Christophe Wirth

Mechanistic insight from the crystal structure of mitochondrial complex I

Energy conversion in complex 1 ATP, the energy source of the cell, is synthesized by a protein residing in the mitochondrial inner membrane. The synthesis is driven by a proton gradient generated by redox reactions that transfer electrons between a series of enzymes in the membrane. The largest complex in this electron transfer chain is the 1-MD complex 1. It couples electron transfer from NADH to ubiquinone to the translocation of four protons. Zickermann et al. report the crystal structure of a complex comprising the 14 central subunits and the largest accessory subunit of mitochondrial complex 1 from a yeast-genetic model at 3.6 Å resolution. The structure identifies four potential proton translocation pathways and gives insight into how energy from the redox reactions is transmitted to drive proton pumping. Science, this issue p. 44 The x-ray structure of a protein complex shows how this enzyme pumps protons across the mitochondrial membrane. Proton-pumping complex I of the mitochondrial respiratory chain is among the largest and most complicated membrane protein complexes. The enzyme contributes substantially to oxidative energy conversion in eukaryotic cells. Its malfunctions are implicated in many hereditary and degenerative disorders. We report the x-ray structure of mitochondrial complex I at a resolution of 3.6 to 3.9 angstroms, describing in detail the central subunits that execute the bioenergetic function. A continuous axis of basic and acidic residues running centrally through the membrane arm connects the ubiquinone reduction site in the hydrophilic arm to four putative proton-pumping units. The binding position for a substrate analogous inhibitor and blockage of the predicted ubiquinone binding site provide a model for the “deactive” form of the enzyme. The proposed transition into the active form is based on a concerted structural rearrangement at the ubiquinone reduction site, providing support for a two-state stabilization-change mechanism of proton pumping.

Dual Function of Sdh3 in the Respiratory Chain and TIM22 Protein Translocase of the Mitochondrial Inner Membrane

The mitochondrial inner membrane harbors the complexes of the respiratory chain and translocase complexes for precursor proteins. We have identified a further subunit of the carrier translocase (TIM22 complex) that surprisingly is identical to subunit 3 of respiratory complex II, succinate dehydrogenase (Sdh3). The membrane-integral protein Sdh3 plays specific functions in electron transfer in complex II. We show by genetic and biochemical approaches that Sdh3 also plays specific functions in the TIM22 complex. Sdh3 forms a subcomplex with Tim18 and is involved in biogenesis and assembly of the membrane-integral subunits of the TIM22 complex. We conclude that the assembly of Sdh3 with different partner proteins, Sdh4 and Tim18, recruits it to two different mitochondrial membrane complexes with functions in bioenergetics and protein biogenesis, respectively.

Coupling of Mitochondrial Import and Export Translocases by Receptor-Mediated Supercomplex Formation

The mitochondrial outer membrane harbors two protein translocases that are essential for cell viability: the translocase of the outer mitochondrial membrane (TOM) and the sorting and assembly machinery (SAM). The precursors of β-barrel proteins use both translocases-TOM for import to the intermembrane space and SAM for export into the outer membrane. It is unknown if the translocases cooperate and where the β-barrel of newly imported proteins is formed. We established a position-specific assay for monitoring β-barrel formation in vivo and in organello and demonstrated that the β-barrel was formed and membrane inserted while the precursor was bound to SAM. β-barrel formation was inhibited by SAM mutants and, unexpectedly, by mutants of the central import receptor, Tom22. We show that the cytosolic domain of Tom22 links TOM and SAM into a supercomplex, facilitating precursor transfer on the intermembrane space side. Our study reveals receptor-mediated coupling of import and export translocases as a means of precursor channeling.

