Reconstitution of an Arginine-Agmatine Antiporter in an Oriented System

Abstract

The arginine-dependent extreme acid resistance system plays a critical role for enteric bacteria to survive the harsh gastric environment. At the center of this multi-protein system is an arginine-agmatine antiporter called AdiC. To maintain cytoplasmic pH, AdiC imports arginine and exports its decarboxylated product agmatine, resulting in a net extrusion of one proton in each turnover. An obstacle to quantifying AdiC's transport mechanism is the random orientation of AdiC protein in reconstituted liposomes. To overcome this problem, we introduced a mutation, S26C, near the substrate-binding site. This mutant exhibits similar substrate recognition and pH-dependent activity as wild-type protein, but loses function completely upon reaction with MTS reagents. The membrane-impermeant MTSES can then be used as a cleanly sided inhibitor to “silence” those S26C-AdiC proteins whose extracellular portion projects from the external side of the liposome. Alternatively, the membrane-permeant MTSEA and membrane-impermeant reducing reagent, TCEP, can be used together to inhibit proteins in the opposite orientation. With this approach, we demonstrate that the activity of AdiC increases to a plateau at pH 4 as the extracellular side is acidified. Interestingly, the optimal pH for the cytoplasmic side is 5.5 and further acidification strongly inhibits AdiC's function. These findings recapitulate the gastric environment where the extracellular and cytoplasmic pH of bacteria are 2-4 and 5-6, respectively. Steady-state kinetic analysis found that for both sides of AdiC, the Michaelis-Menten constants of arginine and agmatine are in the same range. Competition experiments show that argininamide, which mimics the carboxylate-protonated form of arginine, suppresses arginine transport more effectively from the cytoplasmic side. This oriented system allows more precise analysis of AdiC-mediated substrate transport than has been previously available and permits comparison to the situation experienced by the bacterial membrane under acid stress.