Fig. 1: The communication hinge senses nucleotide binding and is crucial for activity.

A Structure of B. subtilis BmrA (PDB ID: 6R81²⁹), an archetypical type IV ABC half transporter (protomers shown in pink and green). The zoom shows the Walker A motif (blue) and the Walker A helix leading into the ‘hinge’ consisting of a conserved arginine residue in the Walker A helix (R^WA, purple), a conserved bulky hydrophobic residue (φ, orange) and a conserved arginine pointing towards intracellular domain 2 (R^ICD2, yellow) (Structural overlay of this region in other ABC transporters shown in Supplementary Fig. 2A). B High degree of conservation within the Walker A and hinge residues (purple, orange and yellow) in both human and bacterial type IV ABC transporters (top). Residue numbers are based on the sequence of B. subtilis BmrA. Note that in bacterial transporters with at least 50% identity with BmrA, residue φ is a strictly conserved Trp residue (based on 226 type IV NBD sequences, bottom). Sequence logo was created using WebLogo3⁶⁴. C Nucleotides are sensed by the communication hinge residues. Comparison of NMR spectra of ¹⁵N-labeled isolated NBDs of BmrA, LmrA and MsbA in the apo state (purple, orange or yellow resonances, respectively) and in the presence of 10 mM ATP (blue) or ADP (teal) reveals chemical shift perturbations of the backbone (and in the case of tryptophan also side chain, φ_sc) amide resonances of the three hinge residues. Shown is a zoom into the respective ¹H, ¹⁵N-HSQC NMR spectra (Supplementary Fig. 2C) to highlight the three hinge residues. D, E The hinge residues are crucial for transporter function. ATPase activity of full-length BmrA variants reconstituted in MSP1E3D1 nanodiscs prepared with E. coli total lipid extract (D) and fluorescence-based transport assay with doxorubicin in inside-out vesicles prepared from E. coli cells overexpressing BmrA variants (E). In both cases, values were normalized to the WT set as 100%. For ATPase activity of WT, K380A and R389X constructs, results shown are the mean of three biological triplicates with three technical replicates each. For W413X constructs, results shown are the mean of two biological replicates with three technical replicates each. For R414X constructs, results shown are the mean of one biological replicate with three technical replicates. For transport activity of all constructs, results shown are the mean of three technical replicates. Protein expression and folding were not affected by the mutations (Supplementary Fig. 3).