Multiple transferable resistance (mtr) operon in Neisseria gonhorroeae encodes the RND efflux-system MtrCDE. This tripartite pump is formed by the outer membrane factor (OMF) MtrE, the periplasmic adapter protein (PAP) MtrC, and the inner-membrane protein (IMP) MtrD.
MtrE shares common protomer organisation with other OMF (TolC-family) members (1). It forms a trimeric channel with a 12-stranded β-barrel embedded in the outer membrane, followed by an unique α-barrel domain, which extends into the periplasmic space. At the periplasmic side, each MtrE protomer has two pairs of helical-hairpins (H3/H4 and H7/H8), which contribute to the formation coiled-coil α-barrel and seal the periplasmic end of the channel in the resting state. Within the context of the MtrE trimer, the repetition of these helical pairs results in the formation of three pairs of “inter-protomer” and “intra-protomer groves” which are speculated to form the contact surface for the PAP MtrC, interaction with which is supposed to cause both tripartite complex association and dilation of the MtrE aperture.
Despite recent advances in structural biology of the tripartite complexes (2), the exact mechanism of the PAP engagement and channel opening of the MtrE remains unclear. This is further complicated by the significant divergence of MtrE from the canonical OMF TolC, the gating of which is relatively well-studied.
Here, we have employed an integrative structural biology approach for the identification of the critical gating residues supporting the closed and open state of the MtrE FA19. By combining site-directed mutagenesis and phenotypic characterization of efflux mutants alongside X-ray crystallography and modelling approaches we provide novel structural model of its gating, which appears markedly different from the rest of the TolC family (3). Furthermore, the structure and gating of the MtrE FA19 appears to be radically different from the previously reported FA136 (4).