The conformational transition pathways of ATP-binding cassette transporter BtuCD revealed by targeted molecular dynamics simulation

BtuCD is a member of the ATP-binding cassette transporters in Escherichia coli that imports vitamin B(12) into the cell by utilizing the energy of ATP hydrolysis. Crystal structures of BtuCD and its homologous protein HI1470/1 in various conformational states support the "alternating access" mechanism which proposes the conformational transitions of the substrate translocation pathway at transmembrane domain (TMD) between the outward-facing and inward-facing states. The conformational transition at TMD is assumed to couple with the movement of the cytoplasmic nucleotide-binding domains (NBDs) driven by ATP hydrolysis/binding. In this study, we performed targeted molecular dynamics (MD) simulations to explore the atomic details of the conformational transitions of BtuCD importer. The outward-facing to inward-facing (O→I) transition was found to be initiated by the conformational movement of NBDs. The subsequent reorientation of the substrate translocation pathway at TMD began with the closing of the periplasmic gate, followed by the opening of the cytoplamic gate in the last stage of the conformational transition due to the extensive hydrophobic interactions at this region, consistent with the functional requirement of unidirectional transport of the substrates. The reverse inward-facing to outward-facing (I→O) transition was found to exhibit intrinsic diversity of the conformational transition pathways and significant structural asymmetry, suggesting that the asymmetric crystal structure of BtuCD-F is an intermediate state in this process.     

Ligand Induced Conformational Changes of the Human Serotonin Transporter Revealed by Molecular Dynamics Simulations

The competitive inhibitor cocaine and the non-competitive inhibitor ibogaine induce different conformational states of the human serotonin transporter. It has been shown from accessibility experiments that cocaine mainly induces an outward-facing conformation, while the non-competitive inhibitor ibogaine, and its active metabolite noribogaine, have been proposed to induce an inward-facing conformation of the human serotonin transporter similar to what has been observed for the endogenous substrate, serotonin. The ligand induced conformational changes within the human serotonin transporter caused by these three different types of ligands, substrate, non-competitive and competitive inhibitors, are studied from multiple atomistic molecular dynamics simulations initiated from a homology model of the human serotonin transporter. The results reveal that diverse conformations of the human serotonin transporter are captured from the molecular dynamics simulations depending on the type of the ligand bound. The inward-facing conformation of the human serotonin transporter is reached with noribogaine bound, and this state resembles a previously identified inward-facing conformation of the human serotonin transporter obtained from molecular dynamics simulation with bound substrate, but also a recently published inward-facing conformation of a bacterial homolog, the leucine transporter from Aquifex Aoelicus. The differences observed in ligand induced behavior are found to originate from different interaction patterns between the ligands and the protein. Such atomic-level understanding of how an inhibitor can dictate the conformational response of a transporter by ligand binding may be of great importance for future drug design.     

Asymmetric states of vitamin B₁₂ transporter BtuCD are not discriminated by its cognate substrate binding protein BtuF

BtuCD is an ABC transporter catalyzing the uptake of vitamin B₁₂ across the Escherichia coli inner membrane. A previously reported X-ray structure of BtuCD in complex with the periplasmic vitamin B₁₂-binding protein BtuF revealed asymmetry of the transmembrane BtuC subunits. The functional relevance of this asymmetry has remained uncertain. Here we report the X-ray structure of a catalytically impaired BtuCD mutant in complex with BtuF, where the BtuC subunits adopt a distinct asymmetric conformation. The structure suggests that BtuF does not discriminate between, or impose, asymmetric conformations of BtuCD. It also explains the conformational disorder observed in BtuCDF crystals.Structured summary of protein interactionsBtuF, BtuD and BtuC physically interact by X-ray crystallography (View interaction)     

Nucleotide-free MalK Drives the Transition of the Maltose Transporter to the Inward-facing Conformation

