Cryo-electron microscopy studies on ovine mitochondrial complex I

The main objective of this work is to determine the atomic structure of mammalian respiratory complex I. Mitochondrial complex I (also known as NADH:ubiquinone oxidoreductase) is one of the central enzymes in the oxidative phosphorylation pathway. It couples electron transfer between NADH and ubiquinone to proton translocation across the inner mitochondrial membrane, contributing to cellular energy production. Complex I is the largest and most elaborate protein assembly of the respiratory chain with a total mass of 970 kilodaltons. It consists of 14 conserved ‘core subunits’ and 31 mitochondria-specific ‘supernumerary subunits’. Together they form a giant, Lshaped molecule, with one arm buried in the mitochondrial membrane and another protruding into the mitochondrial matrix. Here, a novel method for the purification of ovine (Ovis aries) complex I was developed and suitable conditions for cryo-EM imaging established, after extensive screening of detergents and additives. Cryo-EM images were acquired with the recently developed direct electron detector and processed using the latest software. This allowed the solution of the nearly complete atomic model of mitochondrial complex I at 3.9 Å resolution. The membrane part of the complex contains 78 transmembrane helices, mostly contributed by conserved antiporter-like subunits responsible for proton translocation. These helices are stabilized by tightly bound lipids (including cardiolipins). The hydrophilic arm harbours flavin mononucleotide and 8 iron–sulfur clusters involved in electron transfer. Supernumerary subunits build a scaffold around the conserved core, strongly stabilizing the complex. Additionally, subunits containing cofactors (NADPH, zinc ion and phosphopantetheine) may play a regulatory role. Two distinct conformations of the complex are observed, which may describe the active and deactive states or reflect conformations occurring during the catalytic cycle of the enzyme. Currently this is the most detailed model of this molecular machine, providing insight into the mechanism, assembly and dysfunction of mitochondrial complex I. It also allows molecular analysis of numerous disease-causing mutations, and so the structure may serve as a stepping-stone for future medical developments

Mammalian mitochondrial complex I structure and disease causing mutations

Complex I has an essential role in ATP production by coupling electron transfer from NADH to quinone with translocation of protons across the inner mitochondrial membrane. Isolated complex I deficiency is a frequent cause of mitochondrial inherited diseases. Complex I has also been implicated in cancer, ageing, and neurodegenerative conditions. Until recently, the understanding of complex I deficiency on the molecular level was limited due to the lack of high-resolution structures of the enzyme. However, due to developments in single particle cryo-electron microscopy (cryo-EM), recent studies have reported nearly atomic resolution maps and models of mitochondrial complex I. These structures significantly add to our understanding of complex I mechanism and assembly. The disease-causing mutations are discussed here in their structural context

Structures of respiratory supercomplex I+III2 reveal functional and conformational crosstalk

The mitochondrial electron transport chain complexes are organized into supercomplexes (SCs) of defined stoichiometry, which have been proposed to regulate electron flux via substrate channeling. We demonstrate that CoQ trapping in the isolated SC I+III2 limits complex (C)I turnover, arguing against channeling. The SC structure, resolved at up to 3.8 Å in four distinct states, suggests that CoQ oxidation may be rate limiting because of unequal access of CoQ to the active sites of CIII2. CI shows a transition between “closed” and “open” conformations, accompanied by the striking rotation of a key transmembrane helix. Furthermore, the state of CI affects the conformational flexibility within CIII2, demonstrating crosstalk between the enzymes. CoQ was identified at only three of the four binding sites in CIII2, suggesting that interaction with CI disrupts CIII2 symmetry in a functionally relevant manner. Together, these observations indicate a more nuanced functional role for the SCs

Purification of ovine respiratory complex i results in a highly active and stable preparation