Structure and function of mitochondrial complex I

Proton-pumping NADH:ubiquinone oxidoreductase (complex I) is the largest and most complicated enzyme of the respiratory chain. Fourteen central subunits represent the minimal form of complex I and can be assigned to functional modules for NADH oxidation, ubiquinone reduction, and proton pumping. In addition, the mitochondrial enzyme comprises some 30 accessory subunits surrounding the central subunits that are not directly associated with energy conservation. Complex I is known to release deleterious oxygen radicals (ROS) and its dysfunction has been linked to a number of hereditary and degenerative diseases. We here review recent progress in structure determination, and in understanding the role of accessory subunits and functional analysis of mitochondrial complex I. For the central subunits, structures provide insight into the arrangement of functional modules including the substrate binding sites, redox-centers and putative proton channels and pump sites. Only for two of the accessory subunits, detailed structures are available. Nevertheless, many of them could be localized in the overall structure of complex I, but most of these assignments have to be considered tentative. Strikingly, redox reactions and proton pumping machinery are spatially completely separated and the site of reduction for the hydrophobic substrate ubiquinone is found deeply buried in the hydrophilic domain of the complex. The X-ray structure of complex I from Yarrowia lipolytica provides clues supporting the previously proposed two-state stabilization change mechanism, in which ubiquinone redox chemistry induces conformational states and thereby drives proton pumping. The same structural rearrangements may explain the active/deactive transition of complex I implying an integrated mechanistic model for energy conversion and regulation. This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt.

The Large Extracellular Loop of Organic Cation Transporter 1 Influences Substrate Affinity and Is Pivotal for Oligomerization

Background: Organic cation transporter OCT1 forms oligomers. Results: The intact structure of the large extracelluar loop of OCT1 is pivotal for oligomerization. Oligomerization increases membrane targeting and does not influence substrate affinities. Conclusion: OCT1 monomers within oligomeric transporter complexes can operate independently, and oligomerization can be changed by extracellular agents. Significance: The reported data are important to understand transport mechanism and effects of mutations. Polyspecific organic anion transporters (OATs) and organic cation transporters (OCTs) of the SLC22 transporter family play a pivotal role in absorption, distribution, and excretion of drugs. Polymorphisms in these transporters influence therapeutic effects. On the basis of functional characterizations, homology modeling, and mutagenesis, hypotheses for how OCTs bind and translocate structurally different cations were raised, assuming functionally competent monomers. However, homo-oligomerization has been described for OATs and OCTs. In the present study, evidence is provided that the large extracellular loops (EL) of rat Oct1 (rOct1) and rat Oat1 (rOat1) mediate homo- but not hetero-oligomerization. Replacement of the cysteine residues in the EL of rOct1 by serine residues (rOct1(6ΔC-l)) or breaking disulfide bonds with dithiothreitol prevented oligomerization. rOct1 chimera containing the EL of rOat1 (rOct1(rOat1-l)) showed oligomerization but reduced transporter amount in the plasma membrane. For rOct1(6ΔC-l) and rOct1(rOat1-l), similar Km values for 1-methyl-4-phenylpyridinium+ (MPP+) and tetraethylammonium+ (TEA+) were obtained that were higher compared with rOct1 wild type. The increased Km of rOct1(rOat1-l) indicates an allosteric effect of EL on the cation binding region. The similar substrate affinity of the oligomerizing and non-oligomerizing loop mutants suggests that oligomerization does not influence transport function. Independent transport function of rOct1 monomers was also demonstrated by showing that Km values for MPP+ and TEA+ were not changed after treatment with dithiothreitol and that a tandem protein with two rOct1 monomers showed about 50% activity with unchanged Km values for MPP+ and TEA+ when one monomer was blocked. The data help to understand how OCTs work and how mutations in patients may affect their functions.

Biogenesis of mitochondrial β-barrel proteins: the POTRA domain is involved in precursor release from the SAM complex

The sorting and assembly machinery (SAM) of mitochondria is essential for the sorting of β-barrel proteins. Different views have been presented on the role of polypeptide transport–associated (POTRA) domains in protein sorting. We show that the mitochondrial POTRA domain promotes the release of precursor proteins from the SAM complex.

A bacterial toxin catalyzing tyrosine glycosylation of Rho and deamidation of Gq and Gi proteins

Entomopathogenic Photorhabdus asymbiotica is an emerging pathogen in humans. Here, we identified a P. asymbiotica protein toxin (PaTox), which contains a glycosyltransferase and a deamidase domain. PaTox mono-O-glycosylates Y32 (or Y34) of eukaryotic Rho GTPases by using UDP–N-acetylglucosamine (UDP-GlcNAc). Tyrosine glycosylation inhibits Rho activation and prevents interaction with downstream effectors, resulting in actin disassembly, inhibition of phagocytosis and toxicity toward insects and mammalian cells. The crystal structure of the PaTox glycosyltransferase domain in complex with UDP-GlcNAc determined at 1.8-Å resolution represents a canonical GT-A fold and is the smallest glycosyltransferase toxin known. 1H-NMR analysis identifies PaTox as a retaining glycosyltransferase. The glutamine-deamidase domain of PaTox blocks GTP hydrolysis of heterotrimeric Gαq/11 and Gαi proteins, thereby activating RhoA. Thus, PaTox hijacks host GTPase signaling in a bidirectional manner by deamidation-induced activation and glycosylation-induced inactivation of GTPases.

