Noncanonical role for the binding protein in substrate uptake by the MetNI methionine ATP Binding Cassette (ABC) transporter

Abstract

The Escherichia coli methionine ABC transporter MetNI exhibits both high-affinity transport toward l-methionine and broad specificity toward methionine derivatives, including d-methionine. In this work, we characterize the transport of d-methionine derivatives by the MetNI transporter. Unexpectedly, the N229A substrate-binding deficient variant of the cognate binding protein MetQ was found to support high MetNI transport activity toward d-selenomethionine. We determined the crystal structure at 2.95 Å resolution of the ATPγS-bound MetNIQ complex in the outward-facing conformation with the N229A apo MetQ variant. This structure revealed conformational changes in MetQ providing substrate access through the binding protein to the transmembrane translocation pathway. MetQ likely mediates uptake of methionine derivatives through two mechanisms: in the methionine-bound form delivering substrate from the periplasm to the transporter (the canonical mechanism) and in the apo form by facilitating ligand binding when complexed to the transporter (the noncanonical mechanism). This dual role for substrate-binding proteins is proposed to provide a kinetic strategy for ABC transporters to transport both high- and low-affinity substrates present in a physiological concentration range.

Keywords: ATP Binding Cassette transporter; alternating access transport mechanism; methionine transporter; transinhibition.

Fig. 1.

In vivo uptake assay of MetNIQ and its variants. (A) Schematic of whole-cell uptake assay for MetNIQ variants. (B) Michaelis–Menten plot of initial velocities for d-semet uptake versus substrate concentrations for MetNIQ and its variants. The corresponding V_max and K_m values are listed in Table 1.

Fig. 2.

Crystal structure of the MetNIQ complex. (A) Side-view representation of the MetNIQ. MetQ subunit is colored slate, MetI subunits are forest and pale yellow, and MetN subunits are cyan and firebrick. C-regulatory (C2) domains are at the C termini of MetN subunits. Conformational changes of (B) the transmembrane MetI subunits, (C) the nucleotide-binding MetN subunits, and (D) the C2 domains between their OWF (colored as described in A) and IWF conformations (PDB ID code 3TUJ) (colored gray). One subunit of MetI (colored forest) or MetN (colored cyan) of the OWF conformation was overlaid to that of the IWF conformation to highlight the differences in the relative placement of the opposite subunit (B and C). Superposition of MetNIQ and MetNI reveals the rotation of the C2 domains around the molecular twofold axis between the two structures (D).

Fig. 3.

Rotation of the C2 domains facilitates ATP-induced dimerization of MetN subunits. Comparison of the C2 domains following superposition of one of the two C2 domain subunits. C2 domains are presented in the locked transinhibited IWF state (colored wheat; PDB ID code 3TUZ), the unlocked IWF state (colored light blue; PDB ID code 3TUJ), and MetNIQ in the OWF state [colored firebrick, structure 3; PDB ID code 6CVL (this study)]. The superimposed subunit is to the right in each panel. This comparison highlights the significant differences in the two subunits between these states. Of note, the separation between the 200-helices (cyan ellipses, Left) in the unlocked OWF MetNIQ (NIQ) and IWF DM states is significantly shorter than in the IWF locked state (CY5) in the presence of methionine. As a consequence, the catalytic H-motif H199 residues from both NBDs are too far apart in the locked state to form the ATPase active form of the NBDs. l-methionine bound to the regulatory sites on the C2 domains are depicted as yellow spheres.

Fig. 4.

Conformational changes in MetQ creates substrate entry pathways in MetNIQ. (A) Superposition of the substrate-bound (colored cyan) and MetNI complexed MetQ (colored slate) shows a 24° twist between the two lobes that potentially provides access to potential substrate entry pathways. (B) Possible substrate entry pathways (shown in gray spheres) to the MetNIQ assembly were calculated by CAVER 3.0.2 pymol plugin with shell radii of 3 Å (43).

Fig. 5.

Translocation pathways of different ABC importers. (A) Surface slab views of the outward-facing conformations of three ABC importers, including MetNIQ (this work), MalFGK2 (PDB ID code 2R6G), and BtuCDF (PDB ID code 4FI3), reveal potential substrate-binding pockets (red box in B) with different sizes and shapes in the translocation pathways. Although MetI subunits form a small cavity, larger cavities are observed in the case of maltose and vitamin B12 transporters. Only maltose (shown in ball-and-stick) was structurally observed in the cavity. (B) Although a scoop loop was proposed to facilitate substrate handoff between the maltose- and vitamin B12-binding proteins with their transporters, no scoop loop is present in the methionine transporter.

Fig. 6.

Conformational changes of the translocation pathway gates of the MetNIQ complex. Structure comparison of the MetI subunits in their outward-facing conformation (A and C) (colored forest and pale yellow) versus inward-facing conformation (B and D) (colored gray) reveals major rearrangements of the periplasmic gate residues, including Y177 and M163 (cyan; shown in ball-and-stick representation) (A and B), and of the cytoplasmic gate residues, including F103, M107, and Y160 (black; shown in ball-and-stick representation) (C and D).

