title: Behavior of diphtheria toxin T domain containing substitutions that block normal membrane insertion at Pro345 and Leu307: control of deep membrane insertion and coupling between deep insertion of hydrophobic subdomains.
abstract: Diphtheria toxin T domain aids the translocation of toxin A chain across membranes. T domain has two hydrophobic layers/subdomains that can insert deeply into membranes: helices TH8 and 9, which form a transmembrane hairpin, and helices TH5-7, which form a nonclassical, nontransmembrane structure. Substitutions were made at Pro345, a residue located near the turn between TH8 and 9. P345 is critical for toxicity and pore formation by the T domain. Fluorescence methods showed that hairpin-disrupting Gly or Glu substitutions at 345 did not insert into lipid bilayers as deeply as the wild-type protein, and consistent with previous studies, these mutations reduced pore formation activity as assayed by a novel biotin-streptavidin-based influx assay. Introducing Pro at positions 347 or 353 not only failed to compensate for substitutions at P345, but also they further disrupted deep insertion and/or pore formation. Substitution of P345 with Asn, a residue that promotes helical hairpin formation almost as well as Pro, resulted in somewhat more normal insertion and pore formation than other substitutions. Importantly, a P345E substitution disrupted deep insertion of TH5-7. This suggests that TH8 and 9 and TH5-7 undergo some sort of coordinated insertion into the lipid bilayer and/or that the membrane-inserted T domain has a distinct tertiary structure in which TH5-7 interact with TH8 and 9 instead of consisting of noninteracting hydrophobic segments. Intriguingly, a L307R substitution in TH6, which disrupted deep insertion of TH7, had only a weak effect on pore formation and deep insertion of TH8 and 9. This suggests that the TH8 and 9 region can insert independently of TH5-7 to some degree and that TH8 and 9 insertion may occur early in T-domain insertion.
authors.affiliation: Department of Biochemistry and Cell Biology, Stony Brook University, State University of New York, Stony Brook, New York 11794-5215, USA.
authors.forename: Gang
authors.initials: G
authors.lastname: Zhao
authors.forename: Erwin
authors.initials: E
authors.lastname: London
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics*; D001896:Boron Compounds / Q000378:metabolism; D004252:DNA Mutational Analysis; D004167:Diphtheria Toxin / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D007930:Leucine / Q000235:genetics* / Q000378:metabolism; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism*; D008565:Membrane Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D013050:Spectrometry, Fluorescence
chemical_list: D001896:Boron Compounds; D004167:Diphtheria Toxin; D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D008565:Membrane Proteins; C085251:4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-3-indacene; D011392:Proline; D007930:Leucine