Structural insights into crista junction formation by the Mic60-Mic19 complex

Mitochondrial cristae membranes are the oxidative phosphorylation sites in cells. Crista junctions (CJs) form the highly curved neck regions of cristae and are thought to function as selective entry gates into the cristae space. Little is known about how CJs are generated and maintained. We show that the central coiled-coil (CC) domain of the mitochondrial contact site and cristae organizing system subunit Mic60 forms an elongated, bow tie–shaped tetrameric assembly. Mic19 promotes Mic60 tetramerization via a conserved interface between the Mic60 mitofilin and Mic19 CHCH (CC-helix-CC-helix) domains. Dimerization of mitofilin domains exposes a crescent-shaped membrane-binding site with convex curvature tailored to interact with the curved CJ neck. Our study suggests that the Mic60-Mic19 subcomplex traverses CJs as a molecular strut, thereby controlling CJ architecture and function

Funktionelle Charakterisierung des MICOS Komplexes Mic60-Mic19 in der Formung von mitochondrialer crista junction

The investigation of mitochondrial structure and function has developed into an active area of research. A lot of progress was made on understanding the role of mitochondria in energy production and apoptosis, but there is still a profound lack of knowledge how mitochondria obtain their specific shape required for proper function. In particular, the formation of invaginations of the inner mitochondrial membrane termed cristae involves a plethora of different proteins, but it has remained unclear how they contribute to the remodeling of cristae membranes. The newly identified mitochondrial contact site and cristae organizing system (MICOS) is crucial for the formation of crista junctions and mitochondrial inner membrane architecture. MICOS contains two core components. Mic10 shows membrane-bending activity, whereas Mic60 (mitofilin) forms contact sites between inner and outer membranes. In my PhD thesis, I show that Mic60 from the thermophile fungus Chaetomium thermophilum dimerizes via its coiled-coil region and deforms liposomes into thin membrane tubules, therefore displaying membrane-shaping activity. Furthermore, a membrane-binding site in the soluble intermembrane space-exposed part of Mic60 was identified. This membrane-binding site is formed by a predicted amphipathic helix between the conserved coiled-coil and mitofilin domains. The mitofilin domain negatively regulates the membrane-shaping activity of Mic60. It is also shown that the mitofilin domain of Mic60 strongly binds to the CHCH domain of Mic19. ITC experiments indicated that this high affinity interaction requires two conserved cysteines within the CHCH domain that form a predicted disulfide bridge. Binding of Mic19 to the mitofilin domain modulates the membrane remodeling activity. The Mic60-Mic19 subcomplex forms tetramers, which involves both the CHCH-mitofilin domain interactions, but also the coiled-coil domains of both proteins. Membrane binding and shaping by the conserved Mic60-Mic19 complex is crucial for crista junction formation, mitochondrial membrane architecture and efficient respiratory activity. Mic60 thus plays a dual role by shaping inner membrane crista junctions and forming contact sites with the outer membrane.Die Existenz von Mitochondrien wurde bereits vor mehr als 100 Jahren bestätigt. Diesem Organell wird eine Vielzahl von wichtigen Aufgaben zugeschrieben, unter Anderem die Regulierung des programmierten Zelltodes (Apoptose), die Energieproduktion, die Verteilung von Phospholipiden und vielen mehr. Die Untersuchung der mitochondrialen Struktur und Funktion hat sich zu einem aktiven Forschungsgebiet entwickelt. Auch wenn in den letzten Jahren große Fortschritte zum Verständnis der mitochondrialen Funktion wie Apoptose und Energieproduktion gemacht wurden, so ist bis heute unklar, wie Mitochondrien ihre spezifische Form zur Erhaltung ihrer Funktion bilden. Insbesondere die typischen Einstülpungen der inneren Membran (cristae) beinhaltet eine Vielzahl von unterschiedlichen Proteinen, jedoch wie diese zur Bildung von Cristae-Membranen beitragen, ist bisher unklar. Ein erst kürzlich entdeckter Proteinkomplex (MICOS) scheint eine entscheidende Komponente bei der Ausbildung von cristae und deren Übergang zur inneren Membran (cristae junctions) zu sein. MICOS enthält zwei Kernkomponenten. Während Mic10 Membran- Deformierungs-Aktivität zeigt, bildet Mic60 (mitofilin) die Kontaktstellen zwischen innerer und äußerer Membran. In dieser Arbeit wird zum ersten Mal gezeigt, dass Mic60, aus dem thermostabilen Pilz Chaetomium thermophilum, über die Coiled-coil Domäne dimerisiert und die ursprünglich runden Liposomen in lange dünne Schläuche verwandelt. Zusätzlich wird gezeigt, dass neben Mic10, auch Mic60 eine aktive Rolle im Umbau von Membranen spielt. Zugleich wurde eine Membranbindestelle zwischen der Coiled-coil und der Mitofilin-Domäne identifiziert, welche durch eine vorhergesagte amphipathische Helix gebildet wird. Die Mitofilin-Domäne scheint die Membran-Deformierungs-Aktivität negativ zu beeinflussen. Es konnte auch gezeigt werden, dass die Mitofilin-Domäne von Mic60 stark an die CHCH-Domäne von Mic19 bindet. Isothermale Titrationskalorimetrie-Experimente zeigen, dass diese hochaffine Wechselwirkung zwei konservierte Cysteine innerhalb der CHCH-Domäne erfordert, die eine vorhergesagte intramolekulare Disulfidbrücke bilden. Die Bindung von Mic19 an die Mitofilin-Domäne fördert die Membrane-Deformierungs-Aktivität. Weitere biochemische Analysen zeigen auch, dass der Mic60-Mic19-Subkomplex Tetramere bildet, die sowohl die CHCH-Mitofilin-Domänen als auch die Coiled- coil beider Proteine einschließt. Die Membranbindung aber auch deren Deformierung durch den konservierten Mic60-Mic19-Komplex ist für die Bildung der Cristae junctions und der mitochondrialen Membranarchitektur entscheidend, und reguliert auch die Aktivität der Atmungskette. Mic60 spielt also eine doppelte Rolle, indem es Cristae junctions aber auch Kontaktstellen zwischen der inneren und äußeren mitochondrialen Membran formt