Fig. 7.

Residue rearrangement and potential substrate-binding cavity along the translocation pathway. (A) Movement of TM3 and TM4 of the MetI subunits form a small cavity (shown in Fig. 5A), whose boundary is defined by residues Y160 and F103 along the translocation pathway. (B) Superposition of MetI subunits in the OWF and IWF conformation reveals a rearrangement of a potential selectivity filter, M-Ω-Ω-M (Ω, aromatic ring residues), along the translocation pathway of the OWF MetNIQ. Side chains in the OWF and IWF conformations are colored cyan and black, respectively.

Fig. 8.

Mechanistic model for the MetNI-catalyzed transport of methionine derivatives. The relationship between the canonical and noncanonical pathways in the MetNI transport cycle is illustrated utilizing available structures of MetNI, MetQ, and MetNIQ represented as cylinders. Methionine, ADP, and ATP are shown as ball-and-sticks. Conformational states of the two C2 domains are shown in cyan and firebrick colored surface representations. State 1 represents a resting state of MetNI (PDB ID code 3TUJ). In the noncanonical pathway, state 2 represents an ATP-bound MetNIQ in the absence of methionine (this study; PDB ID code 6CVL). d-methionine can subsequently access the translocation pathway of the MetNIQ assembly through MetQ to form state 3 (also modeled as PDB ID code 6CVL). The canonical pathway proceeds through state 2′ depicted as a hypothetical pre-T conformation of l-methionine–bound IWF MetNIQ, based on structural studies of the maltose and molybdate transporters (22, 25). The transition from state 3 to state 1 is common to both pathways and associated with methionine transport into the cell. During the transport cycle, the C2 domains are in unlocked conformations that can accommodate the changes between IWF and OWF states. State 4 represents the transinhibited state of MetNI transporter (PDB ID code 3TUZ) with the C2 domains in a locked conformation stabilized by methionine binding and rearrangement of the β-sheet hydrogen bonding pattern at the interface between the C2 domains.

Fig. 9.

Kinetic analysis of canonical and noncanonical substrate uptake by ABC transporters. (A) Kinetic scheme modeling canonical and noncanonical pathways in substrate import by the transporter, E, where E₁, E₂, E_2′, and E₃ represent different states of the transporter as shown in Fig. 8. The methionine ligand (m) binds to Q to form the liganded species Q_m with a dissociation constant K_d. Q_m and Q can bind to E₁ to form E_2′Q_m and E₂Q, with effective dissociation constants K₁ and K₂, respectively. Transport occurs from the state E₃Q•m that is generated either by isomerization of E_2′Q_m with the first-order rate constant k₁ or by binding of m to E₂Q with a second-order rate constant k₂. The steady-state solution to this scheme for the case where E, E_2′Q_m, E₂Q, Q, and Q_m are at equilibrium is described in Materials and Methods. (B) Dependence of the transport rate for a substrate at 10 µM concentration on various parameters of the kinetic scheme in A, as a function of the K_d for binding of substrate to the SBP. Curves i, ii, and iii correspond to k₂ = 5,000, 500, and 0 M⁻¹⋅s⁻¹, with Q_tot = 10⁻⁴ M, K₁ = K₂ = 10⁻⁴ M, and k = k₁ = 0.01 s⁻¹. For substrates with high affinity to the SBP (K_d < ∼10⁻⁶ M) the models are equivalent, whereas for more weakly bound substrates (K_d > ∼10⁻⁴ M) the transport rate depends critically on the value of k₂. Depending on the value of k₂, the rate of transport of a weakly bound substrate can exceed that of a substrate that binds tightly to the SBP (compare curves i and ii). When k₂ = 0 (curve iii), transport does not occur under these conditions, which corresponds to the case where substrate is only delivered to E₁ by binding to the SBP. (C) Dependence of the K_m for transport on the dissociation constant K_d for binding of substrate to the SBP. The red and black circles represent experimental data points for the methionine (this study) and maltose transporter (30), respectively. The curves are generated with the following parameters: i, k = 0.01 s⁻¹, k₁ = 0.1 s⁻¹, and k₂ = 1,000 M⁻¹⋅s⁻¹; ii, k = 0.1 s⁻¹, k₁ = 0.1 s⁻¹, and k₂ = 100 M⁻¹⋅s⁻¹; and iii, k = 0.1 s⁻¹, k₁ = 0.1 s⁻¹, and k₂ = 0 M⁻¹⋅s⁻¹, with Q_tot = 10⁻⁴ M and K₁ = K₂ = 10⁻⁴ M for all curves. Curves were not explicitly fit to the experimental data but reflect parameter values that approximate the data. For this kinetic scheme, K_m reaches a plateau as K_d increases, and depending on the parameter values, this plateau can be tuned to the physiological concentration range. For curve iii, where substrate does not bind to E₂Q, a plateau region is not reached, so substrates with poor affinities for Q (high K_d) would have correspondingly high K_m values.