Outward-facing Conformation Research Articles

Structural Insights into the Catalytic Cycle of a Bacterial Multidrug ABC Efflux Pump

ABC (“ATP-Binding Cassette”) transporters of the type IV subfamily consist of exporters involved in the efflux of many compounds, notably those capable to confer multidrug resistance like the mammalian P-glycoprotein or the bacterial transporter BmrA. They function according to an alternating access mechanism between inward-facing (IF) and outward-facing (OF) conformations, but the extent of physical separation between the two nucleotide-binding domains (NBDs) in different states is still unsettled. Small Angle Neutron Scattering and hydrogen/deuterium exchange coupled to mass spectrometry were used to highlight different conformational states of BmrA during its ATPase cycle. In particular, mutation of the conserved Lysine residue of the Walker-A motif (K380A) captures BmrA in an ATP-bound IF conformation prior to NBD closure. While in the transition-like state induced by vanadate wild-type BmrA is mainly in an OF conformation, the transporter populates only IF conformations in either the apo state or in the presence of ADP/Mg. Importantly, in this post-hydrolytic step, distances between the two NBDs of BmrA seem to be more separated than in the apo state, but they remain shorter than the widest opening found in the related MsbA transporter. Overall, our results highlight the main steps of the catalytic cycle of a homodimeric bacterial multidrug transporter and underline structural and functional commonalities as well as oddities among the type IV subfamily of ABC transporters.

General principles of secondary active transporter function.

Transport of ions and small molecules across the cell membrane against electrochemical gradients is catalyzed by integral membrane proteins that use a source of free energy to drive the energetically uphill flux of the transported substrate. Secondary active transporters couple the spontaneous influx of a "driving" ion such as Na+ or H+ to the flux of the substrate. The thermodynamics of such cyclical non-equilibrium systems are well understood, and recent work has focused on the molecular mechanism of secondary active transport. The fact that these transporters change their conformation between an inward-facing and outward-facing conformation in a cyclical fashion, called the alternating access model, is broadly recognized as the molecular framework in which to describe transporter function. However, only with the advent of high resolution crystal structures and detailed computer simulations, it has become possible to recognize common molecular-level principles between disparate transporter families. Inverted repeat symmetry in secondary active transporters has shed light onto how protein structures can encode a bi-stable two-state system. Based on structural data, three broad classes of alternating access transitions have been described as rocker-switch, rocking-bundle, and elevator mechanisms. More detailed analysis indicates that transporters can be understood as gated pores with at least two coupled gates. These gates are not just a convenient cartoon element to illustrate a putative mechanism but map to distinct parts of the transporter protein. Enumerating all distinct gate states naturally includes occluded states in the alternating access picture and also suggests what kind of protein conformations might be observable. By connecting the possible conformational states and ion/substrate bound states in a kinetic model, a unified picture emerges in which the symporter, antiporter, and uniporter functions are extremes in a continuum of functionality. As usual with biological systems, few principles and rules are absolute and exceptions are discussed as well as how biological complexity may be integrated in quantitative kinetic models that may provide a bridge from the structure to function.

Complexity revealed by membrane curvature induced by ABC transporters

ABCB1 is the best-characterized member of the ABC superfamily of transmembrane proteins. ABCB1 transports structurally diverse substrates, including anticancer drugs. This, in combination with protein overexpression in cancer cells, hampers interactions with the drugs intracellular targets and jeopardizes chemotherapy treatments. The ATP-driven transport mechanism allows ABCB1 to alternate between an inward-facing (IF) conformation, where the protein adopts an inverted conical shape, and an outward-facing (OF) conformation, with a cylinder-like shape.

A plausible model for the outward-facing conformation of YiiP and its transport mechanism

The zinc/proton antiporter YiiP from Escherichia coli, a member of the cation diffusion facilitator family, plays an important role in maintaining cellular Zn2+ homeostasis. The difference between experimental inward-facing (IF) and outward-facing (OF) structures suggested a transport model involving large-scale scissoring of the transmembrane domains (TMDs). However, several experiments on YiiP and its mammalian homolog ZnT8 showed that this scissor-like rotation was not necessary for transport activity.

