A Unique Mechanism of Cholesterol Binding by an RTX Toxin

Abstract

The leukotoxin (LtxA) secreted by Aggregatibacter actinomycetemcomitans is a member of the repeats-in-toxin (RTX) family, and like the other members of the family, is a virulence protein that destroys host cells. LtxA specifically kills human white blood cells, allowing A. actinomycetemcomitans to survive within the host. LtxA has been shown to bind to an alpha-L/beta-2 integrin, lymphocyte function-associated antigen-1 (LFA-1), and upon binding, to form large LtxA/LFA-1 clusters in cholesterol-rich lipid rafts. Here, we have investigated this interaction by studying the binding of LtxA directly to cholesterol. Using surface plasmon resonance (SPR), we showed that the affinity of LtxA for membranes containing 40% cholesterol was four orders of magnitude greater than its affinity for membranes lacking cholesterol (4.6x10−12M vs. 1.9x10−8M). Surprisingly, the affinity was regulated not by an increase in the association rate in the presence of cholesterol but by a decrease in the dissociation rate. We identified two cholesterol recognition/amino acid consensus (CRAC) motifs in the amino acid sequence of LtxA and investigated the role of these cholesterol-binding motifs in the affinity of LtxA for cholesterol. Synthetic peptides corresponding to both CRAC sites interacted strongly with cholesterol, while control peptides did not. However, only the peptide corresponding to the first CRAC site inhibited binding of LtxA to membranes containing 40% cholesterol, and only this peptide inhibited the cytotoxicity of LtxA, demonstrating the requirement for cholesterol binding in the toxic mechanism of LtxA. This represents a unique mechanism of toxin binding to cholesterol in which the toxin's affinity to cholesterol is regulated by the dissociation rate rather than the association rate. The conservation of the first CRAC site among RTX toxins suggests that this is a mechanism shared by this toxin family.