Structural mechanisms of mitochondrial uncoupling protein 1 regulation in thermogenesis.

In mitochondria, the oxidation of nutrients is coupled to ATP synthesis by the generation of a protonmotive force across the mitochondrial inner membrane. In mammalian brown adipose tissue (BAT), uncoupling protein 1 (UCP1, SLC25A7), a member of the SLC25 mitochondrial carrier family, dissipates the protonmotive force by facilitating the return of protons to the mitochondrial matrix. This process short-circuits the mitochondrion, generating heat for non-shivering thermogenesis. Recent cryo-electron microscopy (cryo-EM) structures of human UCP1 have provided new molecular insights into the inhibition and activation of thermogenesis. Here, we discuss these structures, describing how purine nucleotides lock UCP1 in a proton-impermeable conformation and rationalizing potential conformational changes of this carrier in response to fatty acid activators that enable proton leak for thermogenesis.

The Concise Guide to PHARMACOLOGY 2023/24: Transporters.

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and over 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (https://www.guidetopharmacology.org/), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.16182. Transporters are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors and enzymes. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Fifty years of the mitochondrial pyruvate carrier: New insights into its structure, function, and inhibition.

The mitochondrial pyruvate carrier (MPC) resides in the mitochondrial inner membrane, where it links cytosolic and mitochondrial metabolism by transporting pyruvate produced in glycolysis into the mitochondrial matrix. Due to its central metabolic role, it has been proposed as a potential drug target for diabetes, non-alcoholic fatty liver disease, neurodegeneration, and cancers relying on mitochondrial metabolism. Little is known about the structure and mechanism of MPC, as the proteins involved were only identified a decade ago and technical difficulties concerning their purification and stability have hindered progress in functional and structural analyses. The functional unit of MPC is a hetero-dimer comprising two small homologous membrane proteins, MPC1/MPC2 in humans, with the alternative complex MPC1L/MPC2 forming in the testis, but MPC proteins are found throughout the tree of life. The predicted topology of each protomer consists of an amphipathic helix followed by three transmembrane helices. An increasing number of inhibitors are being identified, expanding MPC pharmacology and providing insights into the inhibitory mechanism. Here, we provide critical insights on the composition, structure, and function of the complex and we summarize the different classes of small molecule inhibitors and their potential in therapeutics.

The free-living flagellate Paratrimastix pyriformis uses a distinct mitochondrial carrier to balance adenine nucleotide pools.

Paratrimastix pyriformis is a free-living flagellate thriving in low-oxygen freshwater sediments. It belongs to the group Metamonada along with human parasites, such as Giardia and Trichomonas. Like other metamonads, P. pyriformis has a mitochondrion-related organelle (MRO) which in this protist is primarily involved in one-carbon folate metabolism. The MRO contains four members of the solute carrier family 25 (SLC25) responsible for the exchange of metabolites across the mitochondrial inner membrane. Here, we characterise the function of the adenine nucleotide carrier PpMC1 by thermostability shift and transport assays. We show that it transports ATP, ADP and, to a lesser extent, AMP, but not phosphate. The carrier is distinct in function and origin from both ADP/ATP carriers and ATP-Mg/phosphate carriers, and it most likely represents a distinct class of adenine nucleotide carriers.

Structural basis of purine nucleotide inhibition of human uncoupling protein 1.

Mitochondrial uncoupling protein 1 (UCP1) gives brown adipose tissue of mammals its specialized ability to burn calories as heat for thermoregulation. When activated by fatty acids, UCP1 catalyzes the leak of protons across the mitochondrial inner membrane, short-circuiting the mitochondrion to generate heat, bypassing ATP synthesis. In contrast, purine nucleotides bind and inhibit UCP1, regulating proton leak by a molecular mechanism that is unclear. We present the cryo-electron microscopy structure of the GTP-inhibited state of UCP1, which is consistent with its nonconducting state. The purine nucleotide cross-links the transmembrane helices of UCP1 with an extensive interaction network. Our results provide a structural basis for understanding the specificity and pH dependency of the regulatory mechanism. UCP1 has retained all of the key functional and structural features required for a mitochondrial carrier-like transport mechanism. The analysis shows that inhibitor binding prevents the conformational changes that UCP1 uses to facilitate proton leak.

Activating ligands of Uncoupling protein 1 identified by rapid membrane protein thermostability shift analysis.

