed that the adenine-binding pocket had widened by 0.7 Å (to 8.70 Å) in comparison to the wild-type subunit A (8.07 Å) owing to tryptophan substitution, as a result of which it was able to bind ATP. In contrast, the adenine-binding pocket had narrowed in the F508W mutant. The two mutants presented demonstrate that the exact volume of the adenine ribose binding pocket is essential for nucleotide binding and even minor narrowing makes it unfit for nucleotide binding. In addition, structural and fluorescence data confirmed the viability of the fluorescently active mutant F427W, which had ideal tryptophan spectra for future structure-based time-resolved dynamic measurements of the catalytic subunit A of the ATP-synthesizing enzyme A-ATP synthase.