Substrate-bound and substrate-free outward-facing structures of a multidrug ABC exporter

Multidrug ABC transporters translocate drugs across membranes by a mechanism for which the molecular features of drug release are so far unknown. Here, we resolved three ATP-Mg2+–bound outward-facing conformations of the Bacillus subtilis (homodimeric) BmrA by x-ray crystallography and single-particle cryo–electron microscopy (EM) in detergent solution, one of them with rhodamine 6G (R6G), a substrate exported by BmrA when overexpressed in B. subtilis. Two R6G molecules bind to the drug-binding cavity at the level of the outer leaflet, between transmembrane (TM) helices 1–2 of one monomer and TM5′–6′ of the other. They induce a rearrangement of TM1–2, highlighting a local flexibility that we confirmed by hydrogen/deuterium exchange and molecular dynamics simulations. In the absence of R6G, simulations show a fast postrelease occlusion of the cavity driven by hydrophobicity, while when present, R6G can move within the cavity, maintaining it open.

Elucidating the Structural Features of ABCA1 in its Heterogeneous Membrane Environment.

ATP Binding Cassette Transporter A1 (ABCA1) plays an integral part in Reverse Cholesterol Transport (RCT) and is critical for maintaining lipid homeostasis. One theory of lipid efflux by the transporter (alternating access) proposes that ABCA1 harbours two different conformations that provide alternating access for lipid binding and release. This is followed by sequestration via a direct interaction between ABCA1 and its partner, ApoA1. The other theory (lateral access) proposes that ABCA1 obtains lipids laterally from the membrane to form a temporary extracellular “reservoir”. This reservoir contains an isolated lipid monolayer due to the net accumulation of lipids in the exofacial leaflet. Recently, a full-length Cryo-EM structure of this 2,261-residue transmembrane protein showed its discreetly folded domains and have detected the presence of a tunnel enclosed within the extracellular domains (ECDs) but not in the TMDs, giving it an outward-facing conformation. This structure was hypothesized to substantiate the lateral access theory. Utilizing long time-scale multiple replica atomistic molecular dynamics simulations (MDS), we simulated the structure in a large heterogeneous lipid environment and found that the protein undergoes several large conformational changes in its extremities. We observed that the cavity enclosed within ATP unbound form of ABCA1 is narrow at the distal ends of TMD as well as the ECD region substantiating the “lateral access” theory. We have also characterized ABCA1 and the lipid dynamics along with the protein-lipid interactions in the heterogeneous environment, providing novel insights into understanding ABCA1 conformation at an atomistic level.

Structures of the peptidase-containing ABC transporter PCAT1 under equilibrium and nonequilibrium conditions

ATP-binding cassette (ABC) transporters are ubiquitous molecular pumps that transport a broad range of substrates across biological membranes. Although the structure and function of ABC transporters has been studied extensively, our understanding of their energetics and dynamics remains limited. Here, we present studies of the peptidase-containing ABC transporter 1 (PCAT1), a polypeptide processing and secretion ABC transporter that functions via the classic alternating access mechanism. PCAT1 is a homodimer containing two peptidase (PEP) domains, two transmembrane domains, and two nucleotide-binding domains (NBDs). Using cryo-electron microscopy, we analyzed the structures of wild-type PCAT1 under conditions that either prevent or permit ATP hydrolysis and observed two completely different conformational distributions. In the presence of ATP but absence of Mg2+, PCAT1 adopts an NBD-dimerized, outward-facing conformation. The two PEP domains are dissociated from the transporter core, preventing uncoupled substrate cleavage. The addition of Mg2+ to promote ATP hydrolysis shifts the majority of the particles into NBD-separated, inward-facing conformations. Under this ATP turnover condition, only a small fraction of PCAT1 adopts the NBD-dimerized conformation. These data give rise to two mechanistic conclusions: 1) the ATP-bound, NBD-dimerized conformation is the lowest energy state, and 2) the rate-limiting step in the PCAT1 transport cycle is the formation of the NBD dimer. The thermodynamic conclusion is likely a general property shared by many ABC transporters. The kinetic bottleneck, however, varies from transporter to transporter.

