Scanning the membrane-bound conformation of helix 1 in the colicin E1 channel domain by site-directed fluorescence labeling.

Helix 1 of the membrane-associated closed state of the colicin E1 channel domain was studied by site-directed fluorescence labeling where bimane was covalently attached to a single cysteine residue in each mutant protein. A number of fluorescence properties of the tethered bimane fluorophore were measured in the membrane-bound state of the channel domain, including fluorescence emission maximum, fluorescence quantum yield, fluorescence anisotropy, membrane bilayer penetration depth, surface accessibility, and apparent polarity. The data show that helix 1 is an amphipathic alpha-helix that is situated parallel to the membrane surface. A least squares fit of the various data sets to a harmonic function indicated that the periodicity and angular frequency for helix 1 are typical for an amphipathic alpha-helix (3.7 +/- 0.1 residues per turn and 97 +/- 3.0 degrees, respectively) that is partially bathing into the membrane bilayer. Dual fluorescence quencher analysis also revealed that helix 1 is peripherally membrane-associated, with one face of the helix dipping into the lipid bilayer and the other face projecting toward the solvent. Finally, our data suggest that the helical boundaries of helix 1, at least at the C-terminal region, remain unaffected upon binding to the surface of the membrane in support of a toroidal pore model for this colicin.

Crystal structure of ADP-ribosylated ribosomal translocase from Saccharomyces cerevisiae.

The crystal structure of ADP-ribosylated yeast elongation factor 2 in the presence of sordarin and GDP has been determined at 2.6 A resolution. The diphthamide at the tip of domain IV, which is the target for diphtheria toxin and Pseudomonas aeruginosa exotoxin A, contains a covalently attached ADP-ribose that functions as a very potent inhibitor of the factor. We have obtained an electron density map of ADP-ribosylated translation factor 2 revealing both the ADP-ribosylation and the diphthamide. This is the first structure showing the conformation of an ADP-ribosylated residue and confirms the inversion of configuration at the glycosidic linkage. Binding experiments show that the ADP-ribosylation has limited effect on nucleotide binding affinity, on ribosome binding, and on association with exotoxin A. These results provide insight to the inhibitory mechanism and suggest that inhibition may be caused by erroneous interaction of the translation factor with the codon-anticodon area in the P-site of the ribosome.