Cryo-EM structure of an elusive pre-transport intermediate of the multidrug ABC transporter BmrCD reveals modes of asymmetric drug binding

Abstract

Vectorial substrate efflux by ATP binding cassette (ABC) transporters, which play a major role in multidrug resistance, entails the ATP-powered interconversion of the transporter between stable intermediates. Despite recent progress in structure elucidation of ABC transporters, a number of such intermediates have yet to be visualized and mechanistically interpreted. Here, we combine single particle cryo-EM, Double Electron Electron Resonance (DEER) spectroscopy with Molecular Dynamics simulations to profile and mechanistically frame the conformation of a hitherto unobserved intermediate in the context of BmrCD, a heterodimeric multidrug ABC exporter from Bacillus subtilis. In our cryo-EM structure, BmrCD adopts an inward facing architecture bound to both ATP and the substrate Hoechst-33342 and is capped by an extracellular domain which undergoes ATP-dependent conformational changes. A striking feature of the structure is a symmetric arrangement of the nucleotide-binding domain (NBD) in the presence of ATP whereas binding of Hoechst at two distinct sites in an acidic pocket stabilizes an asymmetric arrangement of the transmembrane domain architecture (TMD). Mutation of residues coordinating Hoechst in the structure abrogates the cooperative stimulation of ATP hydrolysis. In conjunction with previous studies, our findings suggest a mechanistic role for symmetry mismatch between NBDs and TMDs in the conformational cycle of ABC transporters. Moreover, the resolved structures of bimodally-bound drugs are of notable importance for future rational design and optimization of molecules for targeted transport inhibition of ABC transporters.

Structure and transport mechanism of the human cholesterol transporter ABCG1

“…However, our ABCG1 structure revealed that the NPXDF motif (N 320 PADF 324 ) does not participate in the crosslinking of NBDs but provides a helical structure for the approaching of related residues. As shown in Figure 2A, two ATP molecules bind to the Walker A motifs of the separated NBDs, which indicates that the present structure of ABCG1 is under a turnover condition, a rather low-energy state during the transport cycle, as proposed in recent studies (Thaker et al, 2021;Yu et al, 2021).…”