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. 2018 Jan 3;9:44. doi: 10.1038/s41467-017-02110-1

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — A molecular basis of the GDP-state specificity of GtgE. a Putative hubs involved in the GDP-state specificity of GtgE depicted in the superposition of active and inactive Rab32-structures. Gray: Rab32:GDP:GtgE_C45A; transparent wheat: Rab32:GppCH₂p:VARP (PDB ID: 4CYM¹⁷); sticks: Hub 1 (E86_R), hub 2 (Y54_R), hub 3 (comprising of F88_R, E114_G, and K194_G), and GppCH₂p; spheres: C_α-atoms of the cleavage site; magenta: switch regions Rab32:GDP; black and yellow dashed lines: steric clash and hydrogen bonds, respectively. b Hub 3 (F88_R; E114_G; K194_G) of the Rab:GtgE_C45A switch II interface. Structural superposition of MD-simulations of the Rab32:GtgE complex bound to either GDP (gray/blue) or GTP positioned in silico (light/dark green) after 190 ns. Right: van-der-Waals contacts of hub 3 indicated by yellow dashed lines for the GDP (top) and the GTP-complex (bottom), respectively (see Supplementary Fig. 18). c, d Processing of the hub mutants hub 1 E86A_R (c), and hub 2 Y54A_R (d) by GtgE in the GDP and GTP states, analyzed with a gel shift activity assay (see also Supplementary Fig. 15A). e Selected atom pair distances in a GDP-bound and hypothetical GTP-bound Rab32:GtgE complex from 190 ns MD-simulations (left panels) and histograms of respective distance occurrences (right panels), with an ion-pair distance threshold (< 4.5 Å) highlighted in gray. Top: E86_R and R87_R form a stable ion-pair after ca. 120 ns in the hypothetical GTP-bound complex (in green), initiated by an electrostatic repulsion from the γ-phosphate of GTP (see also Supplementary Fig. 18C). In contrast, no stable ion-pair between E86_R and R87_R is observed in the GDP-bound structure (in gray). Bottom: Distances between F88_R of Rab32 and K194_G of GtgE from MD-simulations of the GDP- (in gray) and GTP-bound (in green) Rab32:GtgE complexes. In the GDP-bound form, K194_G forms an interaction with F88_R, while in the GTP-bound complex, K194_G dissociates from F88_R, which may trigger the dissociation of GtgE from Rab32 (see also Supplementary Fig. 18A). The MD-simulations suggest that F88_R may form a decisive element for the GDP-state preference of GtgE toward Rab32. f Mutations in Rab32 hub 3 change GtgEs nucleotide-state selectivity leading to cleavage of Rab32:GppNHp. Rab32 F88G loaded with GDP or GppNHp was treated with cleared E. coli lysate (or 1:100 diluted lysate) overexpressing wild-type GtgE and analyzed by an gel shift assay (see also Supplementary Fig. 18B)