Exploring structure-function relationship of the mitochondrial DNA packaging protein Abf2p and its dialogue with the DNA

Abstract

Mitochondria are intracellular double-membrane bound organelles in eukaryotic cells that act as the major suppliers of adenosine triphosphate (ATP). They possess their own DNA (mtDNA) that codes for components of the oxidative phosphorylation (OXPHOS) pathway. mtDNA is assembled into nucleo-protein structures called nucleoids and maintained differently compared to histone mediated packaging of nuclear DNA. The molecular basis of mtDNA packaging and maintenance remains poorly understood.

In Saccharomyces cerevisiae (budding yeast), mtDNA is a ~80kb linear molecule, packaged by Abf2p, a double-HMG-box DNA binding protein. Abf2p interacts with DNA in a non-sequence- specific manner, but displays a distinct and yet unexplained ‘phased-binding’ at specific AT-rich DNA stretches containing poly-adenine tracts (A-tracts). Molecular details of DNA binding and maintenance by this protein as well as the mechanism behind its ‘phased binding’ behavior remain to be elucidated. In this doctoral thesis, crystal structures of Abf2p in complex with mtDNA derived fragments bearing A-tracts are presented. The structures reveal that Abf2p binds and induces 180ᵒ U-turn bends in the DNA. Additionally, it avoids binding to A-tracts, giving rise to a unique ‘dual binding’ phenomenon where a single protein molecule binds two DNAs simultaneously. To probe the functional roles played by the different protein structural parts, in vitro and in vivo assays were carried out with different truncation constructs of the protein. These revealed that a 12-residue N-terminal helix, unique to this protein, is crucial for its DNA binding activity. The N-helix+HMG-box1 module possesses a considerably higher DNA binding efficiency compared to HMG-box2, the latter binding little or no DNA on its own. This establishes the predominant role played by the N-helix+HMG-box1 module in the DNA binding event and indicates a hierarchy between the two boxes in the same context. The dynamics of the protein and protein-DNA complex were probed via in-solution (Small Angle X-ray Scattering or SAXS) and simulation (Molecular dynamics or MD) techniques that revealed key mechanisms pertaining to the DNA binding event where the N-helix acts like a pin lock to consolidate DNA binding. Combined with the in vitro and in vivo assays, this provides for a model for DNA binding where the N-helix+HMG-box1 module initiates the DNA binding event, the N-helix locks in and concomitantly or subsequently, HMG-box2 is coaxed into a conformation amenable to DNA binding. Thus, key insights into Abf2p DNA binding mechanism were obtained. The SAXS and MD studies additionally showed that Abf2p is an intrinsically highly flexible protein with considerable relative conformational freedom between the two HMG-boxes that forms a compact species on DNA binding. Additional computational analysis of Abf2p binding on A-tract containing DNA revealed a DNA-structure mediated protein positioning mechanism where the narrow minor groove and intrinsic stiffness of the A-tracts prevent Abf2p binding and thus indirectly guide it to neighboring hospitable sites. The said mechanism would play a key role in orchestrating global nucleoid architecture, given that S. cerevisiae mtDNA has a high percentage of A-tracts. Additionally, the crystal structures disclose an inherent capability of the protein to bind separate DNA strands, that would facilitate DNA packaging by this protein and form an essential mechanistic feature of the process. Analysis of thermodynamics of Abf2p/DNA interactions via isothermal titration calorimetry (ITC) showed a two-phase exothermic-endothermic profile in presence of A-tracts, distinct from that in presence of DNA without A-tracts. The findings reported here thus advance our understanding of mtDNA packaging in the yeast mitochondria from a structural and mechanistic point of view

Els mitocondris posseeixen un ADN (ADNmt) que codifica components de la via de la fosforilació oxidativa. L’ADNmt es compacta en unes estructures nucleo-proteiques, els nucleoides, que s’estructuren de manera diferent a l'ADN nuclear. La base molecular de l’empaquetament de l’ADNmt és desconegut.

A Saccharomyces cerevisiae l’ADNmt és una molècula lineal d’uns 80kb empaquetada per la proteïna Abf2p, que conté dos dominis HMG-box d’unió a ADN. Abf2p contacta l'ADN de forma no específica, però també mostra una unió en fase en regions riques en poli-adenina (regions poly-A). Els detalls moleculars d’aquests dos tipus d’unió encara no s'han dilucidat. En aquesta tesi doctoral es presenten les estructures cristal·logràfiques de l’Abf2p en complex amb fragments d'ADNmt derivats de l’ADN de llevat, que demostren que Abf2p uneix i indueix una curvatura de 180ᵒ a l'ADN. A més a més, en els cristalls, l’Abf2p evita la unió a una regió poly-A induïnt un fenomen únic de d’unió d’una molècula de proteïna a dues molècules d’ADN. Per investigar la funció dels diferents dominis d’Abf2p en la unió ADN hem dut a terme assajos in vitro i in vivo amb fragments i amb la proteïna sencera que mostren que una hèlix de 12 residus N-terminal, única per aquesta proteïna, és crucial per a la unió. A més a més hem estudiat la dinàmica del complex proteïna-ADN en solució per mètodes biofísics (SAXS i ITC) que demostren la flexibilitat de la proteïna i que corroboren el condicionament de la regió poly-A en la unió. Finalment, per dinàmica molecular (MD) hem descobert que l'ADN utilitzat per cristal·litzar té unes propietats estructurals en les regions poly-A, amb un solc menor molt estret, que condicionen el posicionament d’Abf2p. Aquest fenomen és clau en l'organització de l'arquitectura global del nucleoide, atès que en S. cerevisiae l’ADNmt té fins al 30% de regions poly-A, atípic en altres genomes. A més, les estructures cristal·lines mostren la capacitat inherent de la proteïna per unir molècules d'ADN independents, que podrien facilitar el seu empaquetament. Els resultats aquí presentats són un avenç en la nostra comprensió de l’empaquetament de l’ADNmt en el llevat.