The Molecular Mechanism of Transport by the Mitochondrial ADP/ATP Carrier.

Mitochondrial ADP/ATP carriers transport ADP into the mitochondrial matrix for ATP synthesis, and ATP out to fuel the cell, by cycling between cytoplasmic-open and matrix-open states. The structure of the cytoplasmic-open state is known, but it has proved difficult to understand the transport mechanism in the absence of a structure in the matrix-open state. Here, we describe the structure of the matrix-open state locked by bongkrekic acid bound in the ADP/ATP-binding site at the bottom of the central cavity. The cytoplasmic side of the carrier is closed by conserved hydrophobic residues, and a salt bridge network, braced by tyrosines. Glycine and small amino acid residues allow close-packing of helices on the matrix side. Uniquely, the carrier switches between states by rotation of its three domains about a fulcrum provided by the substrate-binding site. Because these features are highly conserved, this mechanism is likely to apply to the whole mitochondrial carrier family. VIDEO ABSTRACT.

A structural framework for unidirectional transport by a bacterial ABC exporter.

The ATP-binding cassette (ABC) transporter of mitochondria (Atm1) mediates iron homeostasis in eukaryotes, while the prokaryotic homolog from Novosphingobium aromaticivorans (NaAtm1) can export glutathione derivatives and confer protection against heavy-metal toxicity. To establish the structural framework underlying the NaAtm1 transport mechanism, we determined eight structures by X-ray crystallography and single-particle cryo-electron microscopy in distinct conformational states, stabilized by individual disulfide crosslinks and nucleotides. As NaAtm1 progresses through the transport cycle, conformational changes in transmembrane helix 6 (TM6) alter the glutathione-binding site and the associated substrate-binding cavity. Significantly, kinking of TM6 in the post-ATP hydrolysis state stabilized by MgADPVO4 eliminates this cavity, precluding uptake of glutathione derivatives. The presence of this cavity during the transition from the inward-facing to outward-facing conformational states, and its absence in the reverse direction, thereby provide an elegant and conceptually simple mechanism for enforcing the export directionality of transport by NaAtm1. One of the disulfide crosslinked NaAtm1 variants characterized in this work retains significant glutathione transport activity, suggesting that ATP hydrolysis and substrate transport by Atm1 may involve a limited set of conformational states with minimal separation of the nucleotide-binding domains in the inward-facing conformation.

Uncoupling Proteins and Regulated Proton Leak in Mitochondria.

Higher concentration of protons in the mitochondrial intermembrane space compared to the matrix results in an electrochemical potential causing the back flux of protons to the matrix. This proton transport can take place through ATP synthase complex (leading to formation of ATP) or can occur via proton transporters of the mitochondrial carrier superfamily and/or membrane lipids. Some mitochondrial proton transporters, such as uncoupling proteins (UCPs), transport protons as their general regulating function; while others are symporters or antiporters, which use the proton gradient as a driving force to co-transport other substrates across the mitochondrial inner membrane (such as phosphate carrier, a symporter; or aspartate/glutamate transporter, an antiporter). Passage (or leakage) of protons across the inner membrane to matrix from any route other than ATP synthase negatively impacts ATP synthesis. The focus of this review is on regulated proton transport by UCPs. Recent findings on the structure and function of UCPs, and the related research methodologies, are also critically reviewed. Due to structural similarity of members of the mitochondrial carrier superfamily, several of the known structural features are potentially expandable to all members. Overall, this report provides a brief, yet comprehensive, overview of the current knowledge in the field.

Structural basis for the alternating access mechanism of the cation diffusion facilitator YiiP.

