Structural basis of substrate recognition and permeation by an amino acid antiporter

Kowalczyk L, Ratera M, Paladino A, Bartoccioni P, Errasti-Murugarren E, Valencia E, Portella G, Bial S, Zorzano A, Fita I, Orozco M, Carpena X, Vázquez-Ibar JL, Palacín M.

Structure of Arg-bound AdiC-N101A.

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. It 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.

We have crystallized and solved the structure of an Arg-bound AdiC mutant (N101A) at 3.0 Å resolution in the open-to-out conformation. This structure completes the picture of the major conformational states during the transport cycle of the 5+5 inverted repeat fold-transporters.

The guanidinium group of Arg shows high mobility and delocalization, hampering substrate occlusion and resulting in a low translocation rate. The proper coordination of this group with side chains of Asn101 and Trp293 is required to transit to the occluded state providing, thus, the first clues on the mechanism of substrate-induced fit in a 5+5 inverted repeat fold-transporter.

Arg-bound AdiC-N101A is in the open-to-out conformation.

(A) 2Fo-Fc electron density map (contoured at 1 σ in purple) and Fo-Fc omit electron density map (contoured at 2 σ in green) of the substrate-binding site (Arg is depicted with green carbon atoms). Trp202 and Trp293 are separated, a characteristic of the open-to-out conformation.

(B) Arg coordination in the AdiC-N101A structure. The Arg α-amino group is next to the negatively charged end of TM6a helix dipole, and at hydrogen-bond distance of Ile205 carbonyl group (unwound segment of TM6). The α- carboxy group lies in the vicinity of the positively charged end of TM1b helix dipole and participates in a hydrogen bond with Ser26 side chain (unwound segment of TM1). The guanidinium group is about 4.6 Å away from Trp293 (TM8) and its nitrogen atoms are at hydrogen-bond distance of Ala96 carbonyl group (TM3) and Ser357 side chain (TM10).

(C) Same view as in (B) of the Arg-bound occluded conformation of AdiC (3L1L). (B) and (C) differ in the position of the Arg guanidinium group as well as in the unwound segment of TM1 (Ile23 to Ser26).

Comparison of Arg position in AdiC N101A crystal structure and the docking model.

N101A modeldocking model

Both, the position of Arg found in the docking model (orange carbon atoms) and in the N101A crystal structure (light green carbon atoms) overlap fairly well in the binding site. The represented docked Arg corresponds to pose of the thermodynamically most favorable cluster (56% of all possible rotamers). Polar interactions of docked Arg with the side chains of Ser26 (TM1) and Ser357 (TM10), and with the carbonyl groups of Ala96 (TM3) and Ile205 (TM6) are indicated. TM segments are numbered in italics.

Transport kinetics Substrate binding

VMAX (pmol/μg/min) Apparent Kd

(μM)KM

(μM)

wt

N101A

N101D

W293Y

50

0.7

95

6.5

35

100

105

125

105

nd

112

nd

Functional analysis of AdiC mutants

VMAX

and KM values of each AdiC variant with 4 mM L-arginine inside the proteoliposomes. Apparent dissociation constants (Kd) of L-arginine-AdiC (wild-type and mutants) binding were calculated from isothermal calorimetry (ITC) measurements. No heat flux in the calorimeter was recorded in W293Y and N101A.

Proposed mechanism of Arg recognition and induced fit by AdiC.

Apo (3NCY) Arg-bound (3OB6)

semi-occluded Apooccluded (3L1L)

Periplasmic Arg is recognized by the apo conformation of AdiC (A; 3NCY) and binds with a similar orientation (B; 3OB6) as in the Arg- occluded conformation (D; 3L1L). The proper Arg binding samples the semi-occluded state (C; docked Arg in 3LRB) by stabilizing Trp202 (TM6a) and Phe350 (loop TMs 9–10) interaction. This semi-occluded conformation evolves to the occluded state mainly by pivoting TM6a. Transition from the apo (A) to the semioccluded state (C) is defective in mutant N101A.

Molecular dynamics simulations of Arg in AdiC wt and N101A mutant

Molecular dynamics of Arg in the open-to-out structure of AdiC wt (top panel) and in the mutant N101A (bottom panel). The docked Arg is retained in the vicinity of Trp293, with a tendency to interact with residues of the TM10. Only minor distance changes between the central guanidinium carbon of Arg (CZ) and the carbonyl oxygen of Ser357 (red and orange) or the CD2 carbon of the Trp293 (black and maroon) in each protomer (A) (see top panel). Significant distance changes were measured for AdiC N101A mutant (B) (see bottom panel) showing that the Arg may easily “travel” between Trp202 and Trp293. (B) illustrates distance measured in (A).

wt

N101A

Symmetry based inward-facing model of AdiC

Model of the open-to-in conformation of AdiC. The model of the first 10 TM segments of the open-to-in conformation of AdiC (A, gray cylinders) was built by swapping the conformations of the two 5+5 inverted repeats of the open-to-out structure (orange cylinders) by a nearly pure rotation along the subunit axis of the two repeats (light orange arrow) (see Methods main text). A central slice of the surface protein is also shown. Superposition (B) of the TM segments of the open-to-out structure and the generated open-to-in model (gray cylinders) of AdiC. Both the bundle (TMs 1, 2, 6, and 7; orange) and the hash (TMs 3, 4, 8, and 9; light orange) domains undergo major conformational changes during this transition. TMs 5 and 10 (beige) undergo minor changes. The the subunit axes are shown as orange arrows (A and B).

An Arg internal binding site model (B) can be generated after applying the 5+5 inverted repeat symmetry to the external binding site of the occluded state (A; N22A mutant, 3L1L). Rotation of Arg through the subunit axis (arrow) results in its turning upside down to this new internal cavity (B). Trp293 (TM8) moves along with the guanidinium group of Arg. The α-amino group of Arg in the internal site sits next to position 22. Positive and negative signs indicate the dipole charge of the corresponding TM ends.

Proposed mechanism of Arg translocation by AdiC.

Symmetrical states in the alternative access mechanism of transporters with the inverted repeat fold.

3OB6

BetP (3PO3)