Behavior of the deeply inserted helices in diphtheria toxin T domain: helices 5, 8, and 9 interact strongly and promote pore formation, while helices 6/7 limit pore formation.
Diphtheria toxin T domain aids the membrane translocation of diphtheria toxin A chain. When the isolated T domain is deeply membrane-inserted, helices TH 8-9 form a transmembrane hairpin, while helices TH 5-7 form a deeply inserted nontransmembrane structure. Blocking deep insertion of TH 8-9 blocks deep insertion of TH 5-7 ( Zhao, G., and London, E. ( 2005) Biochemistry 44, 4488- 4498 ). We now examine the effects of blocking the deep insertion of TH 5 and TH 6/7. An A282R/V283R dual substitution in TH 5 prevented its deep insertion, significantly decreased the deep insertion of TH 9, and to a lesser degree that of TH 6/7. Blocking deep insertion of TH 6/7 with a L307R mutation had no effect on the deep insertion of TH 5, similar to its previously characterized lack of effect on the deep insertion of TH 8-9. An I364K mutation in TH 9 blocked TH 8-9 deep insertion and greatly reduced pore formation by the T domain, consistent with the role of TH 8-9 in pore formation. The A282R/V283R mutations also reduced the extent of pore formation, but to a lesser degree, suggesting either that TH 5 is part of the pore or that interactions with TH 5 affect the ability of TH 8-9 to form pores. The L307R mutation enhanced the extent of pore formation, suggesting that deeply inserted TH 6/7 may act as a "cork" that partly blocks the pore. Combined, these results indicate that TH 5, 8, and 9 combine to form a deeply inserted scaffold of more strongly associated helices.
Lai
Bing
B
Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York 11794-5215, USA.
Zhao
Gang
G
London
Erwin
E
D004167:Diphtheria Toxin / Q000737:chemistry* / Q000235:genetics; D057927:Hydrophobic and Hydrophilic Interactions; D008051:Lipid Bilayers / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence
D004167:Diphtheria Toxin; D008051:Lipid Bilayers; D008565:Membrane Proteins; D010446:Peptide Fragments; C039171:diphtheria toxin fragment B