YiiP is a dimeric antiporter from the cation diffusion facilitator family that uses the proton motive force to transport Zn2+ across bacterial membranes. Previous work defined the atomic structure of an outward-facing conformation, the location of several Zn2+ binding sites, and hydrophobic residues that appear to control access to the transport sites from the cytoplasm. A low-resolution cryo-EM structure revealed changes within the membrane domain that were associated with the alternating access mechanism for transport. In the current work, the resolution of this cryo-EM structure has been extended to 4.1 Å. Comparison with the X-ray structure defines the differences between inward-facing and outward-facing conformations at an atomic level. These differences include rocking and twisting of a four-helix bundle that harbors the Zn2+ transport site and controls its accessibility within each monomer. As previously noted, membrane domains are closely associated in the dimeric structure from cryo-EM but dramatically splayed apart in the X-ray structure. Cysteine crosslinking was used to constrain these membrane domains and to show that this large-scale splaying was not necessary for transport activity. Furthermore, dimer stability was not compromised by mutagenesis of elements in the cytoplasmic domain, suggesting that the extensive interface between membrane domains is a strong determinant of dimerization. As with other secondary transporters, this interface could provide a stable scaffold for movements of the four-helix bundle that confers alternating access of these ions to opposite sides of the membrane.

Functional (un)cooperativity in elevator transport proteins.

The activity of enzymes is subject to regulation at multiple levels. Cooperativity, the interconnected behavior of active sites within a protein complex, directly affects protein activity. Cooperativity is a mode of regulation that requires neither extrinsic factors nor protein modifications. Instead, it allows enzymes themselves to modulate reaction rates. Cooperativity is an important regulatory mechanism in soluble proteins, but also examples of cooperative membrane proteins have been described. In this review, we summarize the current knowledge on interprotomer cooperativity in elevator-type proteins, a class of membrane transporters characterized by large rigid-body movements perpendicular to the membrane, and highlight well-studied examples and experimental approaches.

Molecular model of the ferroportin intracellular gate and implications for the human iron transport cycle and hemochromatosis type 4A.

Ferroportin 1 (FPN1) is a major facilitator superfamily transporter that is essential for proper maintenance of human iron homeostasis at the systemic and cellular level. FPN1 dysfunction leads to the progressive accumulation of iron in reticuloendothelial cells, causing hemochromatosis type 4A (or ferroportin disease), an autosomal dominant disorder that displays large phenotypic heterogeneity. Although crystal structures have unveiled the outward- and inward-facing conformations of the bacterial homolog Bdellovibrio bacteriovorus Fpn (or Bd2019) and calcium has recently been identified as an essential cofactor, our molecular understanding of the iron transport mechanism remains incomplete. Here, we used a combination of molecular modeling, molecular dynamics simulations, and Ala site-directed mutagenesis, followed by complementary in vitro functional analyses, to explore the structural architecture of the human FPN1 intracellular gate. We reveal an interdomain network that involves 5 key amino acids and is likely very important for stability of the iron exporter facing the extracellular milieu. We also identify inter- and intradomain interactions that rely on the 2 Asp84 and Asn174 critical residues and do not exist in the bacterial homolog. These interactions are thought to play an important role in the modulation of conformational changes during the transport cycle. We interpret these results in the context of hemochromatosis type 4A, reinforcing the idea that different categories of loss-of-function mutations exist. Our findings provide an unprecedented view of the human FPN1 outward-facing structure and the particular function of the so-called "gating residues" in the mechanism of iron export.-Guellec, J., Elbahnsi, A., Le Tertre, M., Uguen, K., Gourlaouen, I., Férec, C., Ka, C., Callebaut, I., Le Gac, G. Molecular model of the ferroportin intracellular gate and implications for the human iron transport cycle and hemochromatosis type 4A.

The transport mechanism of the mitochondrial ADP/ATP carrier.