Functional mapping of human dynamin-1-like GTPase domain based on x-ray structure analyses.

Human dynamin-1-like protein (DNM1L) is a GTP-driven molecular machine that segregates mitochondria and peroxisomes. To obtain insights into its catalytic mechanism, we determined crystal structures of a construct comprising the GTPase domain and the bundle signaling element (BSE) in the nucleotide-free and GTP-analogue-bound states. The GTPase domain of DNM1L is structurally related to that of dynamin and binds the nucleotide 5'-Guanylyl-imidodiphosphate (GMP-PNP) via five highly conserved motifs, whereas the BSE folds into a pocket at the opposite side. Based on these structures, the GTPase center was systematically mapped by alanine mutagenesis and kinetic measurements. Thus, residues essential for the GTPase reaction were characterized, among them Lys38, Ser39 and Ser40 in the phosphate binding loop, Thr59 from switch I, Asp146 and Gly149 from switch II, Lys216 and Asp218 in the G4 element, as well as Asn246 in the G5 element. Also, mutated Glu81 and Glu82 in the unique 16-residue insertion of DNM1L influence the activity significantly. Mutations of Gln34, Ser35, and Asp190 in the predicted assembly interface interfered with dimerization of the GTPase domain induced by a transition state analogue and led to a loss of the lipid-stimulated GTPase activity. Our data point to related catalytic mechanisms of DNM1L and dynamin involving dimerization of their GTPase domains

Inositol triphosphate-triggered calcium release blocks lipid exchange at endoplasmic reticulum-Golgi contact sites