The complex MalFGK₂ hydrolyzes ATP and alternates between inward- and outward-facing conformations during maltose transport. It has been shown that ATP promotes closure of MalK₂ and opening of MalFG toward the periplasm. Yet, why the transporter rests in a conformation facing the cytosol in the absence of nucleotide and how it returns to this state after hydrolysis of ATP is unknown. The membrane domain MalFG may be naturally stable in the inward-facing conformation, or the ABC domain may catalyze the transition. We address this question by analyzing the conformation of MalFG in nanodiscs and in proteoliposomes. We find that MalFG alone exists in an intermediate state until MalK binds and converts the membrane domain to the inward-facing state. We also find that MalK, if overly-bound to MalFG, blocks the transition of the transporter, whereas suppressor mutations that weaken this association restore transport. MalK therefore exploits hydrolysis of ATP to reverse the conformation of MalFG to the inward-facing conformation, a step essential for release of maltose in the cytosol.     

Holo‐BtuF stabilizes the open conformation of the vitamin B12 ABC transporter BtuCD

While there is evidence that other ABC transporters can tell between empty and loaded substrate binding protein, reconstitution experiments suggest otherwise for the Escherichia coli vitamin B12 importer BtuCD‐F. Here, we address the question of BtuCD‐F substrate sensitivity in a combined protein–protein docking and molecular dynamics simulation approach. Starting from the BtuCD and holo‐BtuF crystal structures, we model two holo‐BtuCD‐F docking complexes differing by a 180° orientation of BtuF. One of these is similar to the apo‐BtuCD‐F crystal structure. Both docking complexes were embedded in a lipid/water environment to investigate their dynamics and BtuCD's conformational response to the presence and absence of BtuF, vitamin B12, and Mg‐ATP in a series of 28 independent MD simulations. We find holo‐BtuF stabilizing the open conformation of BtuCD, whereas the transporter begins to close again when BtuF or vitamin B12 is removed—suggesting BtuCD‐F is capable of substrate sensitivity. We identified BtuC transmembrane helices 3 and 5, the L‐loops and the adjacent helices comprised of BtuC residues 170–180 as hotspots of conformational change. We propose the latter to act as substrate sensors. BtuF‐Trp44 appears to act as a lid on the vitamin B12 binding cleft in BtuF X‐ray structures and protrudes into the BtuCD transport channel in one of our simulations, which might represent an initial step in vitamin B12 uptake. On an average, we observe subunit motions where the nucleotide binding domains approach each other while the transmembrane domains display an opening trend toward the periplasm. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Transmembrane gate movements in the type II ATP-binding cassette (ABC) importer BtuCD-F during nucleotide cycle

ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins that translocate substrates across cell membranes. The alternating access of their transmembrane domains to opposite sides of the membrane powered by the closure and reopening of the nucleotide binding domains is proposed to drive the translocation events. Despite clear structural similarities, evidence for considerable mechanistic diversity starts to accumulate within the importers subfamily. We present here a detailed study of the gating mechanism of a type II ABC importer, the BtuCD-F vitamin B(12) importer from Escherichia coli, elucidated by EPR spectroscopy. Distance changes at key positions in the translocation gates in the nucleotide-free, ATP- and ADP-bound conformations of the transporter were measured in detergent micelles and liposomes. The translocation gates of the BtuCD-F complex undergo conformational changes in line with a "two-state" alternating access model. We provide the first direct evidence that binding of ATP drives the gates to an inward-facing conformation, in contrast to type I importers specific for maltose, molybdate, or methionine. Following ATP hydrolysis, the translocation gates restore to an apo-like conformation. In the presence of ATP, an excess of vitamin B(12) promotes the reopening of the gates toward the periplasm and the dislodgment of BtuF from the transporter. The EPR data allow a productive translocation cycle of the vitamin B(12) transporter to be modeled.     

Distinct gate conformations of the ABC transporter BtuCD revealed by electron spin resonance spectroscopy and chemical cross-linking

BtuCD is a type II ABC importer that catalyzes the translocation of vitamin B12 from the periplasm into the cytoplasm of Escherichia coli. Crystal structures of BtuCD and the related HiF (or Hi1470/71) protein from Haemophilus influenzae have revealed distinct conformations of the transmembrane domains that form inner and outer gates. We used electron spin resonance spectroscopy to study the reaction cycle of BtuCD after labeling the protein at residues located at these gates. The results suggest that BtuCD as a prototype type II ABC importer may have a mechanism that is distinct from that of ABC exporters such as Sav1866 or type I ABC importers such as those specific for molybdate (ModBC) or maltose (MalFGK).