The mitochondrial ADP/ATP carrier imports ADP from the cytosol and exports ATP from the mitochondrial matrix, which are key transport steps for oxidative phosphorylation in eukaryotic organisms. The transport protein belongs to the mitochondrial carrier family, a large transporter family in the inner membrane of mitochondria. It is one of the best studied members of the family and serves as a paradigm for the molecular mechanism of mitochondrial carriers. Structurally, the carrier consists of three homologous domains, each composed of two transmembrane α-helices linked with a loop and short α-helix on the matrix side. The transporter cycles between a cytoplasmic and matrix state in which a central substrate binding site is alternately accessible to these compartments for binding of ADP or ATP. On both the cytoplasmic and matrix side of the carrier are networks consisting of three salt bridges each. In the cytoplasmic state, the matrix salt bridge network is formed and the cytoplasmic network is disrupted, opening the central substrate binding site to the intermembrane space and cytosol, whereas the converse occurs in the matrix state. In the transport cycle, tighter substrate binding in the intermediate states allows the interconversion of conformations by lowering the energy barrier for disruption and formation of these networks, opening and closing the carrier to either side of the membrane in an alternating way. Conversion between cytoplasmic and matrix states might require the simultaneous rotation of three domains around a central translocation pathway, constituting a unique mechanism among transport proteins. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou.

Structural insights into functional lipid-protein interactions in secondary transporters.

BACKGROUND: Structural evidences with functional corroborations have revealed distinct features of lipid-protein interactions especially in channels and receptors. Many membrane embedded transporters are also known to require specific lipids for their functions and for some of them cellular and biochemical data suggest tight regulation by the lipid bilayer. However, molecular details on lipid-protein interactions in transporters are sparse since lipids are either depleted from the detergent solubilized transporters in three-dimensional crystals or not readily resolved in crystal structures. Nevertheless the steady increase in the progress of transporter structure determination contributed more examples of structures with resolved lipids. SCOPE OF REVIEW: This review gives an overview on transporter structures in complex with lipids reported to date and discusses commonly encountered difficulties in the identification of functionally significant lipid-protein interactions based on those structures and functional in vitro data. Recent structures provided molecular details into regulation mechanism of transporters by specific lipids. The review highlights common findings and conserved patterns for distantly related transporter families to draw a more general picture on the regulatory role of lipid-protein interactions. MAJOR CONCLUSIONS: Several common themes of the manner in which lipids directly influence membrane-mediated folding, oligomerization and structure stability can be found. Especially for LeuT-like fold transporters similarities in structurally resolved lipid-protein interactions suggest a common way in which transporter conformations are affected by lipids even in evolutionarily distinct transporters. Lipids appear to play an additional role as joints mechanically reinforcing the inverted repeat topology, which is a major determinant in the alternating access mechanism of secondary transporters. GENERAL SIGNIFICANCE: This review brings together and adds to the repertoire of knowledge on lipid-protein interactions of functional significance presented in structures of membrane transporters. Knowledge of specific lipid-binding sites and modes of lipid influence on these proteins not only accomplishes the molecular description of transport cycle further, but also sheds light into localization dependent differences of transporter function. This article is part of a Special Issue entitled Structural biochemistry and biophysics of membrane proteins.

The use of LeuT as a model in elucidating binding sites for substrates and inhibitors in neurotransmitter transporters.

BACKGROUND: The mammalian neurotransmitter transporters are complex proteins playing a central role in synaptic transmission between neurons by rapid reuptake of neurotransmitters. The proteins which transport dopamine, noradrenaline and serotonin belong to the Neurotransmitter:Sodium Symporters (NSS). Due to their important role, dysfunctions are associated with several psychiatric and neurological diseases and they also serve as targets for a wide range of therapeutic and illicit drugs. Despite the central physiological and pharmacological importance, direct evidence on structure-function relationships on mammalian NSS proteins has so far been unsuccessful. The crystal structure of the bacterial NSS protein, LeuT, has been a turning point in structural investigations. SCOPE OF REVIEW: To provide an update on what is known about the binding sites for substrates and inhibitors in the LeuT. The different binding modes and binding sites will be discussed with special emphasis on the possible existence of a second substrate binding site. It is the goal to give an insight into how investigations on ligand binding in LeuT have provided basic knowledge about transporter conformations and translocation mechanism which can pave the road for a deeper understanding of drug binding and function of the mammalian transporters. MAJOR CONCLUSIONS: The LeuT is a suitable model for the structural investigation of NSS proteins including the possible location of drug binding sites. It is still debated whether the LeuT is a suitable model for the molecular mechanisms behind substrate translocation. GENERAL SIGNIFICANCE: Structure and functional aspects of NSS proteins are central for understanding synaptic transmission. With the purification and crystallization of LeuT as well as the dopamine transporter from Drosophila melanogaster, the application of biophysical methods such as fluorescence spectroscopy, neutron- or x-ray scattering and NMR for understanding its function becomes increasingly available. This article is part of a Special Issue entitled Structural biochemistry and biophysics of membrane proteins.

