Mitochondria are cellular organelles responsible for the production of ATP, the chemical fuel of the cell. Several metabolic cycles occur within this compartment. The function of this organelle necessitates the exchange of several types of metabolites and small molecules between the mitochondrial matrix and the cytoplasm. The project aims at understanding the transport mechanisms at a molecular level, more specifically the role of essential membrane proteins, the mitochondrial carriers.
The first structure of AAC was solved in 2003 by one of the partners of the project. It is still the only atomic resolution structure of such a carrier. It shed the first light on mitochondrial carriers by showing the overall fold and the mechanism of inhibition by atractylosides, well-known poisons. First hypotheses on substrate binding can also be drawn from this structure. Additional and new information came from molecular dynamic simulations based on the structure (collaboration between two partners of this project). A first study has shown the influence of external parameters on substrate binding, illustrating the power of an approach combining theory and experiments. UCP1 and UCP2, also member of the same carrier family, are studied by one of the partner since several years. To understand completely the transport mechanism, the structures of several other conformation adopted by the carriers in action will be needed. Functional characterizations of mutants of the effect of ligands during the transport will help in getting the structure of new conformations, and will also complement the results coming from molecular dynamics simulations.
Structural studies of membrane proteins still remain challenging. In order to succeed, we combine experiments and theory, by coupling functional studies, structural information with molecular dynamics simulations. The firs step consists in producing the proteins in sufficient quantities, and to identify conditions for their stabilization in a native conformation, in order to start their crystallization for structural studies. Two expression systems will be used, E.coli (two of the partners have already preliminary results), and S. cerevisiae (one of the partner has adapted a special vector already successful for the expression of the Serca1a Ca2+ ATPase, another eucaryotic membrane protein). The design of new ligands and mutants optimizing the production of stable proteins will be facilitated by theoretical approaches. This first step will lead to the central part of the project, which aims at linking structures, functional characterizations and molecular dynamics simulations in order to understand the specificity and the mechanisms by which the transport occurs. This part will be based on the existing structure as an initial model, and on the design of transport measurement experiments. Several mutants will be studied, as well as the influence of various ligands. The last step will open the way to the crystallization of the new conformations by classical approaches but also using lipidic phases in order to obtain additional structural information.
The methods implemented to study the transport characteristics of AAC1 combined with theoretical methods following the nucleotide entrance into the cavity of AAC1 and its binding, led in understanding the link between transport at the molecular level, dynamical properties of the carrier, and strength of human diseases linked to six pathological mutations. These results are in press in ACS Chemical Biology (Ravaud et al. 2012) and will illustrate the cover page of the journal.
The expression protocols for all the systems described in the project are now in place. The purification protocols are also established and some of them still need refinements. Two protocols for measurement the transport activity were developed, one is based on the electric current measurement induced by the transport. The first simulations on AAC in the presence of various nucleotides, i.e. GMP, GDP and GTP have been achieved. The first analyses are not sufficient to fully understand the transport specificity. The simulations were coupled to transport measurement of radioactive labeled ATP in E.coli expressing AAC at its membrane. These measurements showed that GDP and GTP are not transported while AMP is partially transported (manuscript submitted).
The large-scale production has started for AAC1 expressed in E.coli and S. cerevisiae. The scale-up for other protocols and proteins will follow soon. New fermentors were installed and will allow to test the expression of various mutants. First crystallization attempts of AAC1 expressed in E.coli and in S. cerevisiae have started and will be intensively pursued.
1. « Mutations of the mitochondrial ADP/ATP carrier causing genetic diseases impair the transport of nucleotides » S. Ravaud, A. Bidon-Chanal, I. Blesneac, P. Machillot, C. Juillan-Binard, F. Dehez, C. Chipot, E. Pebay-Peyroula (2012) ACS Chem Bio, in press
2. « Production of UCP1 a membrane protein from the inner mitochondrial membrane using the cell free expression system in the presence of a fluorinated surfactant » I. Blesneac, S. Ravaud, C. Juillan-Binard, L.-A. Barret, M. Zoonens, A. Polidori, B. Miroux, B. Pucci, E. Pebay-Peyroula (2012) BBA Biomembranes 1818, 798-805.