Accessory NUMM (NDUFS6) subunit harbors a Zn-binding site and is essential for biogenesis of mitochondrial complex I

Significance Respiratory complex I is the largest membrane protein complex in mitochondria and has a central function in energy metabolism. Numerous human diseases are linked with complex I dysfunction or assembly defects. The concerted assembly of more than 40 subunits and the insertion of cofactors is aided by specific chaperones. In addition to eight FeS clusters, complex I comprises a Zn-binding site of unknown function. Combining X-ray structural analysis of complex I crystals with quantum chemical modeling and proteomic and spectroscopic analysis of a purified assembly intermediate, we show that accessory subunit NUMM (human ortholog NDUFS6) binds Zn at the interface of two functional modules of the enzyme complex and is required for a specific step of complex I biogenesis. Mitochondrial proton-pumping NADH:ubiquinone oxidoreductase (respiratory complex I) comprises more than 40 polypeptides and contains eight canonical FeS clusters. The integration of subunits and insertion of cofactors into the nascent complex is a complicated multistep process that is aided by assembly factors. We show that the accessory NUMM subunit of complex I (human NDUFS6) harbors a Zn-binding site and resolve its position by X-ray crystallography. Chromosomal deletion of the NUMM gene or mutation of Zn-binding residues blocked a late step of complex I assembly. An accumulating assembly intermediate lacked accessory subunit N7BM (NDUFA12), whereas a paralog of this subunit, the assembly factor N7BML (NDUFAF2), was found firmly bound instead. EPR spectroscopic analysis and metal content determination after chromatographic purification of the assembly intermediate showed that NUMM is required for insertion or stabilization of FeS cluster N4.

