The Staphylococcal R-Toxin Pore Has a Flexible Conformation † Beatrix Ve ´csey-Semje ´n, ‡,§ Stefan Knapp, | Roland Mo ¨llby, ‡ and F. Gisou van der Goot* ,§ Microbiology and Tumorbiology Center, Karolinska Institutet, 17177 Stockholm, Sweden, Center for Structural Biochemistry, Karolinska Institutet, NOVUM, 14157 Stockholm, Sweden, and De ´ partement de Biochimie, UniVersite ´ de Gene ` Ve, 30 quai E. Ansermet, 1211 Gene ` Ve 4, Switzerland ReceiVed October 15, 1998; ReVised Manuscript ReceiVed January 26, 1999 ABSTRACT: The R-toxin from Staphylococcus aureus undergoes several conformational changes from the time it is released from the bacterium to the moment it forms a channel in the plasma membrane of its target cell. It is initially a soluble monomer, which undergoes membrane binding and oligomerization into a heptameric ring and finally inserts into the lipid bilayer to form a pore. Here we have analyzed the stability of different forms of the R-toxin (monomer as well as heptamers in solution, bound to the membrane and membrane-inserted) by differential scanning calorimetry and limited proteolysis. Data presented here show that, in contrast to both the membrane-bound prepore complex and the monomer in solution, the membrane-inserted R-toxin channel does not undergo cooperative unfolding and is highly susceptible to proteases. These observations suggest that the channel has a looser conformation. Interestingly, resistance to proteases could be recovered upon solubilization of the channel, indicating that the loss of rigid tertiary packing only occurred upon membrane insertion. Far-UV CD data, however, suggest that the transmembrane -barrel must be stably folded and that therefore only the Cap and Rim domains of the channel are loosely packed. All together, our data show that the R-toxin channel is not a rigid complex within the membrane but adopts a rather flexible conformation. R-Toxin is secreted by Staphylococcus aureus as a water- soluble, 33 kDa single-chain, active polypeptide (for reviews, see refs 1-3). After binding to the target cell surface has occurred, possibly via specific receptor molecules, collision between toxin monomers at the membrane surface leads to the formation of a stable heptameric complex (4, 5). This complex is initially nonlytic. R-Toxin can be blocked in a membrane-bound, nonlytic conformation by introducing point mutations (6-8) or by manipulating parameters such as pH and lipid composition (9). It has also been proposed that this nonlytic complex exists in vivo at the surface of R-toxin insensitive cells (10). A subsequent prepore to pore transition is required for channel formation. This transition involves membrane insertion of the central, glycine-rich, loop (11, 12). The loops from the seven subunits of the complex then come together and form a 14-stranded transmembrane -barrel (9) as beautifully illustrated by the X-ray structure of the heptamer in the presence of detergent (5; also see Figure 5). Each step in the mechanism of action, i.e., membrane binding, heptamerization, and membrane insertion, involves conformational changes not only of quaternary structure but also of tertiary and secondary structures. The changes in structure that take place have not been precisely character- ized; however, the following observations have been made. Upon membrane binding and oligomerization, tryptophan residues that are initially buried in the soluble structure become accessible to soluble quenchers, indicating a change in the tertiary packing (9). The structure, however, remains rigid as witnessed by the presence of a well-defined near- ultraviolet circular dichroism (UV CD) 1 signal (9) and a lack of protease sensitivity except for the N-terminal latch (7). Even the central loop, which is very sensitive to proteases in the soluble form (13-15), becomes protected (6, 7, 13). Upon subsequent membrane insertion, an increase in the level of -sheet structure was observed, most likely corresponding to the folding of the central loops into a -barrel (9) which penetrates into the bilayer (7, 11, 15-18). In this membrane- inserted conformation, tryptophan residues are no longer accessible to soluble quenchers but become accessible to quenching by bromide attached in the middle of the acyl chain of the lipid (9). The recently determined structure of the heptamer (5) indeed reveals that many tryptophans are close to the acyl chain-headgroup interface. Also after membrane insertion, the aromatic residues are free to rotate as witnessed by the collapse of the near-UV CD signal (9). This latter observation raises the possibility that the membrane- inserted toxin is not a rigid complex but rather that it has a loose conformation. In this paper, we have probed the conformation of the R-toxin channel by differential scanning calorimetry, binding of the hydrophobic dye bis-ANS, and limited proteolysis. † This work has been supported by a grant from the Swiss National Science Foundation to F.G.v.d.G. and the Karolinska Institutet’s fonder to R.M. * Corresponding author. Telephone and fax: (41) 22 702 6414. E-mail: Gisou.vandergoot@biochem.unige.ch. ‡ Microbiology and Tumorbiology Center, Karolinska Institutet. § Universite ´ de Gene `ve. | Center for Structural Biochemistry, Karolinska Institutet. 1 Abbreviations: DOPG, dioleoylphosphatidylglycerol; DOPC, dio- leoylphosphatidylcholine; HEPES, N-(2-hydroxyethyl)piperazine-N′- 2-ethanesulfonic acid; UV CD, ultraviolet circular dichroism. 4296 Biochemistry 1999, 38, 4296-4302 10.1021/bi982472k CCC: $18.00 © 1999 American Chemical Society Published on Web 03/18/1999