OBJECTIVE: Uncoupling protein 1 (UCP1) catalyses mitochondrial proton leak in brown adipose tissue to facilitate nutrient oxidation for heat production, and may combat metabolic disease if activated in humans. During the adrenergic stimulation of brown adipocytes, free fatty acids generated from lipolysis activate UCP1 via an unclear interaction. Here, we set out to characterise activator binding to purified UCP1 to clarify the activation process, discern novel activators and the potential to target UCP1. METHODS: We assessed ligand binding to purified UCP1 by protein thermostability shift analysis, which unlike many conventional approaches can inform on the binding of hydrophobic ligands to membrane proteins. A detailed activator interaction analysis and screening approach was carried out, supported by investigations of UCP1 activity in liposomes, isolated brown fat mitochondria and UCP1 expression-controlled cell lines. RESULTS: We reveal that fatty acids and other activators influence UCP1 through a specific destabilising interaction, behaving as transport substrates that shift the protein to a less stable conformation of a transport cycle. Through the detection of specific stability shifts in screens, we identify novel activators, including the over-the-counter drug ibuprofen, where ligand analysis indicates that UCP1 has a relatively wide structural specificity for interacting molecules. Ibuprofen successfully induced UCP1 activity in liposomes, isolated brown fat mitochondria and UCP1-expressing HEK293 cells but not in cultured brown adipocytes, suggesting drug delivery differs in each cell type. CONCLUSIONS: These findings clarify the nature of the activator-UCP1 interaction and demonstrate that the targeting of UCP1 in cells by approved drugs is in principle achievable as a therapeutic avenue, but requires variants with more effective delivery in brown adipocytes.

Substrate binding in the mitochondrial ADP/ATP carrier is a step-wise process guiding the structural changes in the transport cycle.

Mitochondrial ADP/ATP carriers import ADP into the mitochondrial matrix and export ATP to the cytosol to fuel cellular processes. Structures of the inhibited cytoplasmic- and matrix-open states have confirmed an alternating access transport mechanism, but the molecular details of substrate binding remain unresolved. Here, we evaluate the role of the solvent-exposed residues of the translocation pathway in the process of substrate binding. We identify the main binding site, comprising three positively charged and a set of aliphatic and aromatic residues, which bind ADP and ATP in both states. Additionally, there are two pairs of asparagine/arginine residues on opposite sides of this site that are involved in substrate binding in a state-dependent manner. Thus, the substrates are directed through a series of binding poses, inducing the conformational changes of the carrier that lead to their translocation. The properties of this site explain the electrogenic and reversible nature of adenine nucleotide transport.

TMEM63C mutations cause mitochondrial morphology defects and underlie hereditary spastic paraplegia.

The hereditary spastic paraplegias (HSP) are among the most genetically diverse of all Mendelian disorders. They comprise a large group of neurodegenerative diseases that may be divided into 'pure HSP' in forms of the disease primarily entailing progressive lower-limb weakness and spasticity, and 'complex HSP' when these features are accompanied by other neurological (or non-neurological) clinical signs. Here, we identified biallelic variants in the transmembrane protein 63C (TMEM63C) gene, encoding a predicted osmosensitive calcium-permeable cation channel, in individuals with hereditary spastic paraplegias associated with mild intellectual disability in some, but not all cases. Biochemical and microscopy analyses revealed that TMEM63C is an endoplasmic reticulum-localized protein, which is particularly enriched at mitochondria-endoplasmic reticulum contact sites. Functional in cellula studies indicate a role for TMEM63C in regulating both endoplasmic reticulum and mitochondrial morphologies. Together, these findings identify autosomal recessive TMEM63C variants as a cause of pure and complex HSP and add to the growing evidence of a fundamental pathomolecular role of perturbed mitochondrial-endoplasmic reticulum dynamics in motor neurone degenerative diseases.

Pathogenic variants of the mitochondrial aspartate/glutamate carrier causing citrin deficiency.

Citrin deficiency is a pan-ethnic and highly prevalent mitochondrial disease with three different stages: neonatal intrahepatic cholestasis (NICCD), a relatively mild adaptation stage, and type II citrullinemia in adulthood (CTLN2). The cause is the absence or dysfunction of the calcium-regulated mitochondrial aspartate/glutamate carrier 2 (AGC2/SLC25A13), also called citrin, which imports glutamate into the mitochondrial matrix and exports aspartate to the cytosol. In citrin deficiency, these missing transport steps lead to impairment of the malate-aspartate shuttle, gluconeogenesis, amino acid homeostasis, and the urea cycle. In this review, we describe the geological spread and occurrence of citrin deficiency, the metabolic consequences and use our current knowledge of the structure to predict the impact of the known pathogenic mutations on the calcium-regulatory and transport mechanism of citrin.

