Structure of a prokaryotic virtual proton pump at 3.2 A resolution

Abstract

To reach the mammalian gut, enteric bacteria must pass through the stomach. Many such organisms survive exposure to the harsh gastric environment (pH 1.5-4) by mounting extreme acid-resistance responses, one of which, the arginine-dependent system of Escherichia coli, has been studied at levels of cellular physiology, molecular genetics and protein biochemistry. This multiprotein system keeps the cytoplasm above pH 5 during acid challenge by continually pumping protons out of the cell using the free energy of arginine decarboxylation. At the heart of the process is a 'virtual proton pump' in the inner membrane, called AdiC, that imports L-arginine from the gastric juice and exports its decarboxylation product agmatine. AdiC belongs to the APC superfamily of membrane proteins, which transports amino acids, polyamines and organic cations in a multitude of biological roles, including delivery of arginine for nitric oxide synthesis, facilitation of insulin release from pancreatic beta-cells, and, when inappropriately overexpressed, provisioning of certain fast-growing neoplastic cells with amino acids. High-resolution structures and detailed transport mechanisms of APC transporters are currently unknown. Here we describe a crystal structure of AdiC at 3.2 A resolution. The protein is captured in an outward-open, substrate-free conformation with transmembrane architecture remarkably similar to that seen in four other families of apparently unrelated transport proteins.

Fig 1. AdiC physiology and function

a. Virtual proton pumping in extreme acid resistance. Schematic of Arg-dependent acid resistance in E. coli, with AdiC-mediated Arg-Agm exchange across the inner membrane coupled to acid-activated Arg-decarboxylase AdiA. Virtual proton is shown in black circle. b. Selection of α-carboxylate for transport. Uptake (fraction of total counts added) of ¹⁴C-Arg at 50 µM external concentration into AdiC-reconstituted liposomes (2 µg/mg lipid) was followed for 30 min, and then either Arg (filled triangles) or Arg-NH₂ (open triangles) was added (arrow) externally to the indicated concentration. Additional Arg uptake experiments used the indicated AdiC mutants (dashed curves). Error bars indicate s.e.m. of triplicate experiments. c. Proper assembly of tandem construct. Size-exclusion profiles of purified homodimeric AdiC (dashed trace) and WT-WT tandem (solid trace) in its final purification step immediately after elution from Co-affinity column. Material eluting between void volume of Superdex-200 column (asterisk) and main peak most likely represents improperly assembled, oligomeric tandems. Identical profile is obtained with WT-MUT tandem in which the second subunit contains the W293L mutation (data not shown). d. A half-dead heterodimer is functionally active. Arg uptake timecourses for WT-WT to WT-MUT tandems reconstituted at 0.2 µg/mg lipid, a low protein density where transporting liposomes carry only one copy to the reconstituted protein,³². WT-WT transport rate is ~70% of normal homodimeric AdiC (data not shown).

Fig 2. Structure of AdiC

a. Sequence of AdiC aligned with two other E. coli virtual proton pump exchangers in the APC superfamily: PotE (ornithine/putrescine) and CadB (lysine/cadaverine). The five TMs lining the central cavity are highlighted, with matched colors representing helices paired by inverse repeat. Conserved aromatic residues discussed in the text are indicated in red. b. Ribbon diagrams of homodimer viewed from membrane, extracellular side up (upper panel) and from extracellular solution (lower panel), with Y93, W202, W293 side chains indicated (pink sticks). N- and C-termini indicated by blue, red spheres. TM 11,12 are indicated as darker hue in each subunit.

Fig 3. Structural alignment of AdiC with four transporter families

Alignments of AdiC (cyan) with four transporters (grey) are shown. a. LeuT, a wide-spectrum amino acid transporter. Left panel: TMs 1–10, showing bound substrate (spacefilled) and conserved aromatics of AdiC (pink sticks); right panel: TM1-5 core. b. Alignments of AdiC TM 1–5 core with BetP (left panel), Mhp1 (center panel), and SGLT1 (right panel). Cα r.m.s.d. with AdiC: LeuT 3.1 Å, SGLT1 4.0 Å, Mhp1 3.4 Å, BetP 3.6 Å.

Fig 4. AdiC cavity in outward-open conformation

a. Oblique stereo view from extracellular side of single AdiC subunit, showing locations of aromatic sidechains (red). b. Closeup view of proposed substrate-binding region, with 2Fo-Fc map contoured at 1.5 σ. Y93, W293 shown in pink sticks.