Structured summary

MINT-6803800: btuF (uniprotkb: P37028), btuC (uniprotkb:P06609) and btuD (uniprotkb:P06611) physically interact (MI:0218) by molecular sieving (MI:0071)     

Dissecting the Conformational Dynamics of the Bile Acid Transporter Homologue ASBTNM

Apical sodium-dependent bile acid transporter (ASBT) catalyses uphill transport of bile acids using the electrochemical gradient of Na⁺ as the driving force. The crystal structures of two bacterial homologues ASBT_NM and ASBT_Yf have previously been determined, with the former showing an inward-facing conformation, and the latter adopting an outward-facing conformation accomplished by the substitution of the critical Na⁺-binding residue glutamate-254 with an alanine residue. While the two crystal structures suggested an elevator-like movement to afford alternating access to the substrate binding site, the mechanistic role of Na⁺ and substrate in the conformational isomerization remains unclear. In this study, we utilized site-directed alkylation monitored by in-gel fluorescence (SDAF) to probe the solvent accessibility of the residues lining the substrate permeation pathway of ASBT_NM under different Na⁺ and substrate conditions, and interpreted the conformational states inferred from the crystal structures. Unexpectedly, the crosslinking experiments demonstrated that ASBT_NM is a monomer protein, unlike the other elevator-type transporters, usually forming a homodimer or a homotrimer. The conformational dynamics observed by the biochemical experiments were further validated using DEER measuring the distance between the spin-labelled pairs. Our results revealed that Na⁺ ions shift the conformational equilibrium of ASBT_NM toward the inward-facing state thereby facilitating cytoplasmic uptake of substrate. The current findings provide a novel perspective on the conformational equilibrium of secondary active transporters.     

Inward- and outward-facing homology modeling of human concentrative nucleoside transporter 3 (hCNT3) predicts an elevator-type transport mechanism

The human SLC28 family of concentrative (Na⁺-dependent) nucleoside transporters has three members, hCNT1, hCNT2 and hCNT3. Previously, we have used heterologous expression in Xenopus laevis oocytes in combination with an engineered cysteine-less hCNT3 protein hCNT3(C-) to undertake systematic substituted cysteine accessibility method (SCAM) analysis of the transporter using the membrane-impermeant thiol reactive reagent p-chloromercuribenzene sulfonate (PCMBS). A continuous sequence of more than 300 individual amino acid residue positions were investigated, including the entire transport domain of the protein, as well as important elements of the corresponding hCNT3 structural domain. We have now constructed 3D structural homology models of hCNT3 based upon inward-facing, intermediates and outward-facing crystal structures of the bacterial CNT Neisseria wadsworthii CNT_NW to show that all previously identified PCMBS-sensitive residues in hCNT3 are located above (ie on the extracellular side of) the key diagonal barrier scaffold domain TM9 in the transporter’s outward-facing conformation. In addition, both the Na⁺ and permeant binding sites of the mobile transport domain of hCNT3 are elevated from below the scaffold domain TM9 in the inward-facing conformation to above TM9 in the outward-facing conformation. The hCNT3 homology models generated in the present study validate our previously published PCMBS SCAM data, and confirm an elevator-type mechanism of membrane transport.     