Key features of inhibitor binding to the human mitochondrial pyruvate carrier hetero-dimer.

OBJECTIVE: The mitochondrial pyruvate carrier (MPC) has emerged as a promising drug target for metabolic disorders, including non-alcoholic steatohepatitis and diabetes, metabolically dependent cancers and neurodegenerative diseases. A range of structurally diverse small molecule inhibitors have been proposed, but the nature of their interaction with MPC is not understood, and the composition of the functional human MPC is still debated. The goal of this study was to characterise the human MPC protein in vitro, to understand the chemical features that determine binding of structurally diverse inhibitors and to develop novel higher affinity ones. METHODS: We recombinantly expressed and purified human MPC hetero-complexes and studied their composition, transport and inhibitor binding properties by establishing in vitro transport assays, high throughput thermostability shift assays and pharmacophore modeling. RESULTS: We determined that the functional unit of human MPC is a hetero-dimer. We compared all different classes of MPC inhibitors to find that three closely arranged hydrogen bond acceptors followed by an aromatic ring are shared characteristics of all inhibitors and represent the minimal requirement for high potency. We also demonstrated that high affinity binding is not attributed to covalent bond formation with MPC cysteines, as previously proposed. Following the basic pharmacophore properties, we identified 14 new inhibitors of MPC, one outperforming compound UK5099 by tenfold. Two are the commonly prescribed drugs entacapone and nitrofurantoin, suggesting an off-target mechanism associated with their adverse effects. CONCLUSIONS: This work defines the composition of human MPC and the essential MPC inhibitor characteristics. In combination with the functional assays we describe, this new understanding will accelerate the development of clinically relevant MPC modulators.

Structure, substrate binding, and symmetry of the mitochondrial ADP/ATP carrier in its matrix-open state.

The mitochondrial ADP/ATP carrier (AAC) performs the first and last step in oxidative phosphorylation by exchanging ADP and ATP across the mitochondrial inner membrane. Its optimal function has been shown to be dependent on cardiolipins (CLs), unique phospholipids located almost exclusively in the mitochondrial membrane. In addition, AAC exhibits an enthralling threefold pseudosymmetry, a unique feature of members of the SLC25 family. Recently, its conformation poised for binding of ATP was solved by x-ray crystallography referred to as the matrix state. Binding of the substrate leads to conformational changes that export of ATP to the mitochondrial intermembrane space. In this contribution, we investigate the influence of CLs on the structure, substrate-binding properties, and structural symmetry of the matrix state, employing microsecond-scale molecular dynamics simulations. Our findings demonstrate that CLs play a minor stabilizing role on the AAC structure. The interdomain salt bridges and hydrogen bonds forming the cytoplasmic network and tyrosine braces, which ensure the integrity of the global AAC scaffold, highly benefit from the presence of CLs. Under these conditions, the carrier is found to be organized in a more compact structure in its interior, as revealed by analyses of the electrostatic potential, measure of the AAC cavity aperture, and the substrate-binding assays. Introducing a convenient structure-based symmetry metric, we quantified the structural threefold pseudosymmetry of AAC, not only for the crystallographic structure, but also for conformational states of the carrier explored in the molecular dynamics simulations. Our results suggest that CLs moderately contribute to preserve the pseudosymmetric structure of AAC.

Exome sequencing identifies a disease variant of the mitochondrial ATP-Mg/Pi carrier SLC25A25 in two families with kidney stones.

BACKGROUND: Calcium kidney stones are common and recurrences are often not preventable by available empiric remedies. Their etiology is multifactorial and polygenic, and an increasing number of genes are implicated. Their identification will enable improved management. METHODS: DNA from three stone-formers in a Southampton family (UK) and two from an Italian family were analyzed independently by whole exome sequencing and selected variants were genotyped across all available members of both pedigrees. A disease variant of SLC25A25 (OMIM 608745), encoding the mitochondrial ATP-Mg/Pi carrier 3 (APC3) was identified, and analyzed structurally and functionally with respect to its calcium-regulated transport activity. RESULTS: All five patients had a heterozygous dominant SLC25A25 variant (rs140777921; GRCh37.p13: chr 9 130868670 G>C; p.Gln349His; Reference Sequence NM_001006641.3). Non-stone formers also carried the variant indicating incomplete penetrance. Modeling suggests that the variant lacks a conserved polar interaction, which may cause structural instability. Calcium-regulated ATP transport was reduced to ~20% of the wild type, showing a large reduction in function. CONCLUSION: The transporter is important in regulating mitochondrial ATP production. This rare variant may increase urine lithogenicity through impaired provision of ATP for solute transport processes in the kidney, and/or for purinergic signaling. Variants found in other genes may compound this abnormality.

