Sided functions of an arginine-agmatine antiporter oriented in liposomes

From the viewpoint of enteric bacteria, the mammalian stomach, where pH can fall as low as 1.5, is a potentially lethal barrier that must be surmounted to colonize the intestinal tract. Many bacteria breach this host defense by activating multiple extreme acid resistance (XAR) systems (1, 2). This study concerns one of these, the arginine-dependent XAR system, which is composed of an arginine-agmatine antiporter, AdiC, and an acid-activated arginine decarboxylase, AdiA (3, 4). To counter acidification of the cytosol during low pH exposure, AdiC imports arginine, which is then decarboxylated by AdiA; this reaction consumes an aqueous proton, which replaces the α-carboxyl of arginine to form agmatine (1-amino-4-guanidino-n-butane). Agmatine is then exported by the same transporter, resulting in net extrusion of one "virtual proton" (5) from the cytosol in each AdiC transport cycle (Fig. 1A). Fig. 1 Orientation of AdiC in liposome membranes Previous studies have revealed basic biochemical, functional, and structural characteristics of AdiC (6–9). The protein is a homodimer in which each subunit acts as a self-contained antiporter that mediates strict one-to-one exchange of arginine for agmatine and is activated by low pH. AdiC's detailed transport mechanism, however, remains unclear. Several central questions are yet to be answered, including how AdiC senses pH changes and how it avoids transporting the protonated-carboxylate form of the substrate, Arg2+, the predominant form of arginine below pH 2.3, as it must do to achieve acid-resistance. Recent high-resolution structures of AdiC in multiple states (7–9) provide opportunities to tackle issues like these. But crystal structures alone are not enough. Close analysis of transport mechanism requires reconstituting purified protein into liposomes for flux measurements. However, AdiC, like many membrane proteins, inserts into liposome membranes randomly, with two populations of protein oriented in opposite directions. This heterogeneous orientation is a severe impediment to examining a transport mechanism, since it precludes assignment of specific functions to defined sides of the protein and makes it impossible to obtain the protein's kinetic parameters in a quantitatively meaningful way. A method to orient AdiC in liposomes is therefore necessary for detailed mechanistic analysis. In some cases, oriented reconstituted systems may be achieved by the application of sided inhibitors, which exclusively silence one of the two protein populations present. However, no sided inhibitor of AdiC has yet been found. Here, guided by crystal structures, we introduce an innocuous serine-to-cysteine substitution at a position that contributes to AdiC's arginine-binding site. This maneuver permits the use of thiol-directed methanethiosulfonate (MTS) reagents to inhibit AdiC in a strictly sided fashion, to yield liposomes with protein reconstituted in either of two orientations, outside-out or inside-out. An initial kinetic analysis reveals the sidedness of the protein's basic transport parameters, as well as the distinct pH-sensitivities of the cytoplasmic and extracellular sides. This work paves the way for future detailed examination of AdiC's transport mechanism and structural correlates thereof.