Structural basis for amino acid export by DMT superfamily transporter YddG

Abstract

The drug/metabolite transporter (DMT) superfamily is a large group of membrane transporters ubiquitously found in eukaryotes, bacteria and archaea, and includes exporters for a remarkably wide range of substrates, such as toxic compounds and metabolites. YddG is a bacterial DMT protein that expels aromatic amino acids and exogenous toxic compounds, thereby contributing to cellular homeostasis. Here we present structural and functional analyses of YddG. Using liposome-based analyses, we show that Escherichia coli and Starkeya novella YddG export various amino acids. The crystal structure of S. novella YddG at 2.4 Å resolution reveals a new membrane transporter topology, with ten transmembrane segments in an outward-facing state. The overall structure is basket-shaped, with a large substrate-binding cavity at the centre of the molecule, and is composed of inverted structural repeats related by two-fold pseudo-symmetry. On the basis of this intramolecular symmetry, we propose a structural model for the inward-facing state and a mechanism of the conformational change for substrate transport, which we confirmed by biochemical analyses. These findings provide a structural basis for the mechanism of transport of DMT superfamily proteins.

Free energy landscape for the entire transport cycle of triose-phosphate/phosphate translocator

“…Recently, we reported the crystal structure of GsGPT from the thermophilic red alga Galdieria sulphuraria (Lee et al, 2017), which is functionally similar to TPT, rather than GPT, despite its name based on the sequence similarity(Linka et al, 2008). GsGPT is composed of 10 α-helical transmembrane (TM) helices (Figure 1a), and adopts the drug metabolite transporter (DMT) superfamily(Jack, Yang, & H. Saier, 2001) fold, similar to that of the recently reported bacterial metabolite transporter, Starkeya novella YddG (SnYddG)(Tsuchiya et al, 2016), in which the N-terminal half (TM1-5) is related to the C-terminal half (TM6-10) by two-fold pseudo-symmetry. We obtained the two crystal structures bound with different substrates, P i and 3-PGA (Figure 1b and Figure 1—figure supplement 1).…”

Applications of sequence coevolution in membrane protein biochemistry

“…Evolutionary couplings of multiple conformations have been described in several types of membrane protein transporters: major facilitator superfamily members GlpT, a bacterial glycerol-3-phosphaste transporter, OCTN1, a human organic cation transporter, and LacY, the bacterial lactose permease [40,108]; Escherichia coli YddG, an aromatic amino acid transporter from the drug/metabolite transporter (DMT) superfamily [109]; and broadly for ABC transporters [110]. In the different conformations of transporters using an alternating-access mechanism [111-113], individual residues alternate between being solvent-accessible and in contact with other residues in the protein.…”

“…This produces conflicting evolutionary couplings, as residues at one vestibule will coevolve due to their proximity in one conformation, while residues in the other vestibule will coevolve due to their proximity in the other conformation (Figure 5A). When signals from multiple conformations are used to build a de novo model, the conflicting coevolving pairs may enforce an occluded structure where both vestibules are closed, as illustrated for the YddG case in Figure 5B [109]. While occluded structures have been observed experimentally [114,115] and may play a role in the transport cycle of some transporters, determining the structure of inward-open and/or outward-open states requires selection or re-weighting of coevolving pairs, such that some are consistent with single conformations while others may be consistent with multiple conformations.…”

“…Structural modeling of these transporters using all evolutionary couplings can generate an occluded structure (middle). (B) The outward-open crystal structure of YddG (gray; PDB ID: 5I20) differs from the EVFold_membrane predicted structure (orange) in that the transmembrane segments of the predicted structure are bent inwards (shown with black arrows), closing off the outer vestibule [109]. …”

Mechanical Deformation Mediated Transmembrane Transport

“…[1,2] Naturally, molecules pass across biological membranes by either active transport [3][4][5][6] or passive diffusion. [19][20][21][22][23][24][25][26][27][28] However, artificial membrane channels created so far mainly rely on complicated chemical synthesis and are inefficient to dynamically control the transport process. [19][20][21][22][23][24][25][26][27][28] However, artificial membrane channels created so far mainly rely on complicated chemical synthesis and are inefficient to dynamically control the transport process.…”