ATP-binding cassette (ABC) transporters couple hydrolysis of ATP at their nucleotide-binding domains (NBDs) with conformational changes in their transmembrane domains (TMDs), altering the accessibility of the substrate binding pocket and allowing substrate transport across the bilayer. Most purified ABC-transporters display a basal ATPase activity that is stimulated by substrate. However, the molecular mechanisms coupling the NBDs and TMDs remain poorly understood. Here, we use luminescence resonance energy transfer (LRET) to follow conformational changes in the human mitochondrial ABCB10 transporter in response to substrate binding. ABCB10 is an essential homodimeric transporter that exports biliverdin, a heme degradation product with antioxidant properties. Using a labeled single-cysteine ABCB10 reconstituted in MSP2N2 nanodiscs, at physiological temperature, we have detected a clear shift in the NBDs conformational equilibrium in response to biliverdin, with the appearance of a more compact conformation once the substrate is added. Also, we found that mutagenesis of substrate-binding pocket arginine residues to alanine leads to substrate-like elevated ATPase activity, even in the absence of biliverdin. This activity is not further stimulated by biliverdin. Interestingly, in apo conditions this mutant adopts a conformational equilibrium that resembles that of the “wild-type” protein in the biliverdin-bound state, suggesting that these arginine residues are critical for the allosteric coupling between TMDs and NBDs. Thus, the RtoA mutation leads to a constitutively stimulated ABCB10 that in the absence of substrate can adopt a conformation similar to the biliverdin-bound state, which is likely important for increasing the ATPase rate. We are currently evaluating alternative substitutions to determine whether the charge or the size of the amino acid has a more critical role in this allosteric coupling associated with substrate-induced ATPase stimulation. R01GM145938.
Zoghbi et al. (Sun,) studied this question.