Acyl protein thioesterases (APT) catalyze the depalmitoylation of membrane bound proteins, including the key cell proliferation and cancer signaling protein Ras. To catalyze protein depalmitoylation, APTs must bind to and deform the cell membrane and then deacylate the thioester linkage from the palmitoylated protein. Recently, a β-tongue region in a flexible, hydrophobic loop was identified in human APT2 that simultaneously binds and deforms the membrane to allow substrate catalysis. In this research, we investigated the structural changes in human APTs upon β-tongue binding to various substrates and membranes to isolate the importance of the three residues in the β-tongue region to APT2 function. Using intrinsic tryptophan fluorescence and isolating individual tryptophans on the APT2 surface, localized structural changes upon ligand and membrane binding were mapped onto the APT2 surface. Since tryptophan fluorescence is dependent on the local environment, shifts in APT2 structure upon ligand and membrane binding were correlated with decreased fluorescence. Substitutions were then made to the β-tongue residues and shifts in tryptophan fluorescence remeasured to determine the importance of the β-tongue residues to electrostatic attraction to the membrane, to membrane attachment, and to catalysis. Our results provide a view of how APTs perform their dual function of membrane binding and catalytic activity using the key membrane binding β-tongue.

Dissecting the interconnected roles of the β-tongue in APT function using intrinsic tryptophan fluorescence

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