Asymmetry in the homodimeric ABC transporter MsbA recognized by a DARPin

Abstract

ABC transporters harness the energy from ATP binding and hydrolysis to translocate substrates across the membrane. Binding of two ATP molecules at the nucleotide binding domains (NBDs) leads to the formation of an outward-facing state. The conformational changes required to reset the transporter to the inward-facing state are initiated by sequential hydrolysis of the bound nucleotides. In a homodimeric ABC exporter such as MsbA responsible for lipid A transport in Escherichia coli, sequential ATP hydrolysis implies the existence of an asymmetric conformation. Here we report the in vitro selection of a designed ankyrin repeat protein (DARPin) specifically binding to detergent-solubilized MsbA. Only one DARPin binds to the homodimeric transporter in the absence as well as in the presence of nucleotides, suggesting that it recognizes asymmetries in MsbA. DARPin binding increases the rate of ATP hydrolysis by a factor of two independent of the substrate-induced ATPase stimulation. Electron paramagnetic resonance (EPR) measurements are found to be in good agreement with the available crystal structures and reveal that DARPin binding does not affect the large nucleotide-driven conformational changes of MsbA. The binding epitope was mapped by cross-linking and EPR to the membrane-spanning part of the transmembrane domain (TMD). Using cross-linked DARPin-MsbA complexes, 8-azido-ATP was found to preferentially photolabel one chain of the homodimer, suggesting that the asymmetries captured by DARPin binding at the TMDs are propagated to the NBDs. This work demonstrates that in vitro selected binders are useful tools to study the mechanism of membrane proteins.

FIGURE 1.

Characterization of DARPin_55: Specificity, epitope mapping and modulation of ATPase activity of MsbA. A, ELISA to determine binding specificity of DARPin_55. DARPin_55 specifically binds to its target protein bMsbA_AviC but not to the control proteins bLmrCD_AviC and bAcrB_AviC. DARPin_110819 specifically recognizing bAcrB_AviC was used as a control. B, ELISA assay to delineate DARPin_55 binding to subdomains of MsbA. DARPin_55 binds to the transmembrane domain (bTMD_AviC) but not to the nucleotide binding domain (bNBD_AviC) of MsbA. bAcrB_AviC is used as a negative control. C, modulation of basal ATPase activity of detergent-solubilized MsbA in complex with DARPin_55. DARPin_55 was added to MsbA (200 nm) in a 2-fold serial dilution series ranging from 50 nm to 1.6 μm leading to a stimulation of the ATPase activity of MsbA by up to 2.1-fold. Addition of the unselected DARPin E3_5 (1 μm) to MsbA does not increase its ATPase activity. DARPin_55 (1 μm) alone does not hydrolyze ATP. D, ATPase activity of MsbA was determined in the presence of DARPin_55 (1 μm) or E3_5 (1 μm) as well as in absence of DARPins at varying ATP concentrations. Addition of DARPin_55 results in a higher maximal rate of ATP hydrolysis (V_max), while the apparent affinity for ATP (K_m) is not affected. E and F, stimulations of the ATPase activity of MsbA by substrates and DARPin_55 are additive. Lipid A was added to detergent-purified MsbA at concentrations of 0, 0.05, 0.1, and 0.2 mg/ml E, daunomycin was added at concentrations of 0, 50, 100, and 200 μm F and ATPase was measured in the absence and presence of DARPin_55 (1 μm). Error bars represent standard deviations.

FIGURE 2.

Intra-MsbA distance measurements by DEER reveal no major conformational changes of the transporter upon binding of wild-type DARPin. A, left, normalized DEER form factors F(t) and fits obtained with DeerAnalysis2010 on detergent-purified MsbA_191R1 alone and in complex with wild-type DARPin_55. Traces were detected in the apo-state and in the AMP-PNP-state (5 mm AMP-PNP and 5 mm MgCl₂) in the absence and in the presence of DARPin_55, as indicated. Right, distance distributions obtained with Tikhonov regularization parameters 100 or 1000. The intra-MsbA distances simulated based on the AMP-PNP x-ray structure (PDB 3B60) are superimposed (red dotted). B, DEER analysis on MsbA_539R1 alone and in complex with DARPin_55 analogous to A.

FIGURE 3.

Methanethiosulfonate-mediated cross-linking of one DARPin to homodimeric MsbA. A–C, MsbA_191C (1 μm) was incubated with increasing amounts of DARPin_55_29C (from 500 nm to 16 μm) in the absence of nucleotides A, in the presence of MgCl₂ (3.5 mm) and AMP-PNP (3 mm) B, or ATP-vanadate (2.5 mm, each). C, protein complexes were cross-linked using the homobifunctional M11M cross-linker (2 mm) for 15 min at 4 °C and separated by non-reducing SDS-PAGE. D, ATPase activities of cross-linked MsbA191C-DARPin_55_29C complexes are equal to those of non-treated MsbA-DARPin complexes and are consistently increased by a factor of two compared with MsbA alone.

FIGURE 4.

Preferential nucleotide binding to one chain of asymmetrically stabilized MsbA as determined by 8-N₃-[α-³²P]ATP photolabeling. A and B, DARPin_55_29C was cross-linked to MsbA_191C using M11M, followed by addition of 8-N₃-[α-³²P]ATP (1 μm) and UV-cross-linking (performed as quadruplicates). The samples were separated by non-reducing SDS-PAGE and analyzed by SYPRO Ruby staining A and autoradiography B. C, band intensities corresponding to cross-linked DARPin-MsbA complex and free MsbA monomer obtained in A and B were quantified.

FIGURE 5.

Determination of the binding epitope of DARPin_55 by DEER. A, left, normalized DEER form factors F(t) and fits obtained with DeerAnalysis2010 on detergent-purified MsbA_191R1 alone and in complex with DARPin_55 spin labeled at position 29 (N-cap). Traces were detected in the apo-state and in the AMP-PNP-state (5 mm AMP-PNP and 5 mm MgCl₂) in the absence and in the presence of DARPin_55_29R, as indicated. Right, distance distributions obtained with Tikhonov regularization parameters 100 or 1000. Assigned MsbA-DARPin distances are marked in the distance distribution. B, DEER analysis on MsbA_65R1 alone and in complex with DARPin_55 spin labeled at position 160 (C-cap) analogous to A. The vicinity of the intra-MsbA and DARPin-MsbA distances in the apo-state prevented a clear assignment of the DEER constraint.

FIGURE 6.

The MsbA-DARPin complex based on cross-linking and DEER data. A, overview of the cross-linking data (supplemental Fig. S8) using the x-ray structure of MsbA (PDB 3B60) and a homology model of DARPin_55 as templates. The black straight lines indicate thiol pairs showing strong cross-links, the black dotted lines denote pairs showing weak cross-links. B, overview of interspin distances between MsbA and DARPin_55. The possible spin label rotamers attached at selected positions in MsbA were simulated using the Matlab program package MMM and are indicated as green clouds. The black straight lines highlight the pairs for which DEER constraints were obtained. All DEER data including the restraints are presented in the table.