Role of cholesterol in substrate recognition by [Formula: see text]-secretase.

$$\gamma$$ -Secretase is an enzyme known to cleave multiple substrates within their transmembrane domains, with the amyloid precursor protein of Alzheimer’s Disease among the most prominent examples. The activity of $$\gamma$$ -secretase strictly depends on the membrane cholesterol content, yet the mechanistic role of cholesterol in the substrate binding and cleavage remains unclear. In this work, we used all-atom molecular dynamics simulations to examine the role of cholesterol in the initial binding of a direct precursor of $$\beta$$ -amyloid polypeptides by $$\gamma$$ -secretase. We showed that in cholesterol-rich membranes, both the substrate and the enzyme region proximal to the active site induce a local membrane thinning. With the free energy methods we found that in the presence of cholesterol the substrate binds favorably to the identified exosite, while cholesterol depletion completely abolishes the binding. To explain these findings, we directly examined the role of hydrophobic mismatch in the substrate binding to $$\gamma$$ -secretase, showing that increased membrane thickness results in higher propensity of the enzyme to bind substrates. Therefore, we propose that cholesterol promotes substrate binding to $$\gamma$$ -secretase by increasing the membrane thickness, which leads to the negative hydrophobic mismatch between the membrane and binding partners.