STRUCTURE OF THE MOSQUITOCIDAL DELTA-ENDOTOXIN CYTB FROM BACILLUS-THURINGIENSIS SP KYUSHUENSIS AND IMPLICATIONS FOR MEMBRANE PORE FORMATION

The delta-endotoxin CytB, found in parasporal inclusions of Bacillus t huringiensis subspecies kyushuensis, is a membrane pore-forming protei n which is lethal to the larvae of Dipteran insects and broadly cytoly tic in vitro. The crystal structure of CytB in the protoxin form has b een 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 molecule s has been refined at 2.6 Angstrom resolution to a crystallographic X- factor of 19.7% and a free X-factor of 26.1%. CytB has a single domain of alpha/beta architecture but a novel connectivity, comprising two o uter layers of alpha-helix hairpins wrapped around a mixed beta-sheet. In the protoxin form, CytB is a dimer Linked by the intertwined N-ter minal strands in a continuous, 12-stranded beta-sheet. Proteolytic pro cessing cleaves the intertwined beta-strands to release the active Cyt B 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 fo ld. Mutations in CytA that inhibit expression map to the dimer contact s and to the tip of helix pair A-B in contact with the sheet, apparent ly preventing correct folding. Mutations that inhibit toxicity map to the edge of the beta-sheet adjoining the helix pair C-D and to the she et face, while mutations on the helix surfaces have no effect. Therefo re segments forming the sheet, rather than the amphiphilic but short h elices, are responsible for membrane binding and pore formation. A con formational change is postulated by which the helix pair C-D peels awa y from the sheet to lie on the membrane surface, while the sheet regio n rearranges to form an oligomeric trans-membrane pore. (C) 1996 Acade mic Press Limited