| Co-Investigator(Kenkyū-buntansha) |
KANEKO Jun Graduate Schoolo of Agricultural Science, Tohoku University, Research Associate, 大学院・農学研究科, 助手 (30221188)
TOMITA Toshio Graduate Schoolo of Agricultural Science, Tohoku University, Associate Professor, 大学院・農学研究科, 助教授 (00126129)
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| Research Abstract |
Crystal Structure of LukF Delineates Conformational Changes Accompanying For-mation of a Transmembrane Channel
LukF Architecture. LukF has the shape of a prolate ellipsoid with dimensions of 72Åx34Åx25. In LukF the amino latch and the pre-stem adopt dramatically different conformations when compared to the corresponding regions of an Hla protomer. Excluding these areas, the fold of LukF is identical to the fold of an Hla protomer. The residues that comprise the amino latch (E2-K16) adopt a β-strand conformation and extend the inner β-sheet of the β-sandwich by one strand in LukF.Measured in terms of solvent accessible surface area, binding amino latch to the LukF core buries 414 Å^2. In striking contrast ot the Hla protomer, the glycine-rich region of LukF forms a three-strand antiparalleled β-sheet that packs against the inner β-sheet of the β-sandwich domain. The pre-stem β-sheet, which includes the seven disordered residues spanning strand 7" and strand 8 (Figures 1C and 10), has an
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a(β)b(β)c(coil)d(β) fold. Hydrophobic residues predominate in the pre-stem interface with the β-sandwich domain : there are 24 van der Walls interactions and the buried surface area is 852Å^2, which is close to the 670 Å^2 buried by the Hla amino latch when bound at a similar site on an adjacent protomer in the heptamer structure. Direct contact between the pre-stem and the amino latch is mediated by a cluster of hydrophobic residues that includes V13, V17, Y117, and F119. In Hla, the residues that occupy the latter two positions are predicted to lie at the interfacial region of the lipid bilayer. The juxtaposition of Y117 and F119 between the pre-stem and the amino latch makes direct "communication" between these two key regions possible.
LukF Surface Properties and Phospholipid Binding Site. The surface of the pre-stem β-sheet facing the LukF core is primarily hydrophobic while the side directed toward the solvent is polar. Like Hla, the LukF rim domain contains a lot of exposed aromatic residues. Located in a cleft lined by W177 and R198 is a binding site for phospholipid head groups. In the Hla heptamer, there is a similar lipid binding site, i.e., W179 and R200 residues of Hla, and the latter residue is critical for binding of Hla to an erythrocyte membrane.
LukF Monomer and Hla Protomer Comparison. Superposition of the LukF and Hla protomer structures reinforces the conclusion that the cores are very similar despite a sequence identity of only 31.7% for the mature polypeptides. The comparison also emphasizes the divergence in conformation at the amino latch and glycine-rich "stem" regions. Fitting of individual domains demonstrated that the r.m.s deviation between Cα positions for the β-sandwich and rim are 2.1 Å and 2.4 Å, respectively. To a first approximation, the β-sandwich and rim domains behave as rigid bodies that adopt different relative conformations in the monomer (LukF) and heptamer (Hla) due to small changes spread over a number of residues at the β sandwich/rim domain juncture. Recently, the 3-dimensional structure of the water-soluble LukF-PV monomer also analysed and was found to be basically similar to that of LukF.
Major conformational differences between LukF and the Hla protomer structures occur in the triangle region. In contrast to the diffuse nature of the conformational differences relating the β- sandwich and rim domains, there are larger differences in main chain phi and psi angles for 4 residues in the triangle region which allow the LukF pre-stem to fold against the inner surface of the β-sandwich domain. In the Hla protomer, these main chain dihedral angles differ by>90゜ and are associated with a triangle conformation in which the polypeptide chain extends from the protomer core. The LukF pre-stem occupies approximately the same site that the amino latch of a neighboring protomer occupies in the oligomeric toxin, based on the analogy with the Hla heptamer. Less
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