Molecular Disruption of the Power Stroke in the ATP-binding Cassette Transport Protein MsbA

ATP-binding cassette transporters affect drug pharmacokinetics and are associated with inherited human diseases and impaired chemotherapeutic treatment of cancers and microbial infections. Current alternating access models for ATP-binding cassette exporter activity suggest that ATP binding at the two cytosolic nucleotide-binding domains provides a power stroke for the conformational switch of the two membrane domains from the inward-facing conformation to the outward-facing conformation. In outward-facing crystal structures of the bacterial homodimeric ATP-binding cassette transporters MsbA from Gram-negative bacteria and Sav1866 from Staphylococcus aureus, two transmembrane helices (3 and 4) in the membrane domains have their cytoplasmic extensions in close proximity, forming a tetrahelix bundle interface. In biochemical experiments on MsbA from Escherichia coli, we show for the first time that a robust network of inter-monomer interactions in the tetrahelix bundle is crucial for the transmission of nucleotide-dependent conformational changes to the extracellular side of the membrane domains. Our observations are the first to suggest that modulation of tetrahelix bundle interactions in ATP-binding cassette exporters might offer a potent strategy to alter their transport activity.     

Conformational coupling of the nucleotide-binding and the transmembrane domains in ABC transporters

Basic architecture of ABC transporters includes two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). Although the transport process takes place in the TMDs, which provide the substrate translocation pathway across the cell membrane and control its accessibility between the two sides of the membrane, the energy required for the process is provided by conformational changes induced in the NBDs by binding and hydrolysis of ATP. Nucleotide-dependent conformational changes in the NBDs, therefore, need to be coupled to structural changes in the TMDs. Using molecular dynamics simulations, we have investigated the structural elements involved in the conformational coupling between the NBDs and the TMDs in the Escherichia coli maltose transporter, an ABC importer for which an intact structure is available both in inward-facing and outward-facing conformations. The prevailing model of coupling is primarily based on a single structural motif, known as the coupling helices, as the main structural element for the NBD-TMD coupling. Surprisingly, we find that in the absence of the NBDs the coupling helices can be conformationally decoupled from the rest of the TMDs, despite their covalent connection. That is, the structural integrity of the coupling helices and their tight coupling to the core of the TMDs rely on the contacts provided by the NBDs. Based on the conformational and dynamical analysis of the simulation trajectories, we propose that the core coupling elements in the maltose transporter involve contributions from several structural motifs located at the NBD-TMD interface, namely, the EAA loops from the TMDs, and the Q-loop and the ENI motifs from the NBDs. These three structural motifs in small ABC importers show a high degree of correlation in motion and mediate the necessary conformational coupling between the core of TMDs and the helical subdomains of NBDs. A comprehensive analysis of the structurally known ABC transporters shows a high degree of conservation of the identified 3-motif coupling elements only in the subfamily of small ABC importers, suggesting a distinct mode of NBD-TMD coupling from the other two major ABC transporter folds, namely large ABC importers and ABC exporters.     

ABC transporters: how small machines do a big job

Transporters from the ATP-binding cassette (ABC) superfamily operate in all organisms, from bacteria to humans, to pump substances across biological membranes. Recent high-resolution views of ABC transporters in different conformational states provide clues as to how ATP might be used to drive the structural reorganizations that accompany membrane transport. Importantly, it now appears that a putative translocation pathway running through the center of the transporter might be gated alternately, either at the inside or the outside of the cytoplasmic membrane, coupling substrate translocation to a cycle of ATP-dependent conformational changes. ATP binding and ATP hydrolysis have distinct roles in this cycle: binding favors the outward-facing orientation, whereas hydrolysis returns the transporter to an inward-facing conformation.     

Zinc binding alters the conformational dynamics and drives the transport cycle of the cation diffusion facilitator YiiP

YiiP is a secondary transporter that couples Zn²⁺ transport to the proton motive force. Structural studies of YiiP from prokaryotes and Znt8 from humans have revealed three different Zn²⁺ sites and a conserved homodimeric architecture. These structures define the inward-facing and outward-facing states that characterize the archetypal alternating access mechanism of transport. To study the effects of Zn²⁺ binding on the conformational transition, we use cryo-EM together with molecular dynamics simulation to compare structures of YiiP from Shewanella oneidensis in the presence and absence of Zn²⁺. To enable single-particle cryo-EM, we used a phage-display library to develop a Fab antibody fragment with high affinity for YiiP, thus producing a YiiP/Fab complex. To perform MD simulations, we developed a nonbonded dummy model for Zn²⁺ and validated its performance with known Zn²⁺-binding proteins. Using these tools, we find that, in the presence of Zn²⁺, YiiP adopts an inward-facing conformation consistent with that previously seen in tubular crystals. After removal of Zn²⁺ with high-affinity chelators, YiiP exhibits enhanced flexibility and adopts a novel conformation that appears to be intermediate between inward-facing and outward-facing states. This conformation involves closure of a hydrophobic gate that has been postulated to control access to the primary transport site. Comparison of several independent cryo-EM maps suggests that the transition from the inward-facing state is controlled by occupancy of a secondary Zn²⁺ site at the cytoplasmic membrane interface. This work enhances our understanding of individual Zn²⁺ binding sites and their role in the conformational dynamics that govern the transport cycle.     

