Membrane Diffusion Mode of Human Perforin-1 and Its Cholesterol Sensitivity

Perforin-1, a pore-forming protein, is essential for cytotoxic T lymphocyte- and natural killer cell-mediated immune responses. Human perforin-1 (hPRF) exists as a surface-bound monomer that diffuses on the membrane before oligomerizing into a prepore complex. Yet this diffusive stage evades direct experimental observation, and how cholesterol regulates hPRF mobility remains unknown. We therefore performed all-atom molecular dynamics (MD) simulations of the hPRF monomer on lipid membranes with 0-60 mol % cholesterol to identify which lipid environments support or hinder efficient hPRF diffusion. The lateral diffusion coefficient of hPRF decreased progressively with cholesterol content, tracking reduced area per lipid up to 40 mol %. Beyond this packing-saturation point, the hPRF diffusion continued to slow, suggesting a contribution from direct protein-lipid interactions. Intermolecular contact analysis identified W431 and F432 as persistent cholesterol-binding sites that drive raft-like codiffusion of hPRF with an ordered lipid assembly rather than independent diffusion. These results demonstrate that cholesterol suppresses hPRF lateral dynamics through both indirect membrane ordering and direct, residue-specific protein-lipid local interactions, providing molecular-level insights into the early stages of perforin-mediated cytotoxicity. This dual mechanism suggests that, beyond limiting hPRF binding, cholesterol-rich effector-cell membranes may also suppress its lateral diffusion, providing an additional layer of self-protection from perforin-mediated lysis.