Crystal structure of the Alcanivorax borkumensis YdaH transporter reveals an unusual topology

Abstract

The potential of the folic acid biosynthesis pathway as a target for the development of antibiotics has been clinically validated. However, many pathogens have developed resistance to these antibiotics, prompting a re-evaluation of potential drug targets within the pathway. The ydaH gene of Alcanivorax borkumensis encodes an integral membrane protein of the AbgT family of transporters for which no structural information was available. Here we report the crystal structure of A. borkumensis YdaH, revealing a dimeric molecule with an architecture distinct from other families of transporters. YdaH is a bowl-shaped dimer with a solvent-filled basin extending from the cytoplasm to halfway across the membrane bilayer. Each subunit of the transporter contains nine transmembrane helices and two hairpins that suggest a plausible pathway for substrate transport. Further analyses also suggest that YdaH could act as an antibiotic efflux pump and mediate bacterial resistance to sulfonamide antimetabolite drugs.

Fig. 1

Structure of the A. borkumensis YdaH transporter. (a) Transmembrane topology of A. borkumensis YdaH. The transporter contains nine transmembrane helices (TMs) and two hairpins (HPs). (b) Ribbon diagram of a dimer of YdaH viewed in the membrane plane. The right subunit of the dimer is colored using a rainbow gradient from the N-terminus (blue) to the C-terminus (red), whereas the left subunit is colored gray. The YdaH dimer forms a bowl-shaped structure with a concave aqueous basin facing the intracellular solution. (c) Surface representation of the YdaH dimer sliced through the middle of the protein. Each protomer forms an internal cavity (red arrow), which is accessible to the cytoplasm. (d) Bottom view of a surface representation of the YdaH dimer, indicating a solvent accessible cavity (red circle) from each protomer of the protein. The two protomers are colored gray and blue.

Fig. 2

Inner and outer cores of YdaH. (a) The inner core of YdaH, comprising TMs 1, 2, 5, 6 and 7 (colored slate), contributes to dimerization as well as formation of a frame-like structure housing the outer core of the protomer. The outer core of YdaH is composed of TMs 3, 4, 8, 9 as well as HPs 1 and 2 (colored yellow). (b) The outer core of YdaH forms a channel (colored purple) spanning approximately from the middle of the inner membrane up to the periplasmic space. This channel was calculated using the program CAVER (http://loschmidt.chemi.muni.cz/caver). The secondary structural elements of the YdaH protomer are in yellow. Residues D180, N390, W400, P418, R426, D429 and N433 are in green sticks. The bound sodium ion is shown as an orange sphere. (c) The bound Na⁺ (orange sphere) is found to coordinate with N390, G394, D429, N433 and a water molecule (red sphere). The F_o-F_c map, showing the bound Na⁺ and H₂O, is contoured at 3.0 σ (blue mesh).

Fig. 3

Accumulation of radioactive p-aminobenzoic acid. (a) Time course of [³H]-PABA accumulation by E. coli BL21(DE3)ΔabgTΔpabA double knockout cells transformed with pET15bΩydaH or pET15b. Cells expressing ydaH (blue curve) show a significant decrease in [³H]-PABA accumulation when compared with cells carrying the empty vector (black curve). Error bars represent standard deviation (n = 3). “*” indicates values of BL21(DE3)ΔabgTΔpabA /pET15bΩydaH cells that are significantly different from the control (BL21(DE3)ΔabgTΔpabA /pET15b) values (P < 0.04; student’s t-test). (b) Mutants of the YdaH transporter. Cells possessing the mutant transporter D180A, N390A, W400A, P418A, D429A or N433A show a significant increase in the level of [³H]-PABA accumulations compared with cells expressing wild-type YdaH. However, cells expressing R426A are able to decrease the [³H]-PABA concentration when compared with cells carrying wild-type YdaH. Error bars represent standard deviation (n = 3). “*” indicates values of BL21(DE3)ΔabgTΔpabA/pET15b and BL21(DE3)ΔabgTΔpabA cells expressing the mutant transporters that are significantly higher than that of BL21(DE3)ΔabgTΔpabA/pET15bΩydaH expressing wild-type YdaH (P < 0.05; student’s t-test).

Fig. 4

Intracellular folic acid concentration. Folic acid concentration in E. coli BL21(DE3)ΔabgTΔpabA double knockout cells expressing YdaH were markedly reduced in comparison with cells transformed with the empty vector. When transformed with plasmid expressing the mutant transporter, D180A, N390, W400A, P418A, D429A and N433A, folic acid production was significantly increased in these cells. However, the level of intracellular folic acid concentration in BL21(DE3)ΔabgTΔpabA cells expressing R426A was nearly identical to that of the double knockout strain carrying wild-type YdaH. Error bars denote standard deviation of biological replicates, n = 4. “*” indicates values of BL21(DE3)ΔabgTΔpabA/pET15b and BL21(DE3)ΔabgTΔpabA cells expressing the mutant transporters that are significantly higher than that of BL21(DE3)ΔabgTΔpabA/pET15bΩydaH expressing wild-type YdaH (P < 0.02; student’s t-test).

Fig. 5

Accumulation of radioactive sulfamethazine. E. coli BL21(DE3)ΔabgTΔpabA cells expressing YdaH show a significant decrease in [³H]-sulfamethazine accumulation when compared with cells carrying the empty vector. When transformed with plasmids expressing the mutant transporters, D180A, N390, W400A, P418A, R426A, D429A and N433A, the levels of intracellular [³H]-sulfamethazine accumulation were much higher than that of cells expressing wild-type YdaH. Error bars depict standard deviation for n = 3. “*” indicates values of BL21(DE3)ΔabgTΔpabA/pET15b and BL21(DE3)ΔabgTΔpabA cells expressing the mutant transporters that are significantly higher than that of BL21(DE3)ΔabgTΔpabA/pET15bΩydaH expressing wild-type YdaH (P < 0.001; student’s t-test).

Fig. 6

Sodium ion enhances sulfamethazine efflux via YdaH. (a) Accumulation of radioactive sulfamethazine in BL21(DE3)ΔabgTΔpabA/pET15bΩydaH cells with difference sodium ion concentrations. Cells showed a significant decrease in [³H]-sulfamethazine accumulation in the presence of Na⁺. Error bars denote standard deviation (n = 3). “*” indicates values of BL21(DE3)ΔabgTΔpabA/pET15b cells (control) that are significantly different from those of BL21(DE3) ΔabgTΔpabA/pET15bΩydaH expressing wild-type YdaH (P < 0.001; student’s t-test). (b) Efflux of radioactive sulfamethazine in BL21(DE3)ΔabgTΔpabA/pET15bΩydaH cells in the presence of sodium or potassium ions. The presence of Na⁺ significantly enhances [³H]-sulfamethazine efflux in BL21(DE3)ΔabgTΔpabA/pET15bΩydaH cells (black, control cells with the empty vector; red, 0 mM NaCl; magenta, 5 mM KCl; blue, 5 mM NaCl). Error bars denote standard deviation (n = 3). “*” indicates values of radioactive counts of intracellular [³H]-sulfamethazine in BL21(DE3)ΔabgTΔpabA/pET15bΩydaH cells with 5 mM NaCl (blue) that are significantly different from those of the control (black) (P < 0.001; student’s t-test).