Characterization of drug-induced human mitochondrial ADP/ATP carrier inhibition.

An increasing number of commonly prescribed drugs are known to interfere with mitochondrial function, causing cellular toxicity, but the underlying mechanisms are largely unknown. Although often not considered, mitochondrial transport proteins form a significant class of potential mitochondrial off-targets. So far, most drug interactions have been reported for the mitochondrial ADP/ATP carrier (AAC), which exchanges cytosolic ADP for mitochondrial ATP. Here, we show inhibition of cellular respiratory capacity by only a subset of the 18 published AAC inhibitors, which questions whether all compound do indeed inhibit such a central metabolic process. This could be explained by the lack of a simple, direct model system to evaluate and compare drug-induced AAC inhibition. Methods: For its development, we have expressed and purified human AAC1 (hAAC1) and applied two approaches. In the first, thermostability shift assays were carried out to investigate the binding of these compounds to human AAC1. In the second, the effect of these compounds on transport was assessed in proteoliposomes with reconstituted human AAC1, enabling characterization of their inhibition kinetics. Results: Of the proposed inhibitors, chebulinic acid, CD-437 and suramin are the most potent with IC50-values in the low micromolar range, whereas another six are effective at a concentration of 100 µM. Remarkably, half of all previously published AAC inhibitors do not show significant inhibition in our assays, indicating that they are false positives. Finally, we show that inhibitor strength correlates with a negatively charged surface area of the inhibitor, matching the positively charged surface of the substrate binding site. Conclusion: Consequently, we have provided a straightforward model system to investigate AAC inhibition and have gained new insights into the chemical compound features important for inhibition. Better evaluation methods of drug-induced inhibition of mitochondrial transport proteins will contribute to the development of drugs with an enhanced safety profile.

A Single Cysteine Residue in the Translocation Pathway of the Mitosomal ADP/ATP Carrier from Cryptosporidium parvum Confers a Broad Nucleotide Specificity.

Cryptosporidiumparvum is a clinically important eukaryotic parasite that causes the disease cryptosporidiosis, which manifests with gastroenteritis-like symptoms. The protist has mitosomes, which are organelles of mitochondrial origin that have only been partially characterized. The genome encodes a highly reduced set of transport proteins of the SLC25 mitochondrial carrier family of unknown function. Here, we have studied the transport properties of one member of the C. parvum carrier family, demonstrating that it resembles the mitochondrial ADP/ATP carrier of eukaryotes. However, this carrier has a broader substrate specificity for nucleotides, transporting adenosine, thymidine, and uridine di- and triphosphates in contrast to its mitochondrial orthologues, which have a strict substrate specificity for ADP and ATP. Inspection of the putative translocation pathway highlights a cysteine residue, which is a serine in mitochondrial ADP/ATP carriers. When the serine residue is replaced by cysteine or larger hydrophobic residues in the yeast mitochondrial ADP/ATP carrier, the substrate specificity becomes broad, showing that this residue is important for nucleotide base selectivity in ADP/ATP carriers.

The SLC25 Carrier Family: Important Transport Proteins in Mitochondrial Physiology and Pathology.

Members of the mitochondrial carrier family (SLC25) transport a variety of compounds across the inner membrane of mitochondria. These transport steps provide building blocks for the cell and link the pathways of the mitochondrial matrix and cytosol. An increasing number of diseases and pathologies has been associated with their dysfunction. In this review, the molecular basis of these diseases is explained based on our current understanding of their transport mechanism.

The mitochondrial ADP/ATP carrier exists and functions as a monomer.