Cryo-EM structures of the ABCA4 importer reveal mechanisms underlying substrate binding and Stargardt disease

ABCA4 is an ATP-binding cassette (ABC) transporter that flips N-retinylidene-phosphatidylethanolamine (N-Ret-PE) from the lumen to the cytoplasmic leaflet of photoreceptor membranes. Loss-of-function mutations cause Stargardt disease (STGD1), a macular dystrophy associated with severe vision loss. To define the mechanisms underlying substrate binding and STGD1, we determine the cryo-EM structure of ABCA4 in its substrate-free and bound states. The two structures are similar and delineate an elongated protein with the two transmembrane domains (TMD) forming an outward facing conformation, extended and twisted exocytoplasmic domains (ECD), and closely opposed nucleotide binding domains. N-Ret-PE is wedged between the two TMDs and a loop from ECD1 within the lumen leaflet consistent with a lateral access mechanism and is stabilized through hydrophobic and ionic interactions with residues from the TMDs and ECDs. Our studies provide a framework for further elucidating the molecular mechanism associated with lipid transport and disease and developing promising disease interventions.

Cryo-EM structure of the sodium-driven chloride/bicarbonate exchanger NDCBE

SLC4 transporters play significant roles in pH regulation and cellular sodium transport. The previously solved structures of the outward facing (OF) conformation for AE1 (SLC4A1) and NBCe1 (SLC4A4) transporters revealed an identical overall fold despite their different transport modes (chloride/bicarbonate exchange versus sodium-carbonate cotransport). However, the exact mechanism determining the different transport modes in the SLC4 family remains unknown. In this work, we report the cryo-EM 3.4 Å structure of the OF conformation of NDCBE (SLC4A8), which shares transport properties with both AE1 and NBCe1 by mediating the electroneutral exchange of sodium-carbonate with chloride. This structure features a fully resolved extracellular loop 3 and well-defined densities corresponding to sodium and carbonate ions in the tentative substrate binding pocket. Further, we combine computational modeling with functional studies to unravel the molecular determinants involved in NDCBE and SLC4 transport.

The ups and downs of elevator-type di-/tricarboxylate membrane transporters.

The divalent anion sodium symporter (DASS) family contains both sodium-driven anion cotransporters and anion/anion exchangers. The family belongs to a broader ion transporter superfamily (ITS), which comprises 24 families of transporters, including those of AbgT antibiotic efflux transporters. The human proteins in the DASS family play major physiological roles and are drug targets. We recently determined multiple structures of the human sodium-dependent citrate transporter (NaCT) and the succinate/dicarboxylate transporter from Lactobacillus acidophilus (LaINDY). Structures of both proteins show high degrees of structural similarity to the previously determined VcINDY fold. Conservation between these DASS protein structures and those from the AbgT family indicates that the VcINDY fold represents the overall protein structure for the entire ITS. The new structures of NaCT and LaINDY are captured in the inward- or outward-facing conformations, respectively. The domain arrangements in these structures agree with a rigid body elevator-type transport mechanism for substrate translocation across the membrane. Two separate NaCT structures in complex with a substrate or an inhibitor allowed us to explain the inhibition mechanism and propose a detailed classification scheme for grouping disease-causing mutations in the human protein. Structural understanding of multiple kinetic states of DASS proteins is a first step toward the detailed characterization of their entire transport cycle.

Cooperative binding ensures the obligatory melibiose/Na+ cotransport in MelB.

MelB catalyzes the obligatory cotransport of melibiose with Na+, Li+, or H+. Crystal structure determination of the Salmonella typhimurium MelB (MelBSt) has revealed a typical major facilitator superfamily (MFS) fold at a periplasmic open conformation. Cooperative binding of Na+ and melibiose has been previously established. To determine why cotranslocation of sugar solute and cation is obligatory, we analyzed each binding in the thermodynamic cycle using three independent methods, including the determination of melting temperature by circular dichroism spectroscopy, heat capacity change (ΔCp), and regulatory phosphotransferase EIIAGlc binding with isothermal titration calorimetry (ITC). We found that MelBSt thermostability is increased by either substrate (Na+ or melibiose) and observed a cooperative effect of both substrates. ITC measurements showed that either binary formation yields a positive sign in the ΔCp, suggesting MelBSt hydration and a likely widening of the periplasmic cavity. Conversely, formation of a ternary complex yields negative values in ΔCp, suggesting MelBSt dehydration and cavity closure. Lastly, we observed that EIIAGlc, which has been suggested to trap MelBSt at an outward-open state, readily binds to the MelBSt apo state at an affinity similar to MelBSt/Na+. However, it has a suboptimal binding to the ternary state, implying that MelBSt in the ternary complex may be conformationally distant from the EIIAGlc-preferred outward-facing conformation. Our results consistently support the notion that binding of one substrate (Na+ or melibiose) favors MelBSt at open states, whereas the cooperative binding of both substrates triggers the alternating-access process, thus suggesting this conformational regulation could ensure the obligatory cotransport.

