Structure of the Mosquitocidal δ-Endotoxin CytB from Bacillus thuringiensissp. kyushuensisand Implications for Membrane Pore Formation

Abstract

The δ-endotoxin CytB, found in parasporal inclusions of Bacillus thuringiensissubspecies kyushuensis, is a membrane pore-forming protein which is lethal to the larvae of Dipteraninsects and broadly cytolytic in vitro. The crystal structure of CytB in the protoxin form has been determined by isomorphous replacement using heavy-atom derivatives of both the wild-type protein and an engineered cysteine mutant. The atomic model comprising residues 19 to 245 and 28 bound water molecules has been refined at 2.6 Å resolution to a crystallographic R-factor of 19.7% and a free R-factor of 26.1%. CytB has a single domain of α/β architecture but a novel connectivity, comprising two outer layers of α-helix hairpins wrapped around a mixed β-sheet. In the protoxin form, CytB is a dimer linked by the intertwined N-terminal strands in a continuous, 12-stranded β-sheet. Proteolytic processing cleaves the intertwined β-strands to release the active CytB as a monomer, as well as removing the C-terminal tail to uncover the three-layered core. The homologous toxin CytA should show the same fold. Mutations in CytA that inhibit expression map to the dimer contacts and to the tip of helix pair A-B in contact with the sheet, apparently preventing correct folding. Mutations that inhibit toxicity map to the edge of the β-sheet adjoining the helix pair C-D and to the sheet face, while mutations on the helix surfaces have no effect. Therefore segments forming the sheet, rather than the amphiphilic but short helices, are responsible for membrane binding and pore formation. A conformational change is postulated by which the helix pair C-D peels away from the sheet to lie on the membrane surface, while the sheet region rearranges to form an oligomeric trans-membrane pore.