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ATP binding cassette (ABC) transporters participate in many processes central to life including cell wall biosynthesis, lipid homeostasis, and drug efflux. Large conformational rearrangements accompany transport and allosterically couple substrate transport in the transmembrane region to dimerization of nucleotide binding domains (NBDs) nearly ~30-40Å away in the cytoplasm. How the two binding sites coordinate remains elusive, particularly in a class of ABC transporters with only one catalytically competent NBD, the asymmetric ABC transporters. The peptide transporter TmrAB from Thermus thermophilus is one such asymmetric transporter, and here we present biochemical evidence that substrate binding couples to ATP binding in nonequivalent ways through each half of the TmrAB heterodimer. Specifically, we show that this unique mechanism depends on a highly conserved electrostatic motif in a region previously termed the peptide sensor. In this conformationally dynamic region, we demonstrate that mutation of an absolutely conserved glycine in the catalytically active TmrA chain (G131A) accelerates ATPase activity while uncoupling substrate binding from ATP hydrolysis. Surprisingly, mutation of the conserved glycine in the non-functional TmrB chain (G116A) resulted in the opposite effect in which ATPase activity is decreased with little effect on substrate binding. Our findings highlight an intrinsic asymmetry extending beyond that of the NBDs in asymmetric ABC transporters and support a mechanism where the peptide sensor in each half of TmrAB plays different roles in substrate transport.

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