The domains of a cholesterol-dependent cytolysin undergo a major FRET-detected rearrangement during pore formation

doi:10.1073/pnas.0500556102

The domains of a cholesterol-dependent cytolysin undergo a major FRET-detected rearrangement during pore formation

Departments of ^*Biochemistry and Biophysics and ^‡Chemistry, Texas A&M University, College Station, TX 77843; ^†Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104; and ^§Department of Medical Biochemistry and Genetics, Texas A&M University System Health Science Center, College Station, TX 77843

Edited by R. John Collier, Harvard Medical School, Boston, MA, and approved April 1, 2005 (received for review January 21, 2005)

Abstract

FRET measurements were used to determine the domain-specific topography of perfringolysin O, a pore-forming toxin, on a membrane surface at different stages of pore formation. The data reveal that the elongated toxin monomer binds stably to the membrane in an “end-on” orientation, with its long axis approximately perpendicular to the plane of the membrane bilayer. This orientation is largely retained even after monomer association to form an oligomeric prepore complex. The domain 3 (D3) polypeptide segments that ultimately form transmembrane β-hairpins remain far above the membrane surface in both the membrane-bound monomer and prepore oligomer. Upon pore formation, these segments enter the bilayer, whereas D1 moves to a position that is substantially closer to the membrane. Therefore, the extended D2 β-structure that connects D1 to membrane-bound D4 appears to bend or otherwise reconfigure during the prepore-to-pore transition of the perfringolysin O oligomer.

Footnotes

↵ ¶ To whom correspondence should be addressed at: College of Medicine, 116 Reynolds Building, College Station, TX 77843-1114. E-mail: ajohnson{at}medicine.tamhsc.edu.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: PFO, perfringolysin O; CDC, cholesterol-dependent cytolysin; Dn, domain n; TMH, transmembrane β-hairpin; AFM, atomic force microscopy; BODIPY, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-S-indacene-3-yl)methyl)iodoacetamide; Rh-PE, lissamine rhodamine 1,2, dihexadecanoyl-sn-glycero-3-phosphoethanolamine; D, donor dye; A, acceptor dye; DA, D plus A; B, blank.