Membrane Lipids Induce Change in Aggregatibacter actinomycetemcomitans Leukotoxin Secondary Structure

Objectives: Aggregatibacter actinomycetemcomitans (Aa), a common inhabitant of the upper aerodigestive tract of humans, is associated with lung and brain abscesses, infective endocarditis, and localized aggressive periodontitis. Aa secretes a repeats-in-toxin (RTX) protein, leukotoxin (LtxA), which kills LFA-1-bearing cells. Although LtxA’s pathological mechanism is not yet fully understood, experimental evidence indicates that it involves an association with and subsequent destabilization of the target cell plasma membrane.  LtxA is a water-soluble protein that is predicted to expose hydrophobic residues sequestered in the protein’s interior to facilitate membrane disruption. We hypothesize that LtxA is able to achieve its dichotomous existence through intrinsic disorder, whereby the same primary structure can adopt different conformations that depend upon the environment. Thus, the objective of this work is to correlate modulations in LtxA secondary structure with specific functional properties of the protein. Methods: LtxA was incubated with model membranes and analyzed using Förster resonance energy transfer (FRET), surface plasmon resonance, and circular dichroism spectroscopy to gain insight into the relationship between toxin structure and membrane-dependent cytotoxic properties. Results: Upon incubation of LtxA with lipids that favor LtxA association, as measured by 75% FRET and a K_D of 4e^-12±3e^-12 M, we observed a 2.1% increase in LtxA α-helical content.  In contrast, when LtxA was incubated with lipids favoring LtxA-induced membrane disruption, as previously determined using DSC and ³¹P NMR, we observed a 2.1% increase in turn content. Conclusions: The distinct structural changes in LtxA that were observed after incubation with these lipids suggest that membrane binding and membrane disruption are separate and potentially independent events.  These studies provide a preliminary insight into the mechanism of cell damage that leads to disease progression by Aa.