AdiC belongs to the amino acid-polyamine-organocation (APC) transporter superfamily, whose members appear throughout the biological world. APC transporters mediate a panoply of biological tasks, such as supplying amino acids for metabolism, arginine for nitric oxide synthesis, and headgroups such as choline, ethanolamine, and inositol for phospholipid synthesis, as well as energizing membranes of certain anaerobic bacteria in the absence of electron transport. In bacterial XAR, AdiC specifically imports arginine from gastric fluid and exports agmatine along with a "virtual proton" (5
) out of the cytosol in each turnover. Specific recognition of extracellular arginine can be understood from x-ray crystal structures of AdiC solved in two arginine-bound states: extracellular-open (9
) and extracellular-occluded (8
). In these structures, the aromatic side chain of Trp293 appears to stabilize arginine binding by a cation-π interaction with the substrate's guanidinium group. Mutagenesis of a variety of APC transporters have identified aromatic interactions as crucial for substrate binding, and indeed mutations of Trp293 in AdiC itself lead to a complete loss of transport activity (6
A less understood molecular interaction in AdiC's crystal structures is a hydrogen bond between the α-carboxylate of arginine and the hydroxyl group of Ser26, a residue conserved among APC virtual proton pump exchangers (7
). Although it is natural to imagine that this interaction would provide significant binding energy for extracellular amino acids, we find with some surprise that Cys () or Ala (Fig. S4
) substitutions at Ser26 do not undermine arginine transport. Viewed from an evolutionary perspective, however, these results appear less puzzling, since in the stomach, where amino acids are available, the external-facing conformation of AdiC faces no evolutionary pressure to differentiate amino acids from substrates with other chemical groups attached to the α-carbon. A vivid example seen here is that agmatine, which lacks the α-carboxylate, has a similar extracellular transport Km
(~50 µM) as arginine.
No AdiC structure has yet been solved in the cytoplasmic-open conformation. With the sided system developed here, we may begin to gain insight into ligand binding and specificity for AdiC's intracellular side. Kinetic analysis reveals that arginine and agmatine have a similar Km (~100 µM) for this side of the protein, a result that appears surprising at first glance, as transport of arginine from the cytoplasm would produce a futile cycle useless for acid resistance, and one might therefore expect intracellular selectivity for agmatine. We should remember, however, that cytoplasmic arginine is kept low by robust decarboxylation and thus presents no problem as a cytoplasmic competitor for agmatine.
In the gastric fluid, the two sides of AdiC operate under very different conditions. Extracellular pH can fall as low as 1.5, while cytoplasmic pH is maintained in the range 4.5–5.5 by the XAR response (13
). Therefore, the two sides of AdiC must deal with distinct environmental challenges. With a pKa
of 2.3, arginine's carboxylate presents a problem of molecular recognition, since the outward-facing conformation must select the minor, deprotonated α-carboxylate form, Arg+
, over the preponderant protonated form, Arg2+
, to achieve acid resistance (6
). Previous work showed that AdiC transports ArgNH2
, a mimic of Arg2+
, with substantially lower efficiency than Arg+
, but the sidedness of this selectivity could not be determined because of the random protein orientation in the liposomes (6
). Now with the oriented system, we find that extracellular ArgNH2
is substantially less effective than intracellular as a competitive inhibitor of Arg+
transport. In other words (assuming that ArgNH2
serves as a valid analogue of Arg+2
), the outward-facing conformation is more sensitive than the inward-facing to the protonation state of arginine, a circumstance resonant with the biological imperatives that AdiC must satisfy.
Previous work (6
) established that AdiC transport activity is stimulated by acidification, but the random protein orientation in that work made it impossible to evaluate the sidedness of this effect. Now with the oriented system, we find that both sides of the protein sense pH, each with its own characteristic pH profile. The major, extracellular pH-sensor leads to ~30-fold stimulation upon acidification from neutrality, while intracellular acidification produces a biphasic response with 3-fold stimulation followed by inhibition below pH 4. We do not currently understand the molecular basis for this pH-dependent transport activity, but the differing pH profiles for the two sides of the protein imply that at least two pH sensors are involved, one for each side of the membrane. This situation makes biological sense. Under non-stressed conditions, near neutrality on both sides of the bacterial inner membrane, AdiC transport activity is low, and only upon extracellular acidification does the transporter spring into action. We estimate that the two sensors working together in response to typical acid stress conditions - extracellular and cytoplasmic pH of 3 and 5.5, respectively - accelerate the rate of virtual proton pumping ~50-fold (Fig. S5
). It is also notable that the bell-shaped cytoplasmic pH profile for transport is similar in shape to the pH-dependence of the acid-activated arginine decarboxylase AdiA (14
) - a satisfying example of a sophisticated, multi-protein system in which all components are poised to respond to a stress condition in a coordinated way.
Our results also offer a cautionary note regarding the logic sometimes mustered to argue for H+
-coupled mechanisms in transport proteins. For instance, in a recent description of the structure and mechanism of ApcT, a wide-spectrum APC amino acid transporter, it was shown (16
) that substrate uptake is increased ~3-fold in the presence of a pH gradient (pH 4-outside /pH 7-inside), as compared with symmetrical pH 4 conditions. This result was taken to mean that a proton is co-transported with the amino acid, i.e., that ApcT is a H+
-coupled amino acid symporter. However, as shown here, AdiC, which does not co-transport protons (6
), is also stimulated by a similar pH gradient (pH 3-outside / 5.5-inside, vs symmetrical pH 5.5). Thus, the type of evidence adduced to identify ApcT as a H+
-coupled symporter neither supports nor refutes that conclusion.
In this study, we developed an oriented reconstituted system for AdiC and used it to extract sided kinetic parameters for substrate transport and other functional properties of AdiC. Many unresolved problems and ambiguities concerning this protein remain for future examination. For example, the precise role of Ser26 in substrate selectivity is still in question, despite the implication from crystal structures alone, of a strong H-bond donated to substrate by this side chain. In addition, the specific influences of pH on substrate transport have not yet been dissected mechanistically, nor have the protein's pH sensors been identified. The sided system described here will be a useful tool for future approaches to these problems.