Vesicular traffic and membrane contact sites between organelles enable the exchange of proteins, lipids, and metabolites. Recruitment of tethers to contact sites between the endoplasmic reticulum (ER) and the plasma membrane is often triggered by calcium. Here we reveal a function for calcium in the repression of cholesterol export at membrane contact sites between the ER and the Golgi complex. We show that calcium efflux from ER stores induced by inositol-triphosphate [IP3] accumulation upon loss of the inositol 5-phosphatase INPP5A or receptor signaling triggers depletion of cholesterol and associated Gb3 from the cell surface, resulting in a blockade of clathrin-independent endocytosis (CIE) of Shiga toxin. This phenotype is caused by the calcium-induced dissociation of oxysterol binding protein (OSBP) from the Golgi complex and from VAP-containing membrane contact sites. Our findings reveal a crucial function for INPP5A-mediated IP3 hydrolysis in the control of lipid exchange at membrane contact sites

Structural and functional analysis of the NLRP4 pyrin domain

NLRP4 is a member of the nucleotide-binding and leucine-rich repeat receptor (NLR) family of cytosolic receptors and a member of an inflammation signaling cascade. Here, we present the crystal structure of the NLRP4 pyrin domain (PYD) at 2.3 Å resolution. The NLRP4 PYD is a member of the death domain (DD) superfamily and adopts a DD fold consisting of six α-helices tightly packed around a hydrophobic core, with a highly charged surface that is typical of PYDs. Importantly, however, we identified several differences between the NLRP4 PYD crystal structure and other PYD structures that are significant enough to affect NLRP4 function and its interactions with binding partners. Notably, the length of helix α3 and the α2−α3 connecting loop in the NLRP4 PYD are unique among PYDs. The apoptosis-associated speck-like protein containing a CARD (ASC) is an adaptor protein whose interactions with a number of distinct PYDs are believed to be critical for activation of the inflammatory response. Here, we use co-immunoprecipitation, yeast two-hybrid, and nuclear magnetic resonance chemical shift perturbation analysis to demonstrate that, despite being important for activation of the inflammatory response and sharing several similarities with other known ASC-interacting PYDs (i.e., ASC2), NLRP4 does not interact with the adaptor protein ASC. Thus, we propose that the factors governing homotypic PYD interactions are more complex than the currently accepted model, which states that complementary charged surfaces are the main determinants of PYD–PYD interaction specificity

Superposition of the two DNM1L GG structures and dynamin-1 GG.

<p>(<b>A</b>) Overlay of the nucleotide-free DNM1L GG structure in white with the GMP-PNP-bound structure in green (shown without ligands). Side chains that were mutated in our study are shown as stick models with sequence number labels. (<b>B</b>) Overlay of dynamin-1 (PDB code 2X2F) in yellow with the structure of GMP-PNP-bound DNM1L in green. Mutated residues of DNM1L that are equivalent to those of dynamin (see Fig. 3A) are displayed as side chain stick models with dynamin sequence numbers (depicted without ligands).</p

GTPase domain interface model of the DNM1L GG fusion protein and nucleotide-dependent dimerization.

<p>(<b>A</b>) Two chains of DNM1L molecules were superimposed on the GTPase domain dimer of <i>At</i>Drp1A (PDB code 3T34) as molecules A (green) and B (orange). The interface connecting residues Gln34, Ser35, Asp190, and GTP are depicted as stick models. In addition, the movement of the BSE domains between the pre- and postfission states is represented by the extended <i>At</i>Drp1A dimer (white) and the compact DNM1L dimer. The tetramer model (bottom, left) is based on full-length dynamin-1, which may further oligomerize via the stalks and other GTPase domains (green, orange). (<b>B</b>) Close-up view of the interface at Asp190 from molecule B and Gln34, Ser35 and GTP from molecule A. The conformations of the nucleotide-free and GMP-PNP bound structures are displayed. (<b>C</b>) Dimerization ability of the DNM1L GG fusion protein in the presence of different nucleotides. The GG fusion protein (60 µM) was subjected to gel filtration after incubation with different guanine nucleotide analogs (2 mM). Protein standards at 29 and 75 kDa are indicated. The dimeric protein eluted at a retention volume of 9.5 ml and monomeric protein at 11 ml. (<b>D</b>) SDS PAGE analysis of the SEC runs. Lane 1 shows purified GG fusion protein (41 kDa) followed by a molecular weight protein ladder (from top to bottom: 55 kDa, 43 kDa, 34 kDa). Elution volumes are indicated above. (<b>E</b>) Analysis of the DNM1L GMP-PNP complex stability under SEC conditions as in Fig. 8C. SEC elution (red) and further analysis of the peaks by HPLC (blue), with the indicated controls. (<b>F</b>) SEC of GG fusion protein mutants Q34A, S35A and D190A under conditions as in Fig. 8C in the presence of GDP⋅AlF₄⁻. Retention volumes of molecular weight standards are shown above.</p

