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Crystal Structure of the Octameric Pore of Staphylococcal Γ-Hemolysin Reveals the Β-Barrel Pore Formation Mechanism by Two Components

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Crystal Structure of the Octameric Pore of Staphylococcal Γ-Hemolysin Reveals the Β-Barrel Pore Formation Mechanism by Two Components Crystal structure of the octameric pore of staphylococcal γ-hemolysin reveals the β-barrel pore formation mechanism by two components Keitaro Yamashitaa, Yuka Kawaia, Yoshikazu Tanakaa,b,c,1, Nagisa Hiranoc, Jun Kanekod, Noriko Tomitae, Makoto Ohtae, Yoshiyuki Kamiof, Min Yaoa,c, and Isao Tanakaa,c,1 aGraduate School of Life Science , Hokkaido University, Sapporo 060-0810, Japan; bCreative Research Institution Sousei, Hokkaido University, Sapporo 001-0021, Japan; cFaculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810, Japan; dDepartment of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan; eInstitute of Fluid Science, Tohoku University, Sendai 980-8577, Japan; and fDepartment of Biochemical Engineering, Graduate School of Science and Engineering, Yamagata University, Yonezawa 992-8510, Japan Edited by Eric Gouaux, Oregon Health and Science University, Portland, OR, and approved August 25, 2011 (received for review June 27, 2011) Staphylococcal γ-hemolysin is a bicomponent pore-forming toxin heterodimer assembles into an oligomer on the target cell to form composed of LukF and Hlg2. These proteins are expressed as water- a ring-shaped particle called a prepore, in which the β-barrel pore soluble monomers and then assemble into the oligomeric pore is not yet formed (11–14). After forming a stable prepore, the form on the target cell. Here, we report the crystal structure of β-barrel pore is formed. Pore formation requires the binding the octameric pore form of γ-hemolysin at 2.5 Å resolution, which of phosphatidylcholine (PC) head groups to a cleft in the LukF is the first high-resolution structure of a β-barrel transmembrane component surrounded by Trp177 and Arg198 (Trp176 and protein composed of two proteins reported to date. The octameric Arg197 of LukF-PV) (13, 15, 16). The crystal structures of the assembly consists of four molecules of LukF and Hlg2 located alter- monomeric forms of bicomponent β-PFTs [i.e., LukF (15), nately in a circular pattern, which explains the biochemical data LukF-PV (17), and LukS-PV (18)], have been determined. How- accumulated over the past two decades. The structure, in combina- ever, the structures of the pore forms have not been reported tion with the monomeric forms, demonstrates the elaborate mole- at atomic resolution, which has hindered detailed discussion of cular machinery involved in pore formation by two different the complicated molecular mechanism of action of bicomponent molecules, in which interprotomer electrostatic interactions using pore-forming toxins. Although bicomponent PFTs are found in loops connecting β2 and β3 (loop A: Asp43-Lys48 of LukF and Lys37- several species, such as the edible mushroom Pleurotus ostreatus Lys43 of Hlg2) play pivotal roles as the structural determinants for (19), the structures of these pores have not been reported. assembly through unwinding of the N-terminal β-strands (amino- One of the most important issues for staphylococcal bicompo- latch) of the adjacent protomer, releasing the transmembrane stem nent PFTs is the stoichiometry of the class F and S components. domain folded into a β-sheet in the monomer (prestem), and inter- Electron microscopy and cross-linking experiments of purified action with the adjacent protomer. γHL pores on human erythrocyte membranes demonstrated the existence of a heptamer with a 3∶4 or 4∶3 molar ratio of F to S athogenic bacteria secrete various virulence factors to attack components (20, 21), whereas biochemical analyses of pores of Phost cells. The pore-forming toxins (PFTs) are among the engineered covalent γHL heterodimers on erythrocytes and leu- most sophisticated virulence factors, and are expressed as water- kocytes suggested octameric stoichiometry (22). Several reports soluble monomeric proteins that assemble on the membranes of using LUK pores formed on rabbit erythrocytes also demon- the target cells to form bilayer-spanning pores (1). With the strated the existence of an octamer consisting of 4-plus-4 subunits appearance of the pore on the membrane, the cells are killed (23–25). However, hexamer was also proposed based on structure through leakage. It is interesting to note that PFTs are expressed modeling using monomeric structures of PVL (17) and electron not only by bacteria but also by eukaryotes, such as the immune microscopy of LUK pores on leukocytes and erythrocytes (26). proteins perforin and complement C9, suggesting the universality It is also important to determine the significance of using two of these molecules in a wide range of organisms (2). PFTs can be components. Although a role was proposed for the F component classified into two