Assessing the Mechanism by Which a Cholesterol Recognition Amino Acid Consensus (CRAC) Motif Recognizes Membrane Cholesterol

Abstract

The RTX family of toxins include a number of leukotoxins and cytolysins produced by Gram-negative bacteria, that share many structural features, including a cholesterol recognition amino acid consensus (CRAC) motif that regulates the toxins’ binding to cholesterol. Here, we investigate the mechanism by which the CRAC motif recognizes cholesterol and characterize the resulting changes in secondary structure, using a combination of spectroscopic, calorimetric, and microscopic techniques. Our model RTX protein is LtxA, a leukotoxin produced by the oral pathogen, Aggregatibacter actinomycetemcomitans. Initially, we demonstrated that LtxA-mediated cytotoxicity is regulated by the toxin's affinity for cholesterol. With a small peptide derived from the CRAC motif of LtxA, we measured the affinity of the motif for cholesterol and a variety of cholesterol derivatives, while also characterizing the conformational changes resulting from binding of the peptide to each cholesterol derivative. We found that the hydroxyl group of cholesterol has the greatest effect on the affinity of the CRAC peptide, with the other structural features having a significantly smaller impact. Understanding the conformational features of this CRAC peptide and the mechanism by which it recognizes cholesterol will allow us to develop novel therapeutics that target cholesterol. We have demonstrated that CRAC peptides are able to inhibit LtxA cytotoxicity and expect that they could also be used to develop therapeutics to prevent many types of diseases in which cholesterol plays a role.