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.

bioenergetics ͉ transport ͉ fluorescence͞phosphorescence ͉ hydrophobic stacking ͉ sugar binding

One powerful technique that has not been exploited for studying LacY is f luorescence resonance energy transfer (FRET) (13). This sensitive spectroscopic technique yields information regarding inter-and intramolecular distances. Essentially the method analyzes how the lifetime and quantum yield of a fluorescent molecule (the donor) is influenced by another fluorescent molecule (the acceptor) and vice versa. By using Förster theory, which states that the efficiency of energy transfer is inversely proportional to the sixth power of distance, distances between two fluorophors can be measured (14). The sensitivity and range of distances that can be calculated reliably from FRET depends on the spectral characteristics of the dyes and their relative orientations, which together determine R 0 , the distance at which energy transfer efficiency is 50%. The technique is typically most sensitive for distances between 20 and 100 Å (14, 15).Luminescence resonance energy transfer (LRET) is a particular type of FRET where a lanthanide atom (Tb 3ϩ or Eu 3ϩ ) transfers energy to an organic fluorescent acceptor. The technique exploits the remarkable luminescence properties of lanthanides (i.e., millisecond to submillisecond lifetimes, narrow and multiple emission bands in the visible spectrum, and unpolarized emission). As a consequence, lanthanide luminescence overcomes some of the limitations of conventional FRET experiments [e.g., the unpolarized long lifetime allows random orientation of both the donor and acceptor during the energy transfer process, thereby simplifying determination of the orientation factor between donor and acceptor ( 2 )] (14).Current LRET studies use a thiol-reactive chelator to bind a Tb 3ϩ ion at an engineered Cys residue in the protein (16)(17)(18)(19)(20). The chelator binds Tb 3ϩ with high affinity, shielding the cation from nonradiation deexcitation processes (primarily solvent quenching) (19,21). The resulting Tb 3ϩ emission has a high quantum yield, sometimes approaching unity, and a long lifetime. Although these long-lived lanthanide chelates have been used successfully for estimating inter-and intramolecular distances in the range of 45 Å or more (16-20), they are problematic for short distance measurements in the range desired for most intramolecular interactions because: (i) reactive and highly luminescent chelates are not usually available commercially and require synthesis (21); (ii) the bulk of the chelator may perturb the local environment of the protein; (iii) the mobility inherent in the use of a bulky group with a linker makes distance calculations less precise, particularly under 40 Å; and (iv) both the chelator and the fluorophor are usually directed toward Cys residues, sample heterogeneity is introduced.An alternative possibility for binding Tb 3ϩ at a defined position within a protein is to introduce an EF-hand motif, which Abbreviations: LacY, lactose permease; FRET, fluorescence resonance energy transfer; R0, the distance at which energy transfer efficiency is 50%; LRET, lumines...