title: Replacement of C-terminal histidines uncouples membrane insertion and translocation in diphtheria toxin T-domain.
abstract: The translocation (T) domain plays a key role in the action of diphtheria toxin and is responsible for transferring the N-terminus-attached catalytic domain across the endosomal membrane into the cytosol in response to acidification. The T-domain undergoes a series of pH-triggered conformational changes that take place in solution and on the membrane interface, and ultimately result in transbilayer insertion and N-terminus translocation. Structure-function studies along this pathway have been hindered because the protein population occupies multiple conformations at the same time. Here we report that replacement of the three C-terminal histidine residues, H322, H323, and H372, in triple-R or triple-Q mutants prevents effective translocation of the N-terminus. Introduction of these mutations in the full-length toxin results in decrease of its potency. In the context of isolated T-domain, these mutations cause loss of characteristic conductance in planar bilayers. Surprisingly, these mutations do not affect general folding in solution, protein interaction with the membranes, insertion of the consensus transmembrane helical hairpin TH8-9, or the ability of the T-domain to destabilize vesicles to cause leakage of fluorescent markers. Thus, the C-terminal histidine residues are critical for the transition from the inserted intermediate state to the open-channel state in the insertion/translocation pathway of the T-domain.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas, USA.
authors.forename: Mykola V
authors.initials: MV
authors.lastname: Rodnin
authors.forename: Alexander
authors.initials: A
authors.lastname: Kyrychenko
authors.forename: Paul
authors.initials: P
authors.lastname: Kienker
authors.forename: Onkar
authors.initials: O
authors.lastname: Sharma
authors.forename: Mauricio
authors.initials: M
authors.lastname: Vargas-Uribe
authors.forename: R John
authors.initials: RJ
authors.lastname: Collier
authors.forename: Alan
authors.initials: A
authors.lastname: Finkelstein
authors.forename: Alexey S
authors.initials: AS
authors.lastname: Ladokhin
mesh_terms: D018360:Crystallography, X-Ray; D004167:Diphtheria Toxin / Q000737:chemistry*; D055724:Electrophysiological Phenomena; D006639:Histidine / Q000737:chemistry*; D008051:Lipid Bilayers / Q000737:chemistry*; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry; D009154:Mutation / Q000235:genetics*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D013329:Structure-Activity Relationship
chemical_list: D004167:Diphtheria Toxin; D008051:Lipid Bilayers; D050505:Mutant Proteins; D006639:Histidine
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