Topography of helices 5-7 in membrane-inserted diphtheria toxin T domain: identification and insertion boundaries of two hydrophobic sequences that do not form a stable transmembrane hairpin.
The T domain of diphtheria toxin undergoes a low pH-induced conformational change that allows it to penetrate cell membranes. T domain hydrophobic helices 8 and 9 can adopt two conformations, one close to the membrane surface (P state) and a second in which they apparently form a transmembrane hairpin (TM state). We have now studied T domain helices 5-7, a second cluster of hydrophobic helices, using Cys-scanning mutagenesis. After fluorescently labeling a series of Cys residues, penetration into a non-polar environment, accessibility to externally added antibodies, and relative depth in the bilayer were monitored. It was found that helices 5-7 insert shallowly in the P state and deeply in the TM state. Thus, the conformational changes in helices 5-7 are both similar and somehow linked to those in helices 8 and 9. The boundaries of deeply inserting sequences were also identified. One deeply inserted segment was found to span residues 270 to 290, which overlaps helix 5, and a second spanned residues 300 to 320, which includes most of helix 6 and all of helix 7. This indicates that helices 6 and 7 form a continuous hydrophobic segment despite their separation by a Pro-containing kink. Additionally, it is found that in the TM state some residues in the hydrophilic loop between helices 5 and 6 become more highly exposed than they are in the P state. Their exposure to external solution in the TM state indicates that helices 5-7 do not form a stable transmembrane hairpin. However, helix 5 and/or helices 6 plus 7 could form transmembrane structures that are in equilibrium with non-transmembrane states, or be kinetically prevented from forming a transmembrane structure. How helices 5-7 might influence the mechanism by which the T domain aids translocation of the diphtheria toxin A chain across membranes is discussed.
Rosconi
Michael P
MP
Department of Biochemistry, State University of New York at Stony Brook, Stony Brook, New York 11794-5215, USA.
London
Erwin
E
D000595:Amino Acid Sequence; D001896:Boron Compounds / Q000494:pharmacology; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D004167:Diphtheria Toxin / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers / Q000737:chemistry; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010743:Phospholipids / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012709:Serum Albumin / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014867:Water / Q000737:chemistry
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D019086:Bridged Bicyclo Compounds, Heterocyclic; D004167:Diphtheria Toxin; D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D010743:Phospholipids; D012709:Serum Albumin; C021149:bimanes; D014867:Water; D003545:Cysteine