Oxysterols Outcompete Cholesterol Binding to the Membrane-Inserted Cytolysin A Pore Complex

Area of Science:

• Biochemistry
• Molecular Biology
• Computational Biophysics

Background:

• Pore-forming toxins (PFTs) are bacterial proteins crucial for initiating infections via host cell membrane pore formation.
• Cholesterol is a known promoter of PFT activity, but the role of oxysterols, such as 25-hydroxycholesterol (25-HC), remains understudied.
• Oxysterols are implicated in cellular senescence and inflammatory conditions, suggesting potential relevance in disease pathogenesis.

Purpose of the Study:

• To investigate the influence of 25-hydroxycholesterol (25-HC) on the membrane-bound state and activity of alpha-pore-forming toxins (PFTs).
• To elucidate the binding interactions of 25-HC with cytolysin A (ClyA), an alpha-PFT, within a model lipid bilayer.
• To compare the binding dynamics of 25-HC with cholesterol in the context of ClyA pore formation.

Main Methods:

• Utilized all-atom molecular dynamics simulations to model ClyA insertion into a POPC:cholesterol:25-HC membrane.
• Analyzed sterol binding landscapes, including binding hotspots and residence times, for both cholesterol and 25-HC.
• Quantified the impact of 25-HC's hydroxyl group on interactions with ClyA's beta-tongue motifs and N-terminal helix.

Main Results:

• 25-HC effectively replaced cholesterol at binding sites between ClyA's beta-tongue motifs, exhibiting microsecond-scale binding lifetimes.
• While overall sterol occupancy at the N-terminal helix was lower for 25-HC, its binding was more stable compared to cholesterol.
• The hydroxyl group of 25-HC significantly enhanced binding affinity with polar residues in the beta-tongue, increasing binding times severalfold over cholesterol.

Conclusions:

• Enhanced oxysterol interactions with alpha-PFTs like ClyA can significantly modulate their pore-forming activity.
• The findings suggest a distinct mechanism for alpha-PFTs compared to cholesterol-dependent cytolysins, where oxysterols are inhibitory.
• This study highlights the critical role of specific sterol modifications in bacterial toxin function and host-pathogen interactions.