Membrane protein insertion through a mitochondrial β-barrel gate

Making your way through the side of a barrel The mechanism of membrane insertion and assembly of b-barrel proteins is a central question of outer membrane biogenesis of mitochondria, chloroplasts, and Gram-negative bacteria. Höhr et al. developed assays to address this fundamental problem. They systematically mapped precursor proteins transported by the mitochondrial Omp85 channel (Sam50) to elucidate the entire membrane insertion pathway of a precursor in the native membrane environment. Their findings directly demonstrate translocation of precursor proteins through the lumen of the mitochondrial Omp85 channel, signal recognition by β-strand exchange between channel and precursor, and exit through the lateral gate into the membrane. Science, this issue p. eaah6834 The mechanism by which mitochondrial outer membrane proteins are inserted is elucidated. INTRODUCTION The outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts characteristically contain β-barrel membrane proteins. These proteins contain multiple amphipathic β strands that form a closed barrel. This arrangement exposes hydrophobic amino acid residues to the lipid phase of the membrane, with polar residues facing the lumen of the barrel. β-barrel proteins form outer membrane channels for protein import and export, and for metabolite and nutrient exchange. An essential step in the biogenesis of β-barrel proteins is their insertion into the outer membrane. The β-barrel assembly machinery (BAM) of bacteria and the sorting and assembly machinery (SAM) of mitochondria are crucial for the membrane insertion of β-barrel precursors. The core subunits of these machineries, BamA and Sam50, are homologous 16-stranded β-barrel proteins that belong to the outer membrane protein family 85 (Omp85). The β signal located in the last β strand of the precursor initiates protein insertion into the outer membrane; however, the molecular mechanism of β-barrel insertion has not been understood. Controversial models about the role of BAM and SAM have been discussed. These models either favor precursor translocation into the BamA or Sam50 barrel followed by lateral release through an opened β-barrel gate or suggest membrane thinning and precursor insertion at the BamA or Sam50 protein-lipid interface. RATIONALE Structural studies have suggested that BamA and Sam50 harbor a dynamic lateral gate formed between β strands 1 and 16. In addition, BamA and Sam50 have been proposed to induce a thinning of the lipid bilayer near the lateral gate. To determine the translocation pathway during β-barrel membrane insertion, we probed the proximity of β-barrel precursors (Tom40, Por1, VDAC1) to Sam50 in intact mitochondria of the model organism baker’s yeast, Saccharomyces cerevisiae. We engineered precursors and Sam50 variants with cysteine residues at defined positions and mapped the environment of precursors in transit by disulfide-bond scanning and cysteine-specific cross-linking. RESULTS Our findings indicated that during transport of β-barrel precursors by the SAM complex, the lateral gate of Sam50 between β strands 1 and 16 was open and contained accumulated precursor. The β signal of the precursor specifically interacted with β strand 1 of Sam50 and thus replaced the endogenous β signal (β strand 16) of Sam50. Precursor transfer to the lateral gate occurred via the channel lumen of Sam50 and required the conserved loop 6 located in the channel. β hairpin–like elements consisting of two antiparallel β strands of the precursor were translocated and inserted into the lateral gate. The precursor remained associated with the Sam50 gate until the folded full-length β-barrel protein was released into the outer membrane. CONCLUSION Our findings indicate that β-barrel precursors are inserted into the lumen of the Sam50 channel and are released into the mitochondrial outer membrane via the opened lateral gate of Sam50. The carboxy-terminal β signal of the precursor initiates opening of the gate by exchange with the endogenous Sam50 β signal. An increasing number of β hairpin–like loops of the precursor accumulate at the lateral gate. Upon folding at Sam50, the full-length β-barrel protein is laterally released into the outer membrane. Membrane thinning in the vicinity of the lateral gate likely facilitates insertion of the protein into the lipid bilayer. Thus, the membrane-insertion pathway of β-barrel proteins combines elements of both controversially discussed models: transport through the lumen of Sam50 and the lateral gate and subsequent insertion into the thinned membrane next to the gate. Owing to the conservation of both the β signal and Omp85 core machinery, we speculate that β-signal exchange, folding at the gate, and lateral release into the membrane represent a general mechanism for β-barrel protein biogenesis in mitochondria, chloroplasts, and Gram-negative bacteria. β-Barrel protein insertion via the lateral gate of Sam50. β-Barrel precursors are transferred through the Sam50 interior to the lateral gate, which is formed by β strands 1 and 16. Upon gate opening, the β signal of the precursor substitutes for the endogenous Sam50 β signal. A conserved loop of Sam50 promotes β-signal binding to the gate and insertion of subsequent β hairpins. The folded β-barrel protein is released into the outer membrane. Po, polar amino acid residue; G, glycine; Hy, hydrophobic amino acid residue; C, C terminus; IRGF, binding motif. The biogenesis of mitochondria, chloroplasts, and Gram-negative bacteria requires the insertion of β-barrel proteins into the outer membranes. Homologous Omp85 proteins are essential for membrane insertion of β-barrel precursors. It is unknown if precursors are threaded through the Omp85-channel interior and exit laterally or if they are translocated into the membrane at the Omp85-lipid interface. We have mapped the interaction of a precursor in transit with the mitochondrial Omp85-channel Sam50 in the native membrane environment. The precursor is translocated into the channel interior, interacts with an internal loop, and inserts into the lateral gate by β-signal exchange. Transport through the Omp85-channel interior followed by release through the lateral gate into the lipid phase may represent a basic mechanism for membrane insertion of β-barrel proteins.

Protein glutaminylation is a yeast-specific posttranslational modification of elongation factor 1A

Ribosomal translation factors are fundamental for protein synthesis and highly conserved in all kingdoms of life. The essential eukaryotic elongation factor 1A (eEF1A) delivers aminoacyl tRNAs to the A-site of the translating 80S ribosome. Several studies have revealed that eEF1A is posttranslationally modified. Using MS analysis, site-directed mutagenesis, and X-ray structural data analysis of Saccharomyces cerevisiae eEF1A, we identified a posttranslational modification in which the α amino group of mono-l-glutamine is covalently linked to the side chain of glutamate 45 in eEF1A. The MS analysis suggested that all eEF1A molecules are modified by this glutaminylation and that this posttranslational modification occurs at all stages of yeast growth. The mutational studies revealed that this glutaminylation is not essential for the normal functions of eEF1A in S. cerevisiae. However, eEF1A glutaminylation slightly reduced growth under antibiotic-induced translational stress conditions. Moreover, we identified the same posttranslational modification in eEF1A from Schizosaccharomyces pombe but not in various other eukaryotic organisms tested despite strict conservation of the Glu45 residue among these organisms. We therefore conclude that eEF1A glutaminylation is a yeast-specific posttranslational modification that appears to influence protein translation.