Structure and mechanotransmission mechanism of the MacB ABC transporter superfamily

Functional analysis of E. coli MacAB-TolC in vivo. (A) Antibiotic susceptibility comparison between E. coli C43 (DE3) ΔacrABΔmacAB cells expressing E. coli MacA and either wild-type or Glu170Gln E. coli MacB variant. The ratio of the MIC for the wild-type MacB to the Glu170Gln variant is shown graphically for ethidium bromide (EBR), chlorhexidine (CHX), SDS, gentamycin (GEN), tetracycline (TET), ciprofloxacin (CIP), chloramphenicol (CHL), kanamycin (KAN), colistin (COL), bacitracin (BAC), and erythromycin (ERY). Structures of erythromycin, bacitracin, and colistin are shown (Inset). (B) Structure of enterotoxin STII with disulfide bond-forming sulfur atoms (yellow spheres; Protein Data Bank: 1EHS) (32). (C) Detection of secreted enterotoxin in E. coli culture supernatants by SDS/PAGE. Lanes from left to right: C43 wild-type, C43ΔmacAB, C43ΔmacAB expressing plasmid-borne macA (A), macB (B), macAB (AB), or macAB with an ATPase inactivating Glu170Gln substitution (EQ). All strains contain a plasmid-expressing enterotoxin STII. (D) Model for two-step secretion of enterotoxin STII across the E. coli cell envelope. Sec and Dsb represent the general protein secretion machinery and disulfide bond incorporation systems, respectively.

Structure-based mutational analysis of E. coli MacB. Erythromycin resistance of C43 (DE3) ΔacrAB ΔmacAB cells expressing E. coli MacA and variant MacB mapped to a model of one monomer of E. coli MacB. Key indicates the fold decrease in MIC relative to cells expressing wild-type MacAB. (A) The MacB monomer presenting the dimer interface. (B) Close-up view of the periplasmic domain. (C) Top-down view of the MacB periplasmic domain. (D) MICs (µg/mL) for the 70 MacB variants tested. Homology model shown is based on our full-length AaMacB crystal structure and high-resolution structure of the E. coli MacB periplasmic domain.

Mechanotransmission mechanism of MacB. (A) Nucleotide-free (Left) and ATP-bound MacB (Right). (B) Top-down view of the MacB dimer showing the open head of the nucleotide-free form. (C) Equivalent view of the ATP-bound form showing closure of the periplasmic dimer. Domains of MacB colored as in Fig. 1. Both models represent E. coli MacB; the nucleotide-free form is extracted from the cryoEM structure of the MacAB-TolC complex (5NIL), and the ATP-bound form is a homology model generated from our crystal structure of the nucleotide-bound AaMacB (5LIL). A full molecular morph between the two states is presented in Movie S3.

A molecular bellows mechanism for substrate secretion by the MacAB-TolC tripartite efflux pump. (A) Proposed catalytic cycle of the MacAB-TolC efflux pump. MacB (red), MacA (purple), TolC (green) and substrate (blue). (B) Operation of a fireplace bellows.