Protonation of Glu135 Facilitates the Outward-to-Inward Structural Transition of Fucose Transporter

Major facilitator superfamily (MFS) transporters typically need to alternatingly sample the outward-facing and inward-facing conformations, in order to transport the substrate across membrane. To understand the mechanism, in this work, we focused on one MFS member, the L-fucose/H⁺ symporter (FucP), whose crystal structure exhibits an outward-open conformation. Previous experiments imply several residues critical to the substrate/proton binding and structural transition of FucP, among which Glu¹³⁵, located in the periplasm-accessible vestibule, is supposed as being involved in both proton translocation and conformational change of the protein. Here, the structural transition of FucP in presence of substrate was investigated using molecular-dynamics simulations. By combining the equilibrium and accelerated simulations as well as thermodynamic calculations, not only was the large-scale conformational change from the outward-facing to inward-facing state directly observed, but also the free energy change during the structural transition was calculated. The simulations confirm the critical role of Glu¹³⁵, whose protonation facilitates the outward-to-inward structural transition both by energetically favoring the inward-facing conformation in thermodynamics and by reducing the free energy barrier along the reaction pathway in kinetics. Our results may help the mechanistic studies of both FucP and other MFS transporters.     

Reaction of internal forms of the choline carrier of erythrocytes with N-ethylmaleimide: Evidence for a carrier conformational change on complex formation

Summary The choline carrier of human erythrocyte membranes exists in distinguishable outward-facing and inward-facing conformations, and previous studies demonstrated that only the latter reacts with N-ethylmaleimide, producing an irreversible inhibition of transport. We now report experiments to determine the individual reaction rates for the two inward-facing forms: the free carrier and the complex. The pseudo-first-order rate constant for the complex with a substrate analog, di-n-butylaminoethanol, is found to be nearlydouble that for the free carrier, showing that the carrier conformation is altered following addition of a ligand (with 1mm N-ethylmaleimide at pH 6.8, 37°C, the constants are 0.57±0.05 min^–1 and 0.33±0.02 min^–1, respectively). Hence three different conformational states have been distinguished by experiment: (1) the inward-facing free carrier; (2) the inward-facing complex; and (3) the outward-facing carrier.     

The Molecular Mechanism of Ion-Dependent Gating in Secondary Transporters

LeuT-like fold Na-dependent secondary active transporters form a large family of integral membrane proteins that transport various substrates against their concentration gradient across lipid membranes, using the free energy stored in the downhill concentration gradient of sodium ions. These transporters play an active role in synaptic transmission, the delivery of key nutrients, and the maintenance of osmotic pressure inside the cell. It is generally believed that binding of an ion and/or a substrate drives the conformational dynamics of the transporter. However, the exact mechanism for converting ion binding into useful work has yet to be established. Using a multi-dimensional path sampling (string-method) followed by all-atom free energy simulations, we established the principal thermodynamic and kinetic components governing the ion-dependent conformational dynamics of a LeuT-like fold transporter, the sodium/benzyl-hydantoin symporter Mhp1, for an entire conformational cycle. We found that inward-facing and outward-facing states of Mhp1 display nearly the same free energies with an ion absent from the Na2 site conserved across the LeuT-like fold transporters. The barrier separating an apo-state from inward-facing or outward-facing states of the transporter is very low, suggesting stochastic gating in the absence of ion/substrate bound. In contrast, the binding of a Na2 ion shifts the free energy stabilizing the outward-facing state and promoting substrate binding. Our results indicate that ion binding to the Na2 site may also play a key role in the intracellular thin gate dynamics modulation by altering its interactions with the transmembrane helix 5 (TM5). The Potential of Mean Force (PMF) computations for a substrate entrance displays two energy minima that correspond to the locations of the main binding site S1 and proposed allosteric S2 binding site. However, it was found that substrate''s binds to the site S1 ∼5 kcal/mol more favorable than that to the site S2 for all studied bound combinations of ions and a substrate.     

