Towards the structure of the proton channel and the dimerization interface of the yeast mitochondrial F1Fo-ATP synthase. – F1Fo-Struct

This project is divided in two main modules:
- The first one will aim to get structural data on the proton translocating part of the ATP synthase complex. To this end, two approaches are used either by crystallizing the whole enzyme in lipidic sponge phase or in bicelles or by using classical 3D and/or 2D crystallization methods to get crystals of an isolated subcomplex containing the proton channel of the enzyme (subunits i + 6 + 9(10)).
- The second part of the project will focus on the small hydrophobic subunits involved in the ATP synthase dimerization. A production of these proteins, in the presence or not of isotopically labelled amino acids will be performed using different modes of the cell-free expression system. After having determined which mode of production and which detergents or lipids would be the most appropriate for each protein, NMR spectra will be acquired to get data on their structures and the dynamics of their interactions. In combination to this structural and dynamical NMR studies, we will locate each subunit inside the dimeric ATP synthase species. This will be achieved by generating first a precise volume of the dimeric complex by cryo-EM and then by replacing these small subunits in this volume using a specific labeling with nanogold particles. The NMR models of subunits involved in the ATP synthase dimerization and the X-ray models of the whole enzyme and/or the proton channel will then be fitted into the volume of dimers to get a precise model of the dimeric ATP synthase.

Some of the membrane subunits of the yeast ATP-synthase have been produced using the cell-free in vitro expression system. The 1H-spectra obtained with subunit i, e, or with the membrane region of subunit 4 (S4T), produced in the precipitate mode and solubilised in the presence of LMPG (1-myristoyl-2-hydroxy-sn-glycero-3-. [phospho-rac-(1-glycerol)]), have indicated that their structural study by NMR was feasible. During the first 6 months of this project, we have focused on the structure of the membrane part of subunit 4 (S4T). S4T has been uniformly labeled with 15N and 13C. Several multidimensional NMR experiments have been carried out to assign the resonances of the backbone atoms of the protein. The secondary structure of S4T has been deduced from the chemical shifts of the main chain carbon atoms and Cß atoms of each residue. From these values chemical shift indices were derived. The chemical shift index profile, versus the primary structure indicates that three helices are present in S4T, in agreement with secondary structure predictions. The chemical shift indices also depend on the tertiary structure of the protein, and have been used as constraints to establish a first 3D model of S4T. In this model, helices H2 and H3 form a helical hairpin. This model is consistent with the topological, accessibility and crosslinks results that we had on this part of the protein.
New algorithms have been used to increase the quality of the ATP-synthase dimer volume obtained using cryo-electron microscopy. The actual resolution is around 27 Å.

The study of the structure of the membrane part of subunit 4 and the 1H-spectra obtained with the other membrane subunits involved in the ATP-synthase dimerization interface have validated the approach that has been chosen for this project.
The algorithms that have been used to improve the volume of an ATP-synthase dimer have led to a better volume. Although this volume can still be improved, its quality is already sufficient to position the structures of the whole monomeric complex, that should be obtained using the crystallization method in sponge phase. Alternatively, different sub-complexes containing the proton translocating part will be placed into this volume. Finally, the structures obtained by NMR on isolated subunits involved in ATP-synthase dimerization will be located into this volume, with the help of nanogold particle labeling.
Altogether, the data will bring new information on the yeast ATP-synthase complex and help to better understand the dimerization process, that is essential to mitochondral morphology.

Talks

Marie-France Giraud, invited speaker
DYNAMO, Paris 9-12 April 2014. The yeast ATP-synthase: from its atomic structure to its supramolecular organization in the inner mitochondrial membrane. Marie-France Giraud, Daniel Thomas, Reynald Gillet, Evelina Edelweiss, Valentin Gordeliy, James Tolchard, Benoît Odaert, Thomas Meier, Isabelle Larrieu, Patrick Paumard, Corinne Sanchez, Alain Dautant, Daniel Brèthes.

Marie-France Giraud, invited speaker
Biomolecular Science Research Centre, University of Saint-Andrews, Scotland. The yeast ATP-synthase: from its atomic structure to its supramolecular organization.

