Structural basis for allosteric cross-talk between the asymmetric nucleotide binding sites of a heterodimeric ABC exporter

Abstract

ATP binding cassette (ABC) transporters mediate vital transport processes in every living cell. ATP hydrolysis, which fuels transport, displays positive cooperativity in numerous ABC transporters. In particular, heterodimeric ABC exporters exhibit pronounced allosteric coupling between a catalytically impaired degenerate site, where nucleotides bind tightly, and a consensus site, at which ATP is hydrolyzed in every transport cycle. Whereas the functional phenomenon of cooperativity is well described, its structural basis remains poorly understood. Here, we present the apo structure of the heterodimeric ABC exporter TM287/288 and compare it to the previously solved structure with adenosine 5′-(β,γ-imido)triphosphate (AMP-PNP) bound at the degenerate site. In contrast to other ABC exporter structures, the nucleotide binding domains (NBDs) of TM287/288 remain in molecular contact even in the absence of nucleotides, and the arrangement of the transmembrane domains (TMDs) is not influenced by AMP-PNP binding, a notion confirmed by double electron-electron resonance (DEER) measurements. Nucleotide binding at the degenerate site results in structural rearrangements, which are transmitted to the consensus site via two D-loops located at the NBD interface. These loops owe their name from a highly conserved aspartate and are directly connected to the catalytically important Walker B motif. The D-loop at the degenerate site ties the NBDs together even in the absence of nucleotides and substitution of its aspartate by alanine is well-tolerated. By contrast, the D-loop of the consensus site is flexible and the aspartate to alanine mutation and conformational restriction by cross-linking strongly reduces ATP hydrolysis and substrate transport.membrane transport | X-ray crystallography | allosteric communication A BC exporters are found in every organism (1, 2). They minimally consist of four domains and exist as homodimers or heterodimers. Two transmembrane domains (TMDs) span the membrane with a total of 12 transmembrane helices and form the substrate permeation pathway by alternating between inward-and outward-oriented states (Fig. S1A). A pair of nucleotide binding domains (NBDs) is connected to the TMDs via coupling helices and drive conformational cycling of the transporter by binding and hydrolysis of ATP, a process which is linked to NBD dimerization and dissociation (3).In their closed state, the NBDs sandwich two ATP molecules at the dimer interface by composite ATP binding sites involving conserved sequence motifs contributed by both subunits (4, 5). The A-loop and Walker A motif of one NBD and the ABC signature motif of the opposite NBD are involved in nucleotide binding. The Walker B glutamate and the switch-loop histidine constitute a catalytic dyad required for ATP hydrolysis (6, 7). In heterodimeric ABC exporters with asymmetric ATP binding sites, these catalytic residues are noncanonical at the degenerate site and ATP is therefore primarily, if not exclusively, hydrolyzed at the consensus site (8). The Q-and D...

Structural basis for antibacterial peptide self‐immunity by the bacterial ABC transporter McjD

“…The NBDs have moved in a “scissors‐like” motion similar to the transition between inward and outward‐open MsbA. A small degree of disengagement between the NBDs has also been reported for the heterodimeric ABC exporter TM287/288 (Hohl et al , 2012; Hohl et al , 2014), ABCG5/8 (Lee et al , 2016) and LptB 2 FG (Luo et al , 2017). …”

Modulation of Ionic Channels and Insulin Secretion by Drugs and Hormones in Pancreatic Beta Cells

“…The D-loop at NBS1 ties the NBDs together even in the absence of nucleotides, while the D-loop in NBS2 is flexible (Hohl et al, 2014). ATP hydrolysis is necessary to switch SUR1 into a conformation that antagonizes nucleotide binding to the Kir6.2 pore and stimulates channel opening.…”

Structural basis for the mechanism of ABC transporters

“…They have been called the 'engine' of the transporter [3] Thermotoga maritime [4,5] in the presence and absence of nucleotides which demonstrates how the NBDs interact with the ATP and its implications in the transport cycle.…”

“…The crystal structure of the heterodimeric bacterial ABC exporter TM287-TM288 from T. maritime has also been reported in the presence and absence of nucleotides in an inward-facing conformation [4,5]. Despite the clinical importance of human ABC transporters, only one structure has been reported, ABCB10, that is involved in erythropoiesis [23].…”