smFRET Probing Reveals Substrate-Dependent Conformational Dynamics of E. coli Multidrug MdfA

Bacterial multidrug-resistance transporters of the major facilitator superfamily are distinguished by their extraordinary ability to bind structurally diverse substrates, thus serving as a highly efficient tool to protect cells from multiple toxic substances present in their environment, including antibiotic drugs. However, details of the dynamic conformational changes of the transport cycle involved remain to be elucidated. Here, we used the single-molecule fluorescence resonance energy transfer technique to investigate the conformational behavior of the Escherichia coli multidrug transporter MdfA under conditions of different substrates, pH, and alkali metal ions. Our data show that different substrates exhibit distinct effects on both the conformational distribution and transition rate between two major conformations. Although the cationic substrate tetraphenylphosphonium favors the outward-facing conformation, it has less effect on the transition rate. In contrast, binding of the electroneutral substrate chloramphenicol tends to stabilize the inward-facing conformation and decreases the transition rate. Therefore, our study supports the notion that the MdfA transporter uses distinct mechanisms to transport electroneutral and cationic substrates.     

A Conserved Motif in Intracellular Loop 1 Stabilizes the Outward-Facing Conformation of TmrAB

The ATP binding cassette (ABC) family of transporters moves small molecules (lipids, sugars, peptides, drugs, nutrients) across membranes in nearly all organisms. Transport activity requires conformational switching between inward-facing and outward-facing states driven by ATP-dependent dimerization of two nucleotide binding domains (NBDs). The mechanism that connects ATP binding and hydrolysis in the NBDs to conformational changes in a substrate binding site in the transmembrane domains (TMDs) is currently an outstanding question. Here we use sequence coevolution analyses together with biochemical characterization to investigate the role of a highly conserved region in intracellular loop 1 we define as the GRD motif in coordinating domain rearrangements in the heterodimeric peptide exporter from Thermus thermophilus, TmrAB. Mutations in the GRD motif alter ATPase activity as well as transport. Disulfide crosslinking, evolutionary trace, and evolutionary coupling analysis reveal that these effects are likely due to the destabilization of a network in which the GRD motif in TmrA bridges residues of the Q-loop, X-loop, and ABC motif in the NBDs to residues in the TmrAB peptide substrate binding site, thus providing an avenue for conformational coupling. We further find that disruption of this network in TmrA versus TmrB has different functional consequences, hinting at an intrinsic asymmetry in heterodimeric ABC transporters extending beyond that of the NBDs. These results support a mechanism in which the GRD motifs help coordinate a transition to an outward open conformation, and each half of the transporter likely plays a different role in the conformational cycle of TmrAB.     

Structure and mechanism of ABC transporter proteins

ATP-binding cassette (ABC) transporters are ubiquitous membrane proteins that couple the transport of diverse substrates across cellular membranes to the hydrolysis of ATP. The crystal structures of four ABC transporters have recently been determined. They reveal similar arrangements of the conserved ATP-hydrolyzing nucleotide-binding domains, but unrelated architectures of the transmembrane domains, with the notable exception of a common 'coupling helix' that is essential for transmitting conformational changes. The structures suggest a mechanism that rationalizes ATP-driven transport: While binding of ATP appears to trigger an outward-facing conformation, dissociation of the hydrolysis products may promote an inward-facing conformation. This basic scheme can, in principle, explain nutrient import by ABC importers and drug extrusion by ABC exporters.     

Asymmetry in the homodimeric ABC transporter MsbA recognized by a DARPin

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.