Ins and Outs of Rocker Switch Mechanism in Major Facilitator Superfamily of Transporters.

The major facilitator superfamily (MFS) of transporters consists of three classes of membrane transporters: symporters, uniporters, and antiporters. Despite such diverse functions, MFS transporters are believed to undergo similar conformational changes within their distinct transport cycles, known as the rocker-switch mechanism. While the similarities between conformational changes are noteworthy, the differences are also important since they could potentially explain the distinct functions of symporters, uniporters, and antiporters of the MFS superfamily. We reviewed a variety of experimental and computational structural data on a select number of antiporters, symporters, and uniporters from the MFS family to compare the similarities and differences of the conformational dynamics of three different classes of transporters.

The Alternating Access Mechanism in Mammalian Multidrug Resistance Transporters and Their Bacterial Homologs.

Multidrug resistance (MDR) proteins belonging to the ATP-Binding Cassette (ABC) transporter group play a crucial role in the export of cytotoxic drugs across cell membranes. These proteins are particularly fascinating due to their ability to confer drug resistance, which subsequently leads to the failure of therapeutic interventions and hinders successful treatments. One key mechanism by which multidrug resistance (MDR) proteins carry out their transport function is through alternating access. This mechanism involves intricate conformational changes that enable the binding and transport of substrates across cellular membranes. In this extensive review, we provide an overview of ABC transporters, including their classifications and structural similarities. We focus specifically on well-known mammalian multidrug resistance proteins such as MRP1 and Pgp (MDR1), as well as bacterial counterparts such as Sav1866 and lipid flippase MsbA. By exploring the structural and functional features of these MDR proteins, we shed light on the roles of their nucleotide-binding domains (NBDs) and transmembrane domains (TMDs) in the transport process. Notably, while the structures of NBDs in prokaryotic ABC proteins, such as Sav1866, MsbA, and mammalian Pgp, are identical, MRP1 exhibits distinct characteristics in its NBDs. Our review also emphasizes the importance of two ATP molecules for the formation of an interface between the two binding sites of NBD domains across all these transporters. ATP hydrolysis occurs following substrate transport and is vital for recycling the transporters in subsequent cycles of substrate transportation. Specifically, among the studied transporters, only NBD2 in MRP1 possesses the ability to hydrolyze ATP, while both NBDs of Pgp, Sav1866, and MsbA are capable of carrying out this reaction. Furthermore, we highlight recent advancements in the study of MDR proteins and the alternating access mechanism. We discuss the experimental and computational approaches utilized to investigate the structure and dynamics of MDR proteins, providing valuable insights into their conformational changes and substrate transport. This review not only contributes to an enhanced understanding of multidrug resistance proteins but also holds immense potential for guiding future research and facilitating the development of effective strategies to overcome multidrug resistance, thus improving therapeutic interventions.

The molecular features of uncoupling protein 1 support a conventional mitochondrial carrier-like mechanism.