Structure-function map of the modelled DNM1L active site dimer.

<p>All active site and dimerization residues that have been mutated to alanine are represented as stick models, as well as the GTP. The turnover numbers of the respective mutants as determined by the GTPase assay for basal activity are shown, whereby the WT was defined as 100%. Molecule A of the dimer is depicted in green, while the second molecule B is shown in orange, with the corresponding D190A*.</p

Close-up views of the active site cleft in the nucleotide-free and bound structures of the DNM1L GG construct in stereo.

<p>(<b>A</b>) The nucleotide-free form with the most relevant residue side chains of the five GTP binding stretches and citrate (FLC, yellow) displayed as stick models. Electron density of a 2F_o-F_c map is shown in grey and contoured at 1σ. The red sphere designates the catalytic water (<b>C</b>). (<b>B</b>) GMP-PNP complex of the DNM1L GG construct. The nucleotide is shown as stick model, while the red spheres represent water molecules, such as the bridging water (<b>B</b>) and one, which binds to the α-phosphate. The electron density of a 2F_o-F_c map is shown in grey and contoured at 1σ, surrounding the nucleotide and relevant parts of the structure with significant conformational changes with respect to the nucleotide-free form.</p

Detailed views of the GTP-binding elements switch I, switch II, G4 and G5 of DNM1L.

<p>(<b>A</b>) The canonical Mg²⁺ site between O1β and the O2γ is not occupied in the GMP-PNP-DNM1L structure (green). Also, no significant positional shift of Thr59 from the switch I loop takes place between apo- (grey) and nucleotide form (green). The unnatural N3β atom may favour the γ-phosphate conformation rotated by about 60° with respect to the transition state of GTP, shifting it about 2.5 Å away from the catalytic water. Only the nucleotide-free form exhibits the catalytic water molecule (C, grey) bound at the Thr59 carbonyl O (3.15 Å) and connected to switch II via the NH of Gly149 (2.87 Å). The bridging H₂O (B, red) is only present in the nucleotide complex, bound to the Gly149 carbonyl O (3.21 Å). Upon GMP-PNP binding, the Arg53 side chain moves out of the active site, making room for an H₂O, which binds O2 of the α-phosphate. (<b>B</b>) The ribose of GMP-PNP forms bonds with the ether oxygen O4 to the Nζ of Lys216 (3.16 Å), and with the hydroxyl group of O2 to an H₂O (3.25 Å), which is bonded to the carbonyl O of Arg247 (2.45 Å) and the Ser40 Oγ (3.01 Å). Another bond is formed by the ribose O2 to Gln249 NH (2.79 Å). (<b>C</b>) The Lys216 side chain, depicted as thin stick model for clarity, covers the aromatic rings of the guanine part, while the Asp218 carboxylate binds the amino N2 (2.86 Å) and the N1 (3.12 Å). A further interaction from the Asn246 Oδ1 to the N7 (3.54 Å) might be mediated by an unresolved H₂O, which could be bound to the carbonyl O of Gly37, as seen in other dynamin-nucleotide complexes.</p