ABCE1 occludes two nucleotides during 70S splitting.

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ABCE1 occludes two nucleotides during 70S splitting. ABCE1 occludes two nucleotides during 70S splitting. (A) Nucleotide occlusion is assayed at 70S splitting conditions by rapid gel filtration. Autoradiogram of the elution fractions from the nucleotide occlusion assay containing ABCE1 with the respective nucleotides securely trapped within the closed sites. Representative set of two independent experiments. (B) Exchange of the catalytic glutamates facilitates closure of the nucleotide-binding sites and ATP occlusion, hence, all EA variants occlude two nucleotides. As intended, introduction of arginine into the ABC-signature motif prevents nucleotide occlusion in the respective site, leading to one or a background of 0.2 nucleotides per protein for single-SR and double-SR substitutions, respectively. The ATP-to-ADP ratio occluded by the SR variants reflects the ATP turnover rate in the intact site. Elina Nürenberg-Goloub et al. LSA 2018;1:e201800095 © 2018 Nürenberg-Goloub et al.