Crystal Structure of AcrB in Complex with a Single Transmembrane Subunit Reveals Another Twist

Abstract

Bacterial drug resistance is a serious concern for human health. Multidrug efflux pumps export a broad variety of substrates out of the cell and thereby convey resistance to the host. In Escherichia coli, the AcrB:AcrA:TolC efflux complex forms a principal transporter for which structures of the individual component proteins have been determined in isolation. Here, we present the X-ray structure of AcrB in complex with a single transmembrane protein, assigned by mass spectrometry as YajC. A specific rotation of the periplasmic porter domain of AcrB is also revealed, consistent with the hypothesized "twist-to-open" mechanism for TolC activation. Growth experiments with yajc-deleted E. coli reveal a modest increase in the organism's susceptibility to beta-lactam antibiotics, but this effect could not conclusively be attributed to the loss of interactions between YajC and AcrB.

Conserved small protein associates with the multidrug efflux pump AcrB and differentially affects antibiotic resistance

“…Interestingly, AcrB previously was found to associate with YajC, another relatively small TM protein (110 amino acids) in structural studies of AcrB (16). YajC is encoded within an operon that also codes for the RND family member SecDF and is part of the SecDF-YajC complex that facilitates protein secretion via SecYEG.…”

“…YajC is encoded within an operon that also codes for the RND family member SecDF and is part of the SecDF-YajC complex that facilitates protein secretion via SecYEG. The most highly conserved residues of YajC reside in its TM α-helix and were found to interact with a highly conserved binding pocket along the TM surface of AcrB (16). ΔyajC cells were reported to be mildly sensitive to certain β-lactam antibiotics.…”

“…The gradient plate assay also showed that acrZ-SPA fusion is phenotypically wild type. In contrast to the deletion of acrZ, deletions of yajC, which encodes another protein shown to bind AcrB (16), and of mdtC, mdtF, and acrD, encoding three other multidrug transporters, do not result in increased chloramphenicol or tetracycline sensitivity (Fig. S5B).…”

“…As a control, we carried out a similar purification with His 6 -tagged PhoR, an unrelated multitransmembrane protein. This experiment is complicated by the fact that untagged AcrB was found to copurify with other hexahistidine-tagged proteins due to the histidine-rich cluster on its surface (16). Indeed, both AcrB and AcrZ-SPA were found to copurify with PhoR-His 6 from a phoRHis 6 strain carrying wild-type acrAB (Fig.…”

Structural and functional aspects of the multidrug efflux pump AcrB

“…It is postulated that substrate might access the central cavity via these vestibules (Murakami et al, 2002). Indeed, there have been several reports on AcrB/substrate co-crystals with positive densities in the electron density maps derived from (symmetric) R32 crystals that have been interpreted as substrate molecules bound to the inner wall of the AcrB central cavity (Yu et al, 2003a(Yu et al, ,b, 2005Pos et al, 2004;Tornroth-Horsefield et al, 2007;see Figure 1B, C).…”