Abstract
The L-arginine/agmatine antiporter AdiC is a key component of the arginine-dependent extreme acid resistance system of Escherichia coli. Phylogenetic analysis indicated that AdiC belongs to the amino acid/polyamine/organocation (APC) transporter superfamily having sequence identities of 15-17% to eukaryotic and human APC transporters. For functional and structural characterization, we cloned, overexpressed, and purified wild-type AdiC and the point mutant AdiC-W293L, which is unable to bind and consequently transport L-arginine. Purified detergent-solubilized AdiC particles were dimeric. Reconstitution experiments yielded two-dimensional crystals of AdiC-W293L diffracting beyond 6 angstroms resolution from which we determined the projection structure at 6.5 angstroms resolution. The projection map showed 10-12 density peaks per monomer and suggested mainly tilted helices with the exception of one distinct perpendicular membrane spanning alpha-helix. Comparison of AdiC-W293L with the projection map of the oxalate/formate antiporter from Oxalobacter formigenes, a member from the major facilitator superfamily, indicated different structures. Thus, two-dimensional crystals of AdiC-W293L yielded the first detailed view of a transport protein from the APC superfamily at sub-nanometer resolution.
Highlights
Enteric pathogens such as Shigella, Salmonella, Yersinia, spp., and certain Escherichia coli strains can survive the extremely acidic conditions of the human stomach and cause intestinal diseases [1]
Besides low resolution transmission electron microscopy (TEM) data on single detergent-solubilized Ser/Thr exchanger transport proteins (SteT) [23], no structural information is available for other members of the APC transporter superfamily
We have analyzed de novo the phylogenetic relationship of AdiC with known transport proteins
Summary
Chemicals—E. coli polar lipids were purchased from Avanti Polar Lipids (Alabaster, AL) and n-dodecyl--D-maltopyranoside (DDM) from Anatrace (Maumee, OH). The membrane pellet was resuspended in 20 mM Tris-HCl pH 8.0, 150 mM NaCl at a protein concentration between 13 and 25 mg/ml. Purification of AdiC and AdiC-W293L and Determination of Protein Concentration—Frozen E. coli total membranes containing overexpressed AdiC or AdiC-W293L were thawed and solubilized for 2 h at 4 °C under gentle agitation in 1% DDM, 20 mM Tris-HCl, pH 8, 300 mM NaCl, 10% glycerol, 0.01% NaN3. Protein concentration was determined spectrophotometrically measuring the absorbance at 280 nm and using a molar extinction coefficient of 85,830 MϪ1 cmϪ1 (His-tagged AdiC), 91,830 MϪ1 cmϪ1 (untagged AdiC), and 80,330 MϪ1 cmϪ1 (His-tagged AdiC-W293L) These values were calculated from the amino acid sequence of the different AdiC versions using the ProtParam tool from the ExPASy proteomics server. To generate the improved projection map of AdiC-W293L (Fig. 6B), one of the four identical AdiC-W293L dimers in the unit cell was symmetrized with IPLT exploiting the internal, non-crystallographic 2-fold symmetry axis of the dimer
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