Uncoupling protein 1 (UCP1) is an integral membrane protein found in the mitochondrial inner membrane of brown adipose tissue, and facilitates the process of non-shivering thermogenesis in mammals. Its activation by fatty acids, which overcomes its inhibition by purine nucleotides, leads to an increase in the proton conductance of the inner mitochondrial membrane, short-circuiting the mitochondrion to produce heat rather than ATP. Despite 40 years of intense research, the underlying molecular mechanism of UCP1 is still under debate. The protein belongs to the mitochondrial carrier family of transporters, which have recently been shown to utilise a domain-based alternating-access mechanism, cycling between a cytoplasmic and matrix state to transport metabolites across the inner membrane. Here, we review the protein properties of UCP1 and compare them to those of mitochondrial carriers. UCP1 has the same structural fold as other mitochondrial carriers and, in contrast to past claims, is a monomer, binding one purine nucleotide and three cardiolipin molecules tightly. The protein has a single substrate binding site, which is similar to those of the dicarboxylate and oxoglutarate carriers, but also contains a proton binding site and several hydrophobic residues. As found in other mitochondrial carriers, UCP1 has two conserved salt bridge networks on either side of the central cavity, which regulate access to the substrate binding site in an alternating way. The conserved domain structures and mobile inter-domain interfaces are consistent with an alternating access mechanism too. In conclusion, UCP1 has retained all of the key features of mitochondrial carriers, indicating that it operates by a conventional carrier-like mechanism.

Structure of the SLC4 transporter Bor1p in an inward-facing conformation.

Bor1p is a secondary transporter in yeast that is responsible for boron transport. Bor1p belongs to the SLC4 family which controls bicarbonate exchange and pH regulation in animals as well as borate uptake in plants. The SLC4 family is more distantly related to members of the Amino acid-Polyamine-organoCation (APC) superfamily, which includes well studied transporters such as LeuT, Mhp1, AdiC, vSGLT, UraA, SLC26Dg. Their mechanism generally involves relative movements of two domains: a core domain that binds substrate and a gate domain that in many cases mediates dimerization. To shed light on conformational changes governing transport by the SLC4 family, we grew helical membrane crystals of Bor1p from Saccharomyces mikatae and determined a structure at ∼6 Šresolution using cryo-electron microscopy. To evaluate the conformation of Bor1p in these crystals, a homology model was built based on the related anion exchanger from red blood cells (AE1). This homology model was fitted to the cryo-EM density map using the Molecular Dynamics (MD) Flexible Fitting method and then relaxed by all-atom MD simulation in explicit solvent and membrane. Mapping of water accessibility indicates that the resulting structure represents an inward-facing conformation. Comparisons of the resulting Bor1p model with the X-ray structure of AE1 in an outward-facing conformation, together with MD simulations of inward-facing and outward-facing Bor1p models, suggest rigid body movements of the core domain relative to the gate domain. These movements are consistent with the rocking-bundle transport mechanism described for other members of the APC superfamily.

Interrogating the Molecular Basis for Substrate Recognition in Serotonin and Dopamine Transporters with High-Affinity Substrate-Based Bivalent Ligands.

The transporters for the neurotransmitters serotonin and dopamine (SERT and DAT, respectively) are targets for drugs used in the treatment of mental disorders and widely used drugs of abuse. Studies of prokaryotic homologues have advanced our structural understanding of SERT and DAT, but it still remains enigmatic whether the human transporters contain one or two high-affinity substrate binding sites. We have designed and employed 24 bivalent ligands possessing a highly systematic combination of substrate moieties (serotonin and/or dopamine) and aliphatic or poly(ethylene glycol) spacers to reveal insight into substrate recognition in SERT and DAT. An optimized bivalent ligand comprising two serotonin moieties binds SERT with 3,800-fold increased affinity compared to that of serotonin, suggesting that the human transporters have two distinct substrate binding sites. We show that the bivalent ligands are inhibitors of SERT and an experimentally validated docking model suggests that the bivalent compounds bind with one substrate moiety in the central binding site (the S1 site), whereas the other substrate moiety binds in a distinct binding site (the S2 site). A systematic study of nonconserved SERT/DAT residues surrounding the proposed binding region showed that nonconserved binding site residues do not contribute to selective recognition of substrates in SERT or DAT. This study provides novel insight into the molecular basis for substrate recognition in human transporters and provides an improved foundation for the development of new drugs targeting SERT and DAT.