Fig. 1: Functional analysis of inter-domain linker mutants in UraA.
From: Interdomain-linkers control conformational transitions in the SLC23 elevator transporter UraA
A Dimeric UraA structure (PDB:5XLS) with scaffold and transport domain in green or purple, respectively, and the interdomain-linkers in blue. B Topology plot of UraA (center) with color code as in (A). Upper and lower panels depict the sequence conservation of the extracellular and cytoplasmic interdomain-linkers in the UraA-subfamily, respectively, shown as sequence logo. C Absolute differences in dihedral angles between interdomain-linker residues in UraAIO (PDB:3QE7) and UraAOCC (PDB:5XLS). Δφ and Δψ are colored dark and light gray, respectively, and glycine and proline residues flanking the spacer helices are highlighted in red and orange, respectively. D In vivo uptake rates of [3H]-uracil by wild type UraA and interdomain-linker mutants upon expression in E. coli BW25113(ΔuraA) with technical replicates shown as scatter plot and derived mean values ± SEM as bars (control: n = 6, WT: n = 4, G112P, P121G, G320P, and P330G: n = 3). A UraA variant with three alanine substitutions in the substrate binding site (E241A, H245A, and E290A) served as negative control. E Representative size-exclusion chromatograms of decylmaltoside-solubilized UraA variants in the absence of substrate. F Melting temperature of interdomain-linker mutants as determined by differential scanning fluorimetry in absence and presence of 1 mM uracil with technical replicates shown as scatter plot and derived mean values ± SEM as bars (n = 3 for all samples).