For more than 40 years, the oligomeric state of members of the mitochondrial carrier family (SLC25) has been the subject of debate. Initially, the consensus was that they were dimeric, based on the application of a large number of different techniques. However, the structures of the mitochondrial ADP/ATP carrier, a member of the family, clearly demonstrated that its structural fold is monomeric, lacking a conserved dimerisation interface. A re-evaluation of previously published data, with the advantage of hindsight, concluded that technical errors were at the basis of the earlier dimer claims. Here, we revisit this topic, as new claims for the existence of dimers of the bovine ADP/ATP carrier have emerged using native mass spectrometry of mitochondrial membrane vesicles. However, the measured mass does not agree with previously published values, and a large number of post-translational modifications are proposed to account for the difference. Contrarily, these modifications are not observed in electron density maps of the bovine carrier. If they were present, they would interfere with the structure and function of the carrier, including inhibitor and substrate binding. Furthermore, the reported mass does not account for three tightly bound cardiolipin molecules, which are consistently observed in other studies and are important stabilising factors for the transport mechanism. The monomeric carrier has all of the required properties for a functional transporter and undergoes large conformational changes that are incompatible with a stable dimerisation interface. Thus, our view that the native mitochondrial ADP/ATP carrier exists and functions as a monomer remains unaltered.

Structural insight into mitochondrial ß-barrel outer membrane protein biogenesis.

In mitochondria, ß-barrel outer membrane proteins mediate protein import, metabolite transport, lipid transport, and biogenesis. The Sorting and Assembly Machinery (SAM) complex consists of three proteins that assemble as a 1:1:1 complex to fold ß-barrel proteins and insert them into the mitochondrial outer membrane. We report cryoEM structures of the SAM complex from Myceliophthora thermophila, which show that Sam50 forms a 16-stranded transmembrane ß-barrel with a single polypeptide-transport-associated (POTRA) domain extending into the intermembrane space. Sam35 and Sam37 are located on the cytosolic side of the outer membrane, with Sam35 capping Sam50, and Sam37 interacting extensively with Sam35. Sam35 and Sam37 each adopt a GST-like fold, with no functional, structural, or sequence similarity to their bacterial counterparts. Structural analysis shows how the Sam50 ß-barrel opens a lateral gate to accommodate its substrates.

Expression and Purification of Membrane Proteins in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is one of the most popular expression systems for eukaryotic membrane proteins. Here, we describe protocols for the expression and purification of mitochondrial membrane proteins developed in our laboratory during the last 15 years. To optimize their expression in a functional form, different promoter systems as well as codon-optimization and complementation strategies were established. Purification approaches were developed which remove the membrane protein from the affinity column by specific proteolytic cleavage rather than by elution. This strategy has several important advantages, most notably improving the purity of the sample, as contaminants stay bound to the column, thus eliminating the need for a secondary purification step, such as size exclusion chromatography. This strategy also avoids dilution of the sample, which would occur as a consequence of elution, precluding the need for concentration steps, and thus preventing detergent concentration.

The SLC25 Mitochondrial Carrier Family: Structure and Mechanism.

Members of the mitochondrial carrier family (SLC25) provide the transport steps for amino acids, carboxylic acids, fatty acids, cofactors, inorganic ions, and nucleotides across the mitochondrial inner membrane and are crucial for many cellular processes. Here, we use new insights into the transport mechanism of the mitochondrial ADP/ATP carrier to examine the structure and function of other mitochondrial carriers. They all have a single substrate-binding site and two gates, which are present on either side of the membrane and involve salt-bridge networks. Transport is likely to occur by a common mechanism, in which the coordinated movement of six structural elements leads to the alternating opening and closing of the matrix or cytoplasmic side of the carriers.

Thermostability Assays: a Generic and Versatile Tool for Studying the Functional and Structural Properties of Membrane Proteins in Detergents.

There are very few generic methods to assess the stability and functional properties of membrane proteins solubilized in detergent. For this purpose, a thiol-reactive fluorochrome N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM) can be used. An unfolding profile is obtained when the fluorochrome becomes fluorescent on reaction with cysteine residues that have been exposed during thermal denaturation of the protein population. The method was initially developed to optimize the stability of membrane proteins for crystallization studies, but in the course of our work we found many other applications. First, the assay can be used to study the binding of inhibitors, substrates, lipids, and other effectors to membrane proteins. Second, the assay can be used to understand the dynamics of proteins, allowing states to be defined by changes in accessibility of cysteine residues or by changes in specific amino acid interactions. Finally, the assay can be used to study state-dependent domain interactions, for example, as part of regulatory mechanisms. The CPM thermostability assay represents a broadly applicable and versatile tool for a wide range of applications in the functional and structural analysis of membrane proteins.