Only two amino acids are essential for cytolytic toxin recognition of cholesterol at the membrane surface

Abstract

The recognition and binding of cholesterol is an important feature of many eukaryotic, viral, and prokaryotic proteins, but the molecular details of such interactions are understood only for a few proteins. The pore-forming cholesterol-dependent cytolysins (CDCs) contribute to the pathogenic mechanisms of a large number of Gram-positive bacteria. Cholesterol dependence of the CDC mechanism is a hallmark of these toxins, yet the identity of the CDC cholesterol recognition motif has remained elusive. A detailed analysis of membrane interactive structures at the tip of perfringolysin O (PFO) domain 4 reveals that a threonine-leucine pair mediates CDC recognition of and binding to membrane cholesterol. This motif is conserved in all known CDCs and conservative changes in its sequence or order are not well tolerated. Thus, the Thr-Leu pair constitutes a common structural basis for mediating CDC-cholesterol recognition and binding, and defines a unique paradigm for membrane cholesterol recognition by surface-binding proteins.

Fig. 1.

Hemolytic and binding activity of PFO L1–3 loop mutants. Residues within loops L1, L2, and L3 were systematically substituted with alanine and/or glycine and assayed for changes in hemolytic activity (A) The change in hemolytic activity is shown as the ratio of the HD₅₀ (concentration of PFO or mutant required for 50% hemolysis, see Methods for details) for each mutant to that for wild-type PFO (HD₅₀ = 0.34 nM). Hence, bar height is inversely correlated with activity (n = 4 for each mutant hemolytic analysis). Binding to cholesterol-rich liposomes was assessed by SPR. (B) Percent binding of wild-type PFO is calculated by the equation , where RU^WT is the change in resonance units (RU) induced by passing 100 μL wild-type PFO (900 nM) and RU^MUT is the change in resonance units for each mutant at the same concentration. In those cases where the mutant bound better than wild-type PFO, the percent change was calculated from (n = 3 for each binding assay). Shown in C is the location of the Thr-490•Leu-491 pair in the lower half of PFO domain 4 (for the complete PFO structure refer to Fig. S1).

Fig. 2.

Alanine or glycine substitution of the Thr–Leu pair of PFO, PLY, and SLO abolishes binding. (A) Binding of PFO, PLY, SLO, and their derivatives to cholesterol-rich liposomes was measured by SPR (Left column). Binding of the same proteins to human RBCs was shown by flow cytometry (Right column). (B) A series of dot blots with twofold dilutions of cholesterol or epicholesterol (starting at 65 nmols, indicated by the arrow) probed with the same proteins and detected by antibody. Comparisons between binding of wild type and its derivatives can be made, but binding comparisons between CDCs are not valid due to differences in reactivity of the antibodies used for their detection. c, cholesterol; e, epicholesterol; TL→AA and TL→GG, the double alanine and double glycine mutants, respectively, of the Thr–Leu pair. The binding analyses are representative of three experiments. GMF, geometric mean fluorescence.

Fig. 3.

Structural requirements of the cholesterol recognition motif. SPR analysis of binding for the various PFO mutants to cholesterol-rich liposomes is shown in the Left column. Flow cytometric analysis of binding to human erythrocytes is shown in the Right column. The SPR and flow cytometry results are representative of three or more experiments.

Fig. 4.

Binding of PFO mutants to immobilized cholesterol. The EC₅₀ for PFO^T490S, PFO^L491I, and PFO^L491V were compared to the EC₅₀ for PFO. Upper panel is a representative dot blot that shows binding of each toxin to the various cholesterol concentrations (see Fig. 3 and Methods for details). In the table Below are the EC₅₀ values and standard errors (n = 7) calculated from densitometric analysis of the dots and the fold increase in EC₅₀ for each mutant. The double mutants PFO^{T490S•L491I}, PFO^{T490S•L491V}, and PFO^{T490L•L491T} did not exhibit detectable binding to the immobilized cholesterol so an EC₅₀ value was not determined (ND). The absolute concentrations of bound cholesterol on the PVDF membrane are not known; the values are used only to compare the relative binding of PFO and its derivatives (EC₅₀Mut/EC₅₀WT).

Fig. 5.

The Thr–Leu pair is necessary to maintain ILY-membrane contact during pore formation. Binding by flow cytometry of wild-type ILY and monomer-locked ILY (ILY^ML) to human erythrocytes (A) is compared with the Thr–Leu glycine-substituted mutant in nonlocked (ILY^{T517G•L518G}) and monomer-locked background (ILY^{T517G•L518G(ML)}) (B). Also shown are the corresponding analyses of nonlocked and monomer-locked versions of PFO and PFO^{T490G•L491G} (A and B, Right). (C) SDS/PAGE and Western blot analysis showing ILY, ILY^ML, and ILY^{T517G•L518G(ML)} are present in both pellet (P) and supernatant (S) fractions (due to receptor saturation with excess toxin) whereas the ILY double glycine mutant is found exclusively in the supernatant fraction. The flow cytometry and SDS/PAGE analyses are representative of three experiments. *ILY^ML-induced erythrocyte agglutination at this and higher concentrations, which depressed the fluorescence signal.