Molecular basis of substrate-induced permeation by an amino acid antiporter

Abstract

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.

Structural bases for the interaction and stabilization of the human amino acid transporter LAT2 with its ancillary protein 4F2hc

“…The interaction between 4F2hc-ED and LAT2 raises the question of whether the heavy subunit might have an impact on the LAT2 transport cycle. The structural paradigms of HAT light subunits, the bacterial APC transporters, present the LeuT-fold (14)(15)(16)(17). Secondary transporters, upon the binding of an external substrate, transit to inward-facing conformations to release the substrate inside the cell.…”

“…According to our homology models of human LAT2 ( Fig. S3 A and B), which are based on the atomic structure of the bacterial L-arginine/agmatine exchanger AdiC (17), seven of the nine endogenous cysteine residues in LAT2 are located in TMDs and internal loops (Fig. S3 A and C).…”

“…The conserved cysteine residue involved in the intersubunit disulfide bridge is located between TMD3 and TMD4 (11). Structure-function studies (12,13) suggest that the structures of the light subunit of HATs should be similar to those of remote bacterial amino acid/polyamine/ organocation (APC) transporters (∼20% amino acid sequence identities), which present the LeuT-fold (14)(15)(16)(17). In contrast, there is no structural information about the interaction between the two HAT subunits.…”

Substrate-Induced Ubiquitylation and Endocytosis of Yeast Amino Acid Permeases

“…Upon Arg binding to AdiC in the OF open conformation, the indole group of W293 establishes a cationinteraction with the side chain of the substrate (34), and proper orientation of the two groups is crucial to initiating the transition to the next conformation, namely, the occluded state, in which Arg is sandwiched between the indole groups of W293 and W202. The role of W293 in the transition of AdiC to the OF occluded state is illustrated by the observation that the W293Y substitution mostly affects the Arg translocation rate (ϳ85% reduction of V max ) and only slightly reduces its apparent binding affinity (36). In the 3D model of Gap1, the position equivalent to W293 of AdiC is also occupied by a tryptophan, W179.…”

Molecular mechanism of pH-dependent substrate transport by an arginine-agmatine antiporter

“…Structural and biochemical characterization of AdiC and GadC has provided important insights into substrate recognition and transport for these conserved amino acid antiporters (9,(13)(14)(15)(16). Considerable effort has focused on identifying pH sensorspresumably one or more amino acids-in these membrane transporters (9).…”