Posters:
INSERM workshop, April 2013. Various strategies to get structural data on the yeast ATP-synthase.
Marie-France Girauda, Daniel Thomas, Reynald Gillet, Valentin Gordeliy, Benoît Odaert, Thomas Meier, Isabelle Larrieu, Alain Dautant, Patrick Paumard, Corinne Sanchez, Daniel Brèthes.

NMR structural study of the membrane domain of the yeast ATP6synthase subunit 4.
Alexandre Barrasa, Isabelle Larrieu, Corinne Sanchez, Alain Dautant, Daniel Brèthes, Benoît Odaert, Marie-France Girauda.

EUROMAR, Zürich, 29th-3rdJuly 2014, Initial steps in describing the F1Fo ATP synthase dimer interface and modelling the small hydrophobic subunits of the Fo region with solution state NMR. James Tolchard, Daniel Brethes, Erick Dufourc, Marie-France Giraud, Benoit Odaert.

14th Naples Workshop on Bioactive Peptides
NMR structural investigation of the membrane proteins involved in the dimerization of the mitochondrial ATP-synthase. J. Tolchard, A. Barras, D. Brèthes, MF. Giraud and B. Odaert

ATP is the universal fuel molecule of any cell. The F1Fo-ATP synthase is a key enzyme of the energetic metabolism since it is responsible of most of the cellular synthesis of ATP. This 600 kDa complex is composed in yeast by 17 distinct subunits. It uses the energy of an electrochemical proton gradient to synthesize ATP. When protons are conducted across its membrane region, this conduction drives the rotation of a rotor part. This rotor part protrudes in the hydrophilic catalytic sector and induces conformational changes that lead to ATP synthesis.
The yeast enzyme, like mammalian ATP synthases, is not only involved in ATP synthesis but also in the organization of the inner mitochondrial membrane: ATP synthase dimers constitute the building blocks of large oligomers that are involved in mitochondrial cristae morphology. Three ATP synthase subunits are essential for ATP synthase dimerization: su g, su e and the N-terminal extremity of subunit 4 including its two transmembrane segments (Nter 4).
Despite the tremendous structural work performed on this complex, only 3D structures of sub-complexes have been solved and structural data are still missing on the channel region, made by subunit 6 and a ring of 10 subunits 9 (9(10) ring). Hence proton conduction is still a mystery. Besides, no information has been obtained on the structure and the organization of the small hydrophobic subunits e, g and the N-terminal part of subunit 4 that create the interfaces for ATP synthase oligomerization.
This project will be divided in two main modules:
- The first one will aim to get structural data on the channel part of the ATP synthase complex. To this end, two approaches will be used either by crystallizing the whole enzyme in lipidic sponge phase or in bicelles or by using classical 3D and/or 2D crystallization methods to get crystals of an isolated sub-complex containing the proton channel of the enzyme (subunits i + 6 + 9(10)).
- The second part of the project will focus on the small hydrophobic subunits involved in the ATP synthase dimerization. A production of these proteins, in the presence or not of isotopically labelled amino acids will be performed using different modes of the cell-free expression system. After having determined which mode of production and which detergents or lipids would be the most appropriate for each protein, NMR spectra will be acquired to get data on their structures and the dynamics of their interactions. In combination to this structural and dynamical NMR studies, we will locate each subunit inside the dimeric ATP synthase species. This will be achieved by generating first a precise 3D volume of the dimeric complex by cryo-EM and then by replacing these small subunits in the envelope using a specific labelling with nanogold particles.
The NMR models of subunits involved in the ATP synthase dimerization and the X-ray models of the whole enzyme and/or the proton channel will then be fitted into the 3D volume of dimers to get a precise model of the dimeric ATP synthase.
This project will combine a lot of varied and complementary techniques (2D, 3D crystallization, X-ray crystallography, NMR, Cryo-EM, membrane protein expression) that are mandatory if we now want to decipher the intricate mechanism of proton translocation and thus the energetic coupling of this fantastic nanomotor that is the ATP synthase. Getting structural data on the small subunits involved in the dimerization of this complex will help understanding how the basic building blocks essential to the mitochondrial morphology are stabilized.