Family resemblances: A common fold for some dimeric ion-coupled secondary transporters

Abstract

Membrane transporter proteins catalyze the passage of a broad range of solutes across cell membranes, allowing the uptake and efflux of crucial compounds. Because of the difficulty of expressing, purifying, and crystallizing integral membrane proteins, relatively few transporter structures have been elucidated to date. Although every membrane transporter has unique characteristics, structural and mechanistic similarities between evolutionarily diverse transporters have been identified. Here, we compare two recently reported structures of membrane proteins that act as antimicrobial efflux pumps, namely MtrF from Neisseria gonorrhoeae and YdaH from Alcanivorax borkumensis, both with each other and with the previously published structure of a sodium-dependent dicarboxylate transporter from Vibrio cholerae, VcINDY. MtrF and YdaH belong to the p-aminobenzoyl-glutamate transporter (AbgT) family and have been reported as having architectures distinct from those of all other families of transporters. However, our comparative analysis reveals a similar structural arrangement in all three proteins, with highly conserved secondary structure elements. Despite their differences in biological function, the overall "design principle" of MtrF and YdaH appears to be almost identical to that of VcINDY, with a dimeric quaternary structure, helical hairpins, and clear boundaries between the transport and scaffold domains. This observation demonstrates once more that the same secondary transporter architecture can be exploited for multiple distinct transport modes, including cotransport and antiport. Based on our comparisons, we detected conserved motifs in the substrate-binding region and predict specific residues likely to be involved in cation or substrate binding. These findings should prove useful for the future characterization of the transport mechanisms of these families of secondary active transporters.

Figure 1.

Structure of the VcINDY, MtrF, and YdaH transporters. Cartoon representation of the VcINDY (A), MtrF (B), and YdaH (C) dimer structures shown from the extracellular side of the membrane (left). The scaffold domain is colored cyan and dark blue, with the dark blue regions indicating helices involved in oligomerization. The transport domain containing the substrate-binding site(s) is colored green. The helices forming each domain are detailed in the topology diagrams to the right. HPs 1 and 2 correspond to HPin and HPout, respectively, in the nomenclature of Mancusso et al. (2012).

Figure 2.

Structural comparison of the VcINDY, MtrF, and YdaH transporters. (A) A structural alignment of the whole structure of each protomer is shown from the extracellular side of the membrane, with the helices colored according to the topology. The MtrF-VcINDY, YdaH-VcINDY, and MtrF-YdaH alignments are shown on the left, middle, and right, respectively. The structural superimpositions of the scaffold-oligomerization (B) and transport (C) domain are shown from a view parallel to the membrane plane. The scaffold-oligomerization domains exhibit the most significant differences, which are highlighted in red. The structural alignments were obtained using the program Fr-TM-align. The RMSD and TM-score of the structural alignment are given in each case, along with the number of aligned residues and the length of the shortest input.

Figure 3.

Ligand pathways and binding sites in the three inward-facing structures. Cartoon representation of the MtrF (A), YdaH (B), and VcINDY (C) dimer structures viewed along the membrane plane. The subunits on the right are colored with separate colors for each helix. Ligands are shown as spheres, with their presumed pathways illustrated using arrows. The position of the dimers in the membrane was determined for the x-ray structures using the Orientations of Proteins in Membranes (OPM) server. Each of the three transporters forms an upside-down bowl-shaped structure with a concave aqueous basin facing the intracellular side. VcINDY has a citrate molecule and a Na⁺ ion modeled in the binding site of each protomer, whereas YdaH has a Na⁺ ion in each protomer. No substrate was detected in the MtrF structure. To the right of each dimer, the transport domain is shown with a surface representation of the binding pocket in white, indicating that the binding sites vary in size in the presence of substrates. (D) Comparison of the tilt angle of TM5b in VcINDY (light blue and pink) with either TM3b of YdaH (left; dark blue and red) or TM3b of MtrF (right; dark blue and red). The binding pockets were superimposed using the first three helix turns of HP2b, and the angle of TM3b relative to TM5b was calculated. The position of the ligands is shown for VcINDY (transparent spheres) and YdaH (purple sphere).

Figure 4.

Sequence analysis of the three transporters. Sequence alignment between MtrF and VcINDY (A), YdaH and VcINDY (B), and MtrF and YdaH (C), extracted from the structure alignment obtained with the Fr-TM-align program. The sequence identity and similarity are given in each case. The alignment is colored according to the chemical properties of the residues: pale yellow, aliphatic (Ala, Ile, Leu, Met, and Val) and cysteine; cyan, polar uncharged (Asn, Gln, Ser, and Thr); yellow-orange, aromatic (Phe, Trp, and Tyr); red, acidic (Asp and Glu); purple, basic (Lys, Arg, and His); pink, Gly and Pro. The secondary structure (helix) assignments were obtained with DSSP and are indicated by blue rectangles. Black rectangles mark the motifs involved in the substrate-binding sites.

Figure 5.

Sequence logos for key motifs in homologues of VcINDY, MtrF, and YdaH. The conservation of motifs for VcINDY (A), MtrF (B), and YdaH (C) was evaluated separately over a set of close homologues of each protein (≥90% identity). The motifs for YdaH are identical to that of MtrF because the set of homologues contain >90% of the same sequences. Multiple sequence alignments of the homologue sequences were built in each case, and logos were created with WebLogo (version 3.4; Crooks et al., 2004). The secondary structure assignments are indicated by blue (helix) and gray (loop) rectangles. Yellow triangles indicate the residues involved in sodium binding according to the structural data.

Figure 6.

Known and predicted binding ion sites in the ion transporter superfamily fold. Two Na⁺-binding sites have been reported based on the experimental electron density; these ions are shown as opaque purple spheres, and residues coordinating those ions directly are indicated by labels in bold font. (A) In VcINDY (PDB accession no. 4F35), a density was identified at the region labeled Na2. (B) In YdaH (PDB accession no. 4R0C), a density was identified at the region labeled Na3. The corresponding sites in VcINDY (Na3) and YdaH (Na2), shown as transparent spheres, were predicted after superposing YdaH on VcINDY with Fr-TM-align. Putative coordinating residues (Asn, Gln, Asp, Glu, Thr, or Ser within 8 Å of the ion) for the predicted YdaH-Na2 and VcINDY-Na3 site regions are indicated with spheres and labeled. The ion nomenclature follows that adopted previously for NaPi-II (see Results). The protein is shown as ribbons, and residues of interest are highlighted using spheres at the position of the Cα atom. (C) The equivalent region in MtrF (PDB accession no. 4R1I), which is a putative H⁺-coupled transporter. (D) Structural model of human NaPi-IIa, with ions placed according to the results from VcINDY and YdaH shown as purple spheres. For NaPi-IIa, biochemical and electrophysiological evidence supports a role in phosphate or sodium binding for the residues shown as spheres (*). During the transport cycle of NaPi-IIa, an additional sodium binds before Na2 and Na3, at a site named Na1 (not depicted). The structures of VcINDY, YdaH, and MtrF are oriented with the extracellular side toward the top of the page, whereas NaPi-IIa is oriented with the cytoplasmic side toward the top of the page, because it inserts in the membrane in the opposite direction from the other transporters.