Evolutionary mix-and-match with MFS transporters II

Abstract

One fundamentally important problem for understanding the mechanism of coupling between substrate and H(+) translocation with secondary active transport proteins is the identification and physical localization of residues involved in substrate and H(+) binding. This information is exceptionally difficult to obtain with the Major Facilitator Superfamily (MFS) because of the broad sequence diversity of the members. The MFS is the largest and most diverse group of transporters, many of which are clinically important, and includes members from all kingdoms of life. A wide range of substrates is transported, in many instances against a concentration gradient by transduction of the energy stored in an H(+) electrochemical gradient using symport mechanisms, which are discussed herein. Crystallographic structures of MFS members indicate that a deep central hydrophilic cavity surrounded by 12 mostly irregular transmembrane helices represents a common structural feature. An inverted triple-helix structural symmetry motif within the N- and C-terminal six-helix bundles suggests that the proteins may have arisen by intragenic multiplication. In the work presented here, the triple-helix motifs are aligned in combinatorial fashion so as to detect functionally homologous positions with known atomic structures of MFS members. Substrate and H(+)-binding sites in symporters that transport substrates, ranging from simple ions like phosphate to more complex peptides or disaccharides, are found to be in similar locations. It also appears likely that there is a homologous ordered kinetic mechanism for the H(+)-coupled MFS symporters.

Keywords: bioenergetics; membrane transport; sequence alignment.

Fig. 1.

LacY structure. (A) Side view of the inward-facing conformation of LacY (PDB ID code: 2V8N). Helices are shown as rods; the numbers of helices in the N-terminal six-helix bundle are colored green, and the numbers of C-terminal six-helix bundle are colored orange. Critical residues are indicated (green, residues involved in sugar binding exclusively; orange, residues involved in sugar affinity and H+ translocation; magenta, residues involved in H+ translocation exclusively; yellow, weakly salt-bridged residues; details are provided in the main text). The water-accessible surface of the cavity is shown as a light blue surface [calculated using the Computed Atlas of Surface Topography of proteins (CASTp) Web tool with a probe size of 1.4 Å]. (B) Cytoplasmic view; the color-coding is the same as in A.

Fig. 2.

Schematic alignment of the helix-triplets in consecutive order in the sequence. (A) Helix-triplets (represented by colored boxes) from FucP, PiPT, XylE, and PepT are aligned with LacY (helices 1–3, blue; helices 4–6, green; helices 7–9, orange; helices 10–12, yellow). The flags indicate the loops within symmetry motifs. Helix-triplets from LacY are aligned with FucP (B), PiPT (C), and PepT (D) (helices 1–3, blue; helices 4–6, green; helices 7–9, orange; helices 10–12, yellow). The alignments are oriented with the LacY cytoplasmic side to the top. The flags indicate the loops within symmetry motifs (white, cytoplasmic loop; gray, periplasmic loop). The numbers on the flags indicate the two helices that are connected by the respective loop. See SI Appendix, Fig. S1 for a schematic representation.

Fig. 3.

Overall architecture of the substrate binding sites compared with LacY. (A) C-terminal six-helix bundle of XylE (colored in light olive) is superposed on the N-teminal six-helix bundle of LacY (side chains shown colored in light blue). The ligand of XylE, xylose, is shown as a gold ball-and-stick model. (B) C-terminal six-helix bundle of PiPT (colored in light olive) is superposed on the N-teminal six-helix bundle of LacY (side chains shown colored in light blue). The ligand of PiPT, phosphate, is shown as an orange ball-and-stick model. Oxygen and nitrogen atoms are colored red and blue respectively. The superposition of XylE and PiPT is provided in SI Appendix, Fig. S2.

Fig. 4.

Functional alignment of XylE (A), PiPT (B), and PepT (C) to LacY helix-triplets (stereo-view). The Cα atom trace is show as wire and colored according to the helix-triplet (Fig. 2 and Table 1) except for PiPT in B. Significantly equivalent residue pairs are shown as sticks in the same color. Labels of LacY residues are indicated in blue.

Fig. 5.

Transport cycle of LacY. Overview of the postulated steps in the transport model. Inward-facing (blue) and outward-facing (green) conformations are separated by the apo-intermediate conformational cluster (gray) or by the occluded-intermediate conformational cluster (orange). Substrate (S) and H⁺ are indicated. Steps are numbered consecutively: Substrate translocating transitions are indicated by blue arrows (steps 5–8) and transitions recycling the outward-open cavity are indicated by red arrows (steps 1–4). All steps are reversible (indicated by double-headed arrows). The blue-shaded area demarcates the equilibrium-exchange reaction. Examples of experimental coordinates (transporter and PDBID) associated with respective conformations are indicated.

Fig. 6.

Conformational changes in the substrate binding site of PepT (see SI Appendix, Fig. S3 for stero-view version of this figure). (A) Inward-open, substrate-bound state of PepT_Gk (PDB ID code: 4IKZ). (B) Inward-open, substrate-free state of PepT_Gk (PDB ID code: 4IKV). The position of the substrate detected in the substrate-bound state is indicated as white profile. (C) Inward-occluded conformation of PepT_So (PDB ID code: 2XUT). The colors of the bars at the bottom indicate the mechanistic affiliation of the respective states with regard to the mechanistic model shown in Fig. 5.