Acyl protein thioesterases catalyze the depalmitoylation of key signaling proteins attached to the plasma membrane. Recently, human APTs were shown to use a two-step catalytic mechanism involving initial membrane binding of an exposed flexible loop followed by substrate binding and cleavage. This two-step catalytic mechanism is likely conserved as a bacterial homologue of human APTs is also regulated by the large-scale movement of an essential dynamic loop upon membrane binding. However, the causes for the movement of this dynamic loop in APTs are unknown. Herein, we investigated factors that trigger loop movement in human and bacterial APTs. In particular, APTs were assayed with inhibitors structurally similar to long chain lipids and to membrane-mimicking surfactants to determine if these factors induce loop movement. Loop movement was detected by measuring the change in intrinsic tryptophan fluorescence of a central tryptophan on the dynamic loop before and after exposure to these conditions. Although inhibitors and substrate mimics induced minor changes in loop dynamics, larger shifts were measured with various membrane-mimicking surfactants, suggesting that APT loop dynamics are mainly regulated by proximity to the plasma membrane. Ultimately, understanding the factors controlling APT function could provide novel mechanisms for controlling cell signaling.

Investigation of structural factors controlling loop dynamics in acyl protein thioesterases

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