Structure and mechanism of the human NHE1-CHP1 complex

Sodium/proton exchanger 1 (NHE1) is an electroneutral secondary active transporter present on the plasma membrane of most mammalian cells and plays critical roles in regulating intracellular pH and volume homeostasis. Calcineurin B-homologous protein 1 (CHP1) is an obligate binding partner that promotes NHE1 biosynthetic maturation, cell surface expression and pH-sensitivity. Dysfunctions of either protein are associated with neurological disorders. Here, we elucidate structures of the human NHE1-CHP1 complex in both inward- and inhibitor (cariporide)-bound outward-facing conformations. We find that NHE1 assembles as a symmetrical homodimer, with each subunit undergoing an elevator-like conformational change during cation exchange. The cryo-EM map reveals the binding site for the NHE1 inhibitor cariporide, illustrating how inhibitors block transport activity. The CHP1 molecule differentially associates with these two conformational states of each NHE1 monomer, and this association difference probably underlies the regulation of NHE1 pH-sensitivity by CHP1.

Rare Opportunities for Insights Into Serotonergic Contributions to Brain and Bowel Disorders: Studies of the SERT Ala56 Mouse.

Altered structure, expression, and regulation of the presynaptic serotonin (5-HT) transporter (SERT) have been associated with multiple neurobehavioral disorders, including mood disorders, obsessive-compulsive disorder (OCD), and autism spectrum disorder (ASD). Opportunities to investigate mechanistic links supporting these associations were spurred with the identification of multiple, rare human SERT coding variants in a study that established a male-specific linkage of ASD to a linkage marker on chromosome 17 which encompassed the location of the SERT gene (SLC6A4). We have explored the most common of these variants, SERT Ala56, in vitro and in vivo. Results support a tonic elevation of 5-HT transport activity in transfected cells and human lymphoblasts by the variant in vitro that leads to an increased 5-HT clearance rate in vivo when studied in the SERT Ala56 mouse model, along with altered sensitivity to SERT regulatory signaling pathways. Importantly, hyperserotonemia, or an elevated whole blood 5-HT, level, was found in SERT Ala56 mice, reproducing a well-replicated trait observed in a significant fraction of ASD subjects. Additionally, we found multiple biochemical, physiological, and behavioral alterations in the SERT Ala56 mice that can be analogized to those observed in ASD and its medical comorbidities. The similarity of the functional impact of the SERT Ala56 variant to the consequences of p38α MAPK activation, ascribed to the induction of a biased conformation of the transporter toward an outward-facing conformation, has resulted in successful efforts to restore normal behavioral and bowel function via pharmacological and genetic p38α MAPK targeting. Moreover, the ability of the inflammatory cytokine IL-1β to enhance SERT activity via a p38α MAPK-dependent pathway suggests that the SERT Ala56 conformation mimics that of a chronic inflammatory state, supporting findings in ASD of elevated inflammatory cytokine levels. In this report, we review studies of the SERT Ala56 variant, discussing opportunities for continued insight into how chronically altered synaptic 5-HT homeostasis can drive reversible, functional perturbations in 5-HT sensitive pathways in the brain and periphery, and how targeting the SERT regulome, particularly through activating pathways such as those involving IL-1β/p38α MAPK, may be of benefit for neurobehavioral disorders, including ASD.

Structural Determinants of the Neuronal Glycine Transporter 2 for the Selective Inhibitors ALX1393 and ORG25543.

