MYC and MexR interactions with DNA : a Small Angle Scattering perspective

Sammanfattning: Protein-DNA complexes govern transcription, that is, the cellular mechanism that converts the information stored in the DNA into proteins. These complexes need to be highly dynamic to respond to external factors that regulate their functions in agreement with what the cells need at that time. Macromolecular X-ray crystallography is very useful for structural studies of large molecular assemblies, but its general application is limited by the difficulties in crystallising highly dynamic and transient complexes. Furthermore, crystal lattices constrain the macromolecular conformation and do not entirely reveal the conformational ensemble adopted by protein-DNA complexes in the solution.Small-Angle X-Ray Scattering (SAXS) and Small-Angle Neutron Scattering (SANS) are two complementary techniques known jointly as Small-angle Scattering (SAS). SAS is a powerful tool for analysing the shape and changes of molecules in solution in their native state. It is beneficial if the variability of conformation or disorder complements high-resolution methods such as NMR or crystallography. With SANS, we can explore non-crystallisable protein-DNA complexes in solution without restrictions of artificially symmetrised DNA and limitations of a protein sequence. Neutrons are well-suited probes for studying protein-DNA complexes for the capability of the neutrons to scatter common atoms in biomolecules differentially and can thereby distinguish between hydrogen and deuterium. Together with varying the solvent deuterium ratio, the contrast variation approach can reveal shapes of distinct components within a macromolecular complex.The goal of this thesis is to explore unchartered territories of regulatory protein-DNA interactions by studying such complexes by SAS, with a specific focus on the flexibility of the complexes. In my study of the MexR-DNA complex, I try to elucidate the molecular mechanism by which the MexR repressor regulates the expression of the MexAB-OPrM efflux pump through DNA binding. This pump is one of the multidrug-resistant tools of the pathogen Pseudomonas Aeruginosa (P. Aer.). It can extrude antibacterial drugs from the bacteria enabling them to survive in hostile environments. In the second project, I strive to explore the MYