Structure and biophysical studies of mitochondrial Transcription Factor A in complex with DNA

The mitochondrial transcription factor A, TFAM, has a dual function in the organelle: it activates mitochondrial DNA transcription by binding to the HSP and LSP promoters, while in higher concentrations compacts the mtDNA. In this thesis the mechanism of complex formation between the mitochondrial transcription factor A (TFAM) and its cognate DNA binding sequences is analysed. TFAM is a DNA binding protein that belongs to the HMG-box family. Previous crystallographic works have shown that, by its two HMG-boxes and the intervening linker, TFAM binds to the DNA minor groove of mitochondrial DNA promoters imposing severe DNA distortions. These include two sharp 90-degree kinks that bend the cognate DNA into a U-turn. We here present the crystallographic structure of TFAM in complex with site Y, which together with site X are protein binding sites alternative to the promoter binding regions at the control region of mitochondrial DNA (mtDNA). The structure of the TFAM/site Y complex shows the two HMG-box domains (HMG-box1 and 2) organized in an “L”-shape fold that bends the contacted DNA by 90 degrees. Each HMG-box domain inserts a leucine, Leu 58 from HMG-box1 and Leu182 from HMG-box2, into a base-pair step from respective DNA contacted regions. The two DNA steps are separated by a DNA helix turn. Each insertion disrupts the DNA stacking and, together with additional interactions, facilitates the 90o DNA bending, the two bends resulting in the U-turn conformation. A structural comparison between available TFAM/DNA complexes shows that the linker between HMG-domains is instrumental for the protein to adapt to a conformation variability induced by the different DNA sequences. In addition, while all other crystal structures are unambiguous in the assigned DNA sequence, TFAM/site Y electron density maps indicated a surprising DNA disorder that suggested to trace the DNA in an alternative, not predicted, orientation. Thus in order to better characterize the binding mechanism of TFAM to the DNA and the role of the DNA properties in this process, we further studied the TFAM/site Y, TFAM/site X and TFAM/LSP complexes by molecular dynamics (MD) simulations, Abstract 2 isothermal titration calorimetry (ITC) and electrophoretic mobility shift assays (EMSA). All these techniques showed a recurrent result, which is that TFAM has a clear preference in binding and bending site Y over site X and LSP. The three DNAs present intrinsic distortions that facilitate binding, which occurs by a mechanism in all cases endothermic and spontaneous and TFAM presents similar affinities to all of them. However, site Y is intrinsically more rigid but easier to distort into the shape found in the crystal, it competes better for TFAM binding, and the enthalpy and entropy of binding are much higher than for the other two sequences. These results suggest a specific binding and bending mechanism significantly dependent on the DNA sequence. Finally, by multi-angle laser light scattering (MALLS) and analytical ultracentrifugation the multimerization ability of TFAM detected by EMSA and size exclusion chromatography was analysed. The results indicate multimerization of the protein either alone or on the DNA in a cooperative manner at increased complex concentrations, which is consistent with the alternative function of TFAM as an mtDNA packaging protein. Altogether, our results suggest that the DNA sequence properties mediate TFAM binding, involving either specific interactions at the mtDNA control region, or non-specific contacts during mtDNA compaction. For this latter, the regulation of TFAM binding exerted by the DNA sequence might be combined with regulation of protein multimerization processes, all together determining mtDNA compaction, which is essential for cell life.