Characterization of Aquifex aeolicus F1FO ATP synthase and its heterologous production in Escherichia coli

  • This work presents a biochemical, functional and structural characterization of Aquifex aeolicus F1FO ATP synthase obtained using both a native form (AAF1FO) and a heterologous form (EAF1FO) of this enzyme. F1FO ATP synthases catalyze the synthesis of ATP from ADP and inorganic phosphate driven by ion motive forces across the membrane and therefore play a key cellular function. Because of their central role in supporting life, F1FO ATP synthases are ubiquitous and have been remarkably conserved throughout evolution. For their biological importance, F1FO ATP synthases have been extensively studied for many decades and many of them were characterized from both a functional and a structural standpoint. However, important properties of ATP synthases – specifically properties pertaining to their membrane embedded subunits – have yet to be determined and no structures are available to date for the intact enzyme complex. Therefore, F1FO ATP synthases are still a major focus of research worldwide. Our research group had previously reported an initial characterization of AAF1FO and had indicated that this enzyme presents unique features, i.e. a bent central stalk and a putatively heterodimeric peripheral stalk. Based on such a characterization, this enzyme revealed promising for structural and functional studies on ATP synthases and became the focus of this doctoral thesis. Two different lines of research were followed in this work. First, the characterization of AAF1FO was extended by bioinformatic, biochemical and enzymatic analyses. The work on AAF1FO led to the identification of a new detergent that maintains a higher homogeneity and integrity of the complex, namely the detergent trans-4-(trans-4’-propylcyclohexyl)cyclohexyl-α-D-maltoside (α-PCC). The characterization of AAF1FO in this new detergent showed that AAF1FO is a proton-dependent, not a sodium ion-dependent ATP synthase and that its ATP hydrolysis mechanism needs to be triggered and activated by high temperatures, possibly inducing a conformational switch in subunit γ. Moreover, this approach suggested that AAF1FO may present unusual features in its membrane subunits, i.e. short N-terminal segments in subunits a and c with implications for the membrane insertion mechanism of these subunits. Investigating on these unique features of A. aeolicus F1FO ATP synthase could not be done using A. aeolicus cells, because these require a harsh and dangerous environment for growth and they are inaccessible to genetic manipulations. Therefore, a second approach was pursued, in which an expression system was created to produce the enzyme in the heterologous host E. coli. This second approach was experimentally challenging, because A. aeolicus F1FO ATP synthase is a 500-kDa multimeric membrane enzyme with a complicated and still not entirely determined stoichiometry and because its encoding genes are scattered throughout A. aeolicus genome, rather than being organized in one single operon. However, an artificial operon suitable for expression was created in this work and led to the successful production of an active and fully assembled form of Aquifex aeolicus F1FO ATP synthase. Such artificial operon was created using a stepwise approach, in which we expressed and studied first individual subunits, then subcomplexes, and finally the entire F1FO ATP synthase complex. We confirmed experimentally that subunits b1 and b2 form a heterodimeric subcomplex in the E. coli membranes, which is a unique case among ATP synthases of non-photosynthetic organisms. Moreover, we determined that the b1b2 subcomplex is sufficient to recruit the soluble F1 subcomplex to the membranes, without requiring the presence of the other membrane subunits a and c. The latter subunits can be produced in our expression system only when the whole ATP synthase is expressed, but not in isolation nor in the context of smaller FO subcomplexes. These observations led us to propose a novel mechanism for the assembly of ATP synthases, in which first the F1 subcomplex attaches to the membrane via subunit b1b2, and then cring and subunits a assemble to complete the FO subcomplex. Furthermore, we could purify the heterologous ATP synthase (EAF1FO) to homogeneity by chromatography and electro-elution. Enzymatic assays showed that the purified form of EAF1FO is as active as AAF1FO. Peptide mass fingerprinting showed that EAF1FO is composed of the same subunits as AAF1FO and all soluble and membrane subunits could be identified. Finally, single-particle electron microscopy analysis revealed that the structure of EAF1FO is identical to that of AAF1FO. Therefore, the EAF1FO expression system serves as a reliable platform for investigating on properties of AAF1FO. Specifically, in this work, EAF1FO was used to study the membrane insertion mechanism of rotary subunit c. Subunits c possess different lengths and levels of hydrophobicity across species and by analyzing their N-terminal variability, four phylogenetic groups of subunits c were distinguished (groups 1 to 4). As a member of group 2, the subunit c from A. aeolicus F1FO ATP synthase is characterized by an N-terminal segment that functions as a signal peptide with SRP recognition features, a unique case for bacterial F1FO ATP synthases. By accurately designing mutants of EAF1FO, we determined that such a signal peptide is strictly necessary for membrane insertion of subunit c and we concluded that A. aeolicus subunit c inserts into E. coli membranes using a different pathway than E. coli subunit c. Such a property may be common to other ATP synthases from extremophilic organisms, which all cluster in the same phylogenetic group. In conclusion, the successful production of the fully assembled and active F1FO ATP synthase from A. aeolicus in E. coli reported in this work provides a novel genetic system to study A. aeolicus F1FO ATP synthase. To a broader extent, it will also serve in the future as a solid reference for designing strategies aimed at producing large multi-subunit complexes with complicated stoichiometry.