NADH-ubiquinone oxidoreductase (complex I) is the largest (∼1 MDa) and the least characterized complex of the mitochondrial electron transport chain. Because of the ease of sample availability, previous work has focused almost exclusively on bovine complex I. However, only medium resolution structural analyses of this complex have been reported. Working with other mammalian complex I homologues is a potential approach for overcoming these limitations. Due to the inherent difficulty of expressing large membrane protein complexes, screening of complex I homologues is limited to large mammals reared for human consumption. The high sequence identity among these available sources may preclude the benefits of screening. Here, we report the characterization of complex I purified from Ovis aries (ovine) heart mitochondria. All 44 unique subunits of the intact complex were identified by mass spectrometry. We identified differences in the subunit composition of subcomplexes of ovine complex I as compared with bovine, suggesting differential stability of inter-subunit interactions within the complex. Furthermore, the 42-kDa subunit, which is easily lost from the bovine enzyme, remains tightly bound to ovine complex I. Additionally, we developed a novel purification protocol for highly active and stable mitochondrial complex I using the branched-chain detergent lauryl maltose neopentyl glycol. Our data demonstrate that, although closely related, significant differences exist between the biochemical properties of complex I prepared from ovine and bovine mitochondria and that ovine complex I represents a suitable alternative target for further structural studies

Tubulin acetyltransferase alpha TAT1 destabilizes microtubules Independently of its acetylation activity

Acetylation of\u3b1-tubulin at lysine 40 (K40) is a well-conserved posttranslational modification that marks long-lived microtubules but has poorly understood functional significance. Recently,\u3b1TAT1, a member of the Gcn5-relatedN-acetyltransferase superfamily, has been identified as anF-tubulin acetyltransferase in ciliated organisms. Here, we explored the function of\u3b1TAT1 with the aim of understanding the consequences of\u3b1TAT1-mediated microtubule acetylation.Wedemonstrate that\u3b1-tubulin is the major target of \u3b1TAT1 but that\u3b1TAT1 also acetylates itself in a regulatory mechanism that is required for effective modification of tubulin.Wefurther show that in mammalian cells,\u3b1TAT1 promotes microtubule destabilization and accelerates microtubule dynamics. Intriguingly, this effect persists in anFTAT1 mutant with no acetyltransferase activity, suggesting that interaction of\u3b1TAT1 with microtubules, rather than acetylation per se, is the critical factor regulating microtubule stability. Our data demonstrate that\u3b1TAT1 has cellular functions that extend beyond its classical enzymatic activity as an \u3b1-tubulin acetyltransferase \ua92013,American Society for Microbiology

Atomic structure of the entire mammalian mitochondrial complex i

Mitochondrial complex I (also known as NADH:ubiquinone oxidoreductase) contributes to cellular energy production by transferring electrons from NADH to ubiquinone coupled to proton translocation across the membrane. It is the largest protein assembly of the respiratory chain with a total mass of 970 kilodaltons. Here we present a nearly complete atomic structure of ovine (Ovis aries) mitochondrial complex I at 3.9 Å resolution, solved by cryo-electron microscopy with cross-linking and mass-spectrometry mapping experiments. All 14 conserved core subunits and 31 mitochondria-specific supernumerary subunits are resolved within the L-shaped molecule. The hydrophilic matrix arm comprises flavin mononucleotide and 8 iron-sulfur clusters involved in electron transfer, and the membrane arm contains 78 transmembrane helices, mostly contributed by antiporter-like subunits involved in proton translocation. Supernumerary subunits form an interlinked, stabilizing shell around the conserved core. Tightly bound lipids (including cardiolipins) further stabilize interactions between the hydrophobic subunits. Subunits with possible regulatory roles contain additional cofactors, NADPH and two phosphopantetheine molecules, which are shown to be involved in inter-subunit interactions. We observe two different conformations of the complex, which may be related to the conformationally driven coupling mechanism and to the active-deactive transition of the enzyme. Our structure provides insight into the mechanism, assembly, maturation and dysfunction of mitochondrial complex I, and allows detailed molecular analysis of disease-causing mutations