Fig. 1

BsGpsB:BsPBP1 interactions require conserved arginines in the α-helical cytoplasmic minidomain of BsPBP1. a BsGpsB interacts with the first 16 amino acids of BsPBP1. SPR sensorgrams of full-length BsGpsB against immobilised full-length BsPBP1 (black) and BsPBP1_17-914 (red). BsGpsB does not interact with the BsPBP1_17-914 coated chip, even when 25 μM GpsB is injected. b Cartoon (left) and surface representation (right) of the crystal structure of the BsGpsB_5-64:BsPBP1_1-17 complex. BsGpsB_5-64 is coloured cyan and BsPBP1_1-17 is coloured green and the likely plane of the bacterial membrane is shown as a red box. c The BsGpsB_5-64:BsPBP1_1-17 complex is dependent upon a conserved SRxxR(R/K) motif in BsPBP1. Key interfacial residues in the BsGpsB_5-64:BsPBP1_1-17 complex are shown as sticks and coloured (and labelled) blue and green, respectively. The carbonyl oxygens of BsGpsB^Ile13, BsGpsB^Leu14 and BsGpsB^Lys16 are labelled with their respective red numerals. Hydrogen bonds are shown as black dashed lines and the van der Waals’ interactions between BsGpsB^Leu34 and BsPBP1^Arg8 are in yellow. d Mutation of key BsPBP1 interfacial residues in the structure of BsGpsB_5-64:BsPBP1_1-17 leads to a loss of binding of position 16 TAMRA-labelled BsPBP1_1-32 variants to BsGpsB_1-68 as measured by fluorescence polarisation. When the BsPBP1_1-32 peptide was labelled with fluorescein at position 31 the measured affinity was the same as if the fluorophore was at position 16, indicating that the fluorophore itself or its position in the peptide has no impact on the binding interaction. The calculated dissociation constants can be found in Supplementary Table 1