The neuronal glycine transporter GlyT2 modulates inhibitory glycinergic neurotransmission by controlling the extracellular concentration of synaptic glycine and the supply of neurotransmitter to the presynaptic terminal. Spinal cord glycinergic neurons present in the dorsal horn diminish their activity in pathological pain conditions and behave as gate keepers of the touch-pain circuitry. The pharmacological blockade of GlyT2 reduces the progression of the painful signal to rostral areas of the central nervous system by increasing glycine extracellular levels, so it has analgesic action. O-[(2-benzyloxyphenyl-3-fluorophenyl)methyl]-l-serine (ALX1393) and N-[[1-(dimethylamino)cyclopentyl]methyl]-3,5-dimethoxy-4-(phenylmethoxy)benzamide (ORG25543) are two selective GlyT2 inhibitors with nanomolar affinity for the transporter and analgesic effects in pain animal models, although with deficiencies which preclude further clinical development. In this report, we performed a comparative ligand docking of ALX1393 and ORG25543 on a validated GlyT2 structural model including all ligand sites constructed by homology with the crystallized dopamine transporter from Drosophila melanogaster. Molecular dynamics simulations and energy analysis of the complex and functional analysis of a series of point mutants permitted to determine the structural determinants of ALX1393 and ORG25543 discrimination by GlyT2. The ligands establish simultaneous contacts with residues present in transmembrane domains 1, 3, 6, and 8 and block the transporter in outward-facing conformation and hence inhibit glycine transport. In addition, differential interactions of ALX1393 with the cation bound at Na1 site and ORG25543 with TM10 define the differential sites of the inhibitors and explain some of their individual features. Structural information about the interactions with GlyT2 may provide useful tools for new drug discovery.

Mechanism of substrate transport and inhibition of the human LAT1-4F2hc amino acid transporter

LAT1 (SLC7A5) is one of the representative light chain proteins of heteromeric amino acid transporters, forming a heterodimer with its heavy chain partner 4F2hc (SLC3A2). LAT1 is overexpressed in many types of tumors and mediates the transfer of drugs and hormones across the blood-brain barrier. Thus, LAT1 is considered as a drug target for cancer treatment and may be exploited for drug delivery into the brain. Here, we synthesized three potent inhibitors of human LAT1, which inhibit transport of leucine with IC50 values between 100 and 250 nM, and solved the cryo-EM structures of the corresponding LAT1-4F2hc complexes with these inhibitors bound at resolution of up to 2.7 or 2.8 Å. The protein assumes an outward-facing occluded conformation, with the inhibitors bound in the classical substrate binding pocket, but with their tails wedged between the substrate binding site and TM10 of LAT1. We also solved the complex structure of LAT1-4F2hc with 3,5-diiodo-l-tyrosine (Diiodo-Tyr) at 3.4 Å overall resolution, which revealed a different inhibition mechanism and might represent an intermediate conformation between the outward-facing occluded state mentioned above and the outward-open state. To our knowledge, this is the first time that the outward-facing conformation is revealed for the HAT family. Our results unveil more important insights into the working mechanisms of HATs and provide a structural basis for future drug design.

Molecular structures of the eukaryotic retinal importer ABCA4.

The ATP-binding cassette (ABC) transporter family contains thousands of members with diverse functions. Movement of the substrate, powered by ATP hydrolysis, can be outward (export) or inward (import). ABCA4 is a eukaryotic importer transporting retinal to the cytosol to enter the visual cycle. It also removes toxic retinoids from the disc lumen. Mutations in ABCA4 cause impaired vision or blindness. Despite decades of clinical, biochemical, and animal model studies, the molecular mechanism of ABCA4 is unknown. Here, we report the structures of human ABCA4 in two conformations. In the absence of ATP, ABCA4 adopts an outward-facing conformation, poised to recruit substrate. The presence of ATP induces large conformational changes that could lead to substrate release. These structures provide a molecular basis to understand many disease-causing mutations and a rational guide for new experiments to uncover how ABCA4 recruits, flips, and releases retinoids.