families according to the secondary structure in initiating the pore formation process (27), that of the S com- of the transmembrane region in the pore structure; i.e., α-helical ponent remains unclear. PFT (α-PFT) and β-barrel PFT (β-PFTs) (3, 4). In the present study, we determined the crystal structure of Staphylococcus aureus, a ubiquitous and pernicious human the pore form of bicomponent β-PFT, γHL. This is a unique re- pathogen, secretes several β-PFTs including αHL, γ-hemolysin port of the crystal structure of a heterocomponent β-barrel- type (γHL), leukocidin (LUK), and Panton–Valentine leukocidin (PVL) transmembrane protein. This is also a unique bicomponent β- (5). The αHL consists of a single polypeptide, whereas the others PFTof which both monomer- and pore-form structures have been are bicomponent β-PFTs that require the synergistic association determined by X-ray crystallography, which allowed us to discuss of a class F component and a class S component. The γHL, LUK, the pore formation mechanism based on their real structures at and PVL are composed of LukF and Hlg2, LukF and LukS, and LukF-PVand LukS-PV,as class F and S components, respectively. The components of bicomponent β-PFTs are similar to each other Author contributions: Y.T., J.K., Y. Kamio, M.Y., and I.T. designed research; K.Y., Y. Kawai, α Y.T., N.H., and N.T. performed research; K.Y., Y. Kawai, Y.T., N.T., M.O., and M.Y. analyzed and to HL in amino acid sequence: Within a class, S and F pro- data; and K.Y., Y. Kawai, Y.T., J.K., N.T., M.O., Y. Kamio, M.Y., and I.T. wrote the paper; teins are approximately 70% identical, whereas between classes The authors declare no conflict of interest. the identity drops to approximately 30%. Class F proteins are This article is a PNAS Direct Submission. more closely related to αHL (approximately 30%) than class S Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, proteins (approximately 20%) (5, 6). Extensive experiments have www.pdb.org (PDB ID code 3B07). been carried out for more than two decades, and have suggested 1To whom correspondence may be addressed. E-mail: [email protected] or that the pore formation mechanism of bicomponent toxins is as [email protected]. follows (7, 8). The soluble forms of F and S components bind se- This article contains supporting information online at www.pnas.org/lookup/suppl/ quentially to the target cells and form a heterodimer (9, 10). Each doi:10.1073/pnas.1110402108/-/DCSupplemental. 17314–17319 ∣ PNAS ∣ October 18, 2011 ∣ vol. 108 ∣ no. 42 www.pnas.org/cgi/doi/10.1073/pnas.1110402108 Downloaded by guest on September 26, 2021 atomic resolution. Based on the structural differences between A Cap pore and monomer forms in combination with biological data accumulated over the past two decades, we propose a mechanism β 70 Å of pore formation by -PFTs along with the roles of each compo- Rim 93 Å 25.5 Å nent. The electrochemical properties of the pore are also dis- 114 Å cussed from a structural viewpoint. 47 Å Stem Results Molecular Architecture of γHL Pore. Recently, we found that mono- Interface 1 Interface 2 meric αHL in the presence of 2-methyl-2,4-pentanediol (MPD) Amino-latch can spontaneously form a pore structure identical to that in B Pre-stem Cap the biological membrane (28). We applied this method to prepare crystals of γHL pores. Purified monomeric soluble LukF and Stem Hlg2 formed SDS-resistant oligomers in the presence of 20% Rim MPD, suggesting that MPD can induce pore formation of γHL (Fig. S1). Fortunately, crystals were grown from a buffer contain- ing 50% MPD, and the structure was determined by the molecu- LukF monomer LukF protomer lar replacement method at a resolution of 2.5 Å. The revealed Pre-stem structure was an octameric pore form consisting of four mole- Cap cules of each of LukF and Hlg2 (Fig. 1A). Each component was located alternately in a circular pattern along a noncrystallo- graphic fourfold axis. The height and diameter of the octameric Stem structure were 93 and 114 Å, respectively. In analogy to the hep- Rim tameric structure of αHL (29, 30), the protomer was composed of cap, rim, and stem domains (Fig. 1B). The cap domain interacts Hlg2 monomer Hlg2 protomer with those of adjacent protomers, and the rim domain is located beneath the cap domain. The height of the ellipsoidal aqueous C domain composed of cap and rim domains was approximately 70 Å. The protruding stem domains formed a transmembrane MPD β-barrel 25 Å in diameter and 47 Å in height (measured from W177 Cα positions), composed of 16 antiparallel β-strands from eight protomers (four protomers from each of LukF and LukS). The R198 N-terminal amino-latches, which participate in the interprotomer interaction in the heptameric pore structure of αHL (28–30), were disordered. MPD molecules were bound at the base of the Fig. 1. Overall octameric pore structure of γHL. (A) Side and top views of rim domain of four LukF protomers, and recognized by Trp177 the heptamer.
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