Structure of the YajR transporter suggests a transport mechanism based on the conserved motif A

Abstract

The major facilitator superfamily (MFS) is the largest family of secondary active transporters and is present in all life kingdoms. Detailed structural basis of the substrate transport and energy-coupling mechanisms of these proteins remain to be elucidated. YajR is a putative proton-driven MFS transporter found in many Gram-negative bacteria. Here we report the crystal structure of Escherichia coli YajR at 3.15 Å resolution in an outward-facing conformation. In addition to having the 12 canonical transmembrane helices, the YajR structure includes a unique 65-residue C-terminal domain which is independently stable. The structure is unique in illustrating the functional role of "sequence motif A." This highly conserved element is seen to stabilize the outward conformation of YajR and suggests a general mechanism for the conformational change between the inward and outward states of the MFS transporters.

Keywords: charge relay; charge-dipole interaction; membrane potential; protonation.

Fig. 1.

Thermal stability and overall structure of YajR. (A) Thermal denaturation curves of YajR variants. Thermal denaturation of full-length WT YajR, the G51W G69W, and D73R mutants, and truncation YajR-ΔYAM (i.e., residues 1−394 of WT) were measured using the blue fluorescence of the cpm dye (the left vertical axis), and that of the YAM-alone sample, which does not contain any Cys residue, was measured using the green fluorescence of Sypro orange (the right vertical axis). Raw data were analyzed with Prism (GraphPad). Multiple experiments were performed with good reproducibility and representative results are shown. (B) Cartoon representation of the YajR crystal structure. In the TM core, central helices (i.e., TMs 1, 4, 7, and 10), rocker helices (TMs 2, 5, 8, and 11), and supporting helices (TMs 3, 6, 9, and 12) are shown in green, yellow, and red, respectively. The short helix α6–7 is colored in blue and the YAM domain in orange.

Fig. 2.

Functional roles of motif A. (A) Active conformation of motif A from the MFS family. Side chains in motif A and Asp126 are shown as stick models. Cα atoms of conserved Gly residues are shown as spheres. TM11 is colored from cyan at the N-terminal end to red at the C-terminal end. The figure was generated using PyMol. (B) Disulfide-bond formation assay. Protein samples were subjected to SDS/PAGE in the absence and presence of DTT, followed by immunoblot against the His-tag at the C termini of recombinant proteins. A double-point mutation G55C-G355C in the WT background, which presumably forms a disulfide bond in the inward conformation but not in the outward conformation, showed both reduced (Upper band) and oxidized (Lower band) forms. Additional G69W and D73R point mutations in the double Cys-mutation background (marked with an asterisk) showed predominantly oxidized forms. The WT YajR was included as a negative control. The results demonstrated that mutations in the motif A increase occupancy of the inward conformation.

Fig. 3.

Schematic diagram of MFS conformational changes. (A) Changes in a functional cycle of MFS. (B) Changes driven by protonation. Directions of movements are shown in arrows. (C) Alternative mechanism of proton-driven conformational changes. (D) Effects of the interdomain linker on motif A.