Crystal Structures of a Melibiose Symporter MelB with Bound Sugar Substrate

MelB is a well-studied symporter from the major facilitator superfamily of transporters, and its mammalian homologues play important roles in the delivery of essential omega-3 fatty acids into brain and eyes. The bacterial MelB is selective for galactose or galactosides, and catalyzes the obligatory cotransport of the sugar with Na+, Li+, or H+. Previous 3-D crystal structure of the MelBSt of Salmonella thyphimurium shows that MelB adopts a typical MFS-fold, and the thermodynamic ligand binding studies reveal that cooperative binding of Na+ and melibiose is the core mechanism for the obligatory symport. To gain insights into the substrate binding, we solved three sugar-bound crystal structures from two MelBSt mutants that are compromised on Na+ binding. The structures with bound nitrophenyl-α-D-galactoside or melibiose-based detergent dodecyl-β-d-melibioside (DDMB) were refined to a resolution between 3.05 - 3.25 Å. All three structures are nearly identical at an outward-facing conformation; each one contains a á-galactoside molecule in the middle of protein. The binding interactions of galactosyl moiety from each ligand are indistinguishable, revealing a selection pocket, and the non-galactosyl moieties occupy differently. The active site displays a strong salt-bridge/hydrogen-bonding network that provides multiple polar interactions to each of four OH groups on the galactosyl moiety, and the galactosyl ring is sandwiched by two aromatic sidechains via aromatic stacking interactions. There is little or no interaction toward the non-galactosyl moieties, and the entire 12-carbon tail of DDMB lies within the internal cavity. The localized interactions towards the specific galactosyl moiety and the large internal cavity allow MelB to accommodate a variety of galactosides with different structures. The proposed Na+-binding pocket with no bound Na+ seems to lie in a close proximity to the bound sugar. These structures represent a Na+-free, galactoside-bound binary complex of MelB.

Structural Switches Regulating the Alternating Access Mechanism of Human P-Glycoprotein

P-glycoprotein (Pgp) is a biomedically important ATP-binding cassette (ABC) transporter involved in the development of multidrug resistance in cancer cells. In spite of the canonized work on the transporter function, a lack of general consensus remains concerning the structural details of the different conformational states of Pgp. A major limitation in the study of conformational transitions in ABC transporters is that the process is driven by formation of specific ATP interactions with the nucleotide binding domains (NBDs) of the transporter. Using a novel steered molecular dynamics methodology coupling specific ligand-protein and inter-domain salt-bridge interactions with global conformational changes, we successfully generate four different states of human Pgp, well-separated on the conformational landscape. These include the first stable and catalytically active ATP-bound outward-facing (OF) conformation displaying an open extracellular gate stabilized by lipid binding inside the lumen, as well as an APO (nucleotide-free) inward-facing (IF) conformation showing an open cytoplasmic gate and a large central drug-binding cavity capable of recruiting substrates. In addition, we characterize the only available ATP-bound human Pgp structure as the substrate-free occluded (OC) state of the transporter, with the lumen present in a collapsed state, and likely responsible for the basal-ATPase activity of Pgp. Binding of ATP to the APO IF conformation promotes the closure of the cytoplasmic gate as well as the collapse of the central cavity, likely inducing the transition to the substrate-free OC state. Differential formation of specific salt bridges located at the drug-entry portals in the transmembrane domain as well as between the two NBDs may play an important role in allowing transition between the ATP-bound IF and OF states. The study provides key molecular insights into the specific local switches regulating the global conformational transitions in the transport cycle of human Pgp.

Conformational Transition Pathway of GkPOT

GkPOT, a Proton-Coupled coupled Oligopeptide Transporter transporter (POTs) from Geobacillus Kaustophilus, is among the symporter members of the Major Facilitator Superfamily (MFS) of transporters. Functional and structural analysis of various POTs suggest that the uptake of small peptides and peptide- like molecules to the cell across the cell membrane, via the alternating access mechanism, utilizes an inward directed proton electrochemical gradient the source of energy for their active symport function. The transition between the inward-facing (IF) and outward-facing (OF) conformations is important in understanding the structural relationship between large-scale protein conformational changes and substrate translocation that has often times remained elusive due to the short time scales of molecular dynamics simulations (MD) limitations of computational and experimental effort techniques. Using an extensive set of enhanced sampling techniques, we have characterized the conformational transition pathway of GkPOT. We have aimed at describing the large-scale conformational changes between the IF and OF states while in the presence and absence of substrate (dialanine peptide). Nonequlibrium pulling simulations were initiated from the known inward facing IF conformation and steered the system toward the unknown outward facing OF conformation using orientation based collective variables defined on different transmembrane helices. The transition pathways are far from equilibrium and are smoothed with string method with swarms of trajectories (SMwST), thus generating microseconds of biased and unbiased simlulations. Free energy profiles of the conformational transition pathways were obtained from a novel maximum-likelihood estimation method. Our results indicate that the presence of ligand significantly changes the conformational landscape with respect to the apo system which contains a greater degree of conformational variability.

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.