  • Die F1FO ATP-Synthase katalysiert die Synthese von ATP aus ADP und anorganischem Phosphat. Die hierfür benötigte Energie wird durch einen über die Zellmembran bzw. Innere Mitochondrienmembran bestehenden elektrochemischen Ionengradienten geliefert. Die F1FO ATP-Synthase ist sowohl in Bakterien, als auch in Mitochondrien und Chloroplasten zu finden und dabei hoch konserviert. Das Holoenzym besteht aus zwei größeren Subkomplexen, dem hydrophilen F1- und dem hydrophoben FO-Komplex. Der F1-Subkomplex besteht aus den Untereinheiten α, β, γ, δ und ε in der Zusammensetzung 3:3:1:1:1. Der membrangebundene FO-Komplex besteht aus den Untereinheiten a, b und c, in den Stöchiometrien 1:2: (8-15). Die Untereinheiten a und c sind für die Ionentranslokation zuständig. Die Untereinheiten γ und ε verbinden den F1-Subkomplex mit dem c-Ring des FO-Subkomplexes. Neben der Einteilung in den hydrophoben und hydrophilen Teil des Enzyms kann die F1FO ATP-Synthase auch in einen Stator (a, b, δ, α, β) und in einen Rotor (γ, ε, c) gegliedert werden. Bisher konnten atomare Strukturen nur von Subkomplexen oder einzelnen Untereinheiten bestimmt werden, wie zum Beispiel dem bovinen F1-Subkomplex oder den c-Ringen aus Ilyobacter tartaricus, Bacillus pseudofirmus und Arthrospira platensis. Strukturen für den bovinen Stator-Subkomplex konnten ebenfalls bestimmt werden. Allerdings ist noch keine Struktur für das Holoenzym oder den membrangebundenen FOSubkomplex bekannt. Die Struktur des Holoenzyms in atomarer Auflösung könnte detaillierte Einblicke in den Ionen-Transportmechanismus geben, der bis heute nicht komplett geklärt ist. Bisher konnte in unserem Labor gezeigt werden, dass die F1FO ATP-Synthase aus Aquifex aeolicus aufgrund ihrer thermophilen Herkunft ein hoch stabiles Enzym darstellt, das als Holoenzym in seiner aktiven Form aufgereinigt werden konnte. Zusätzlich konnten neue strukturelle Daten für die F1FO ATP-Synthase gewonnen werden, wie etwa eine Deformation des zentralen Stators (γ und ε-Untereinheiten) oder ein möglicher heterodimerer peripherer Stator im Vergleich zum Rinderenzym. Daher stellt die F1FO ATP-Synthase aus A. aeolicus ein interessantes Ziel für weitere strukturelle und funktionelle Studien dar. Es wurden vier Ziele für diese Doktorarbeit formuliert, basierend auf früheren Studien: (i) Ergänzung der bisherigen Charakterisierung der nativen F1FO ATP-Synthase aus A. aeolicus (AAF1FO) durch bioinformatische, biochemische und funktionelle Studien, (ii) Etablierung eines heterologen Expressionssystem für AAF1Fo in E. coli, (iii) Charakterisierung der so exprimierten ATP-Synthase (EAF1FO), (iv) Untersuchung von Eigenschaften der AAF1FO, wie etwa die Rolle des N-Terminus der c-Untereinheit, die nur mit Hilfe eines heterologen Expressionssystems durchgeführt werden können. ...

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Author:Chunli Zhang
Place of publication:Frankfurt am Main
Referee:Bernd LudwigGND, Hartmut MichelORCiDGND
Document Type:Doctoral Thesis
Year of Completion:2013
Year of first Publication:2013
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Granting Institution:Johann Wolfgang Goethe-Universität
Date of final exam:2013/06/24
Release Date:2013/11/20
Page Number:202
Institutes:Biochemie, Chemie und Pharmazie / Biochemie und Chemie
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
Sammlung Biologie / Biologische Hochschulschriften (Goethe-Universität)
Licence (German):License LogoDeutsches Urheberrecht