Our project aims at understanding the mechanisms of action at a molecular level of essential membrane proteins, i.e. mitochondrial carriers. These carriers play a crucial role in importing and exporting in a very specific manner, the substrates or products of all metabolic reactions that occur within mitochondria. The project will focus on three human mitochondrial carriers, the ADP/ATP carrier (AAC), and the uncoupling proteins (UCP1, UCP2). The proteins we are considering, are integral membrane proteins for which structural studies still are a challenge. In order to optimize the success rate, the strategy we propose, combines experiments and theory. For this, experimental functional and structural information coupled to molecular dynamics simulations are planned. To achieve this goal we need to overcome several major bottlenecks of membrane protein chemistry, i.e. large-scale production, stabilization of the protein in its native state and crystallization.
AAC, which imports ADP from the cytosol and exports ATP after being synthezised from ADP and organic phosphate in the mitochondrial matrix is the best known member of the mitochondrial carrier family (MCF). Although MCF carriers are essential in all metabolic pathways occurring in mitochondria, only few teams in the world are devoted to their study at a molecular level. The low level of implication mainly reflects the difficulty of such studies rather than a lack of interest. The first structure of a MCF carrier was solved in 2003 by partner 1. AAC still remains the only high resolution structure available so far. The AAC structure sheds the first light onto the MCF family. It shows the overall fold and the mechanism by which atractylosides severely inhibit the transport and therefore act as strong poisons and opens several hypotheses on substrate binding. New insights arose from molecular dynamics simulations based on the structure (partner 2 in collaboration with partner 1 and others). In particular, insights to the parameters that affect ADP binding were given by P2 and P1 emphasizing on the high potential in combining structural, functional and molecular dynamics simulations for deciphering substrate binding and specificity and transport mechanisms.
The uncoupling protein (UCP) also a member of the MCF is studied since several years by Partner 3. Its sequence comprises the triplicated MCF motif. Therefore, UCP most probably share the same overall fold as AAC. However, the transported substrates are very different: adenine nucleotides for AAC and protons for UCP1. The clue to the question of deciphering substrate specificity and transport mechanisms might be solved by a comparative structure/function analysis at the atomic level, by providing similarities and differences between both carriers.
The project is divided in 3 tasks and several sub-tasks. Most of the sub-tasks implicate more than 2 partners. Task 1 will focus on the production of carriers in membranes by 2 expression systems: E. coli (Partner 1 and 3 already have preliminary results with fusion proteins), and S. cerevisiae (Partner 4 has adapted a special vector and already produced Serca1a Ca2+-ATPase, another eukaryotic membrane protein, with this system). Design of new ligands or mutants that will facilitate the production of stable protein will be facilitated by theoretical approaches. Task1 will provide the samples for task 2 and 3. Task2 is the central core as it addresses the functional aspects linking together results from structures, functional assays and MD simulations in order to understand the specificity of the transport and the transport mechanism. Task3 aims at crystallizing the carriers using classical and lipidic phase approaches in order to get additional structural information that will be valuable for task 2.
Madame Eva PEBAY-PEYROULA (UNIVERSITE GRENOBLE I [Joseph Fourier]) – firstname.lastname@example.org
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
SB2SM CNRS - DELEGATION REGIONALE ILE-DE-FRANCE SECTEUR SUD
IBS UNIVERSITE GRENOBLE I [Joseph Fourier]
SRSMC CNRS - DELEGATION REGIONALE CENTRE-EST
LBPCPM CNRS - DELEGATION REGIONALE ILE-DE-FRANCE SECTEUR PARIS B
Help of the ANR 719,986 euros
Beginning and duration of the scientific project: - 48 Months