Transporters of the amino acid, polyamine and organocation (APC) superfamily play essential roles in cell redox balance, cancer, and aminoacidurias. The bacterial L-arginine/agmatine antiporter, AdiC, is the main APC structural paradigm and shares the “5 + 5 inverted repeat” fold found in other families like the Na + -coupled neurotransmitter transporters. The available AdiC crystal structures capture two states of its transport cycle: the open-to-out apo and the outward-facing Arg + -bound occluded. However, the role of Arg + during the transition between these two states remains unknown. Here, we report the crystal structure at 3.0 Å resolution of an Arg + -bound AdiC mutant (N101A) in the open-to-out conformation, completing the picture of the major conformational states during the transport cycle of the 5 + 5 inverted repeat fold-transporters. The N101A structure is an intermediate state between the previous known AdiC conformations. The Arg + -guanidinium group in the current structure presents high mobility and delocalization, hampering substrate occlusion and resulting in a low translocation rate. Further analysis supports that proper coordination of this group with residues Asn101 and Trp293 is required to transit to the occluded state, providing the first clues on the molecular mechanism of substrate-induced fit in a 5 + 5 inverted repeat fold-transporter. The pseudosymmetry found between repeats in AdiC, and in all fold-related transporters, restraints the conformational changes, in particular the transmembrane helices rearrangements, which occur during the transport cycle. In AdiC these movements take place away from the dimer interface, explaining the independent functioning of each subunit.

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 twodimensional crystals of AdiC-W293L diffracting beyond 6 Å resolution from which we determined the projection structure at 6.5 Å 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 ␣-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.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). To overcome the protective barrier of the gastric acidity, pathogenic and nonpathogenic strains of E. coli have developed acid resistance systems. One of these systems requires arginine to protect E. coli during low pH exposure. This arginine system is composed of an arginine-agmatine exchange transporter and of an acid-activated arginine decarboxylase (2). Acidification of the cytosol is prevented by the consumption of protons through decarboxylation of arginine to agmatine and carbon dioxide. Agmatine is then exported out of the cytosol, and new arginine is imported through the arginineagmatine transporter in a one-to-one exchange stoichiometry (2). This recently identified transport protein is the product of the adiC gene (3, 4). In vitro, AdiC-mediated exchange transport of arginine and agmatine is tightly coupled, electrogenic, and acid-activated (5). AdiC forms stable homodimers in detergent and phospholipid membranes as determined by gel filtration and glutaraldehyde cross-linking experiments (5).The origin of AdiC is somehow controversial as it was assigned to two families of transport proteins, i.e. the amino acid/polyamine/organocation (APC) 7 transporter superfamily (6) and the major facilitator superfamily (MFS) (5, 7). The APC superfamily of transporters consists of nearly 250 members that function as solute-cation symporters and solute-solute antiporters (6). According to hydropathy profile analysis and biochemically established topological features of most prokaryotic and eukaryotic APC superfamily members, both the N and C termini of ...

Adhesion of pathogenic bacteria to target cells is a prerequisite for colonization and further infection. The main adhesins of the emerging sexually transmitted pathogen Mycoplasma genitalium, P140 and P110, interact to form a Nap complex anchored to the cell membrane. Herein, we present the crystal structures of the extracellular region of the virulence factor P110 (916 residues) unliganded and in complex with sialic acid oligosaccharides. P110 interacts only with the neuraminic acid moiety of the oligosaccharides and experiments with human cells demonstrate that these interactions are essential for mycoplasma cytadherence. Additionally, structural information provides a deep insight of the P110 antigenic regions undergoing programmed variation to evade the host immune response. These results enlighten the interplay of M. genitalium with human target cells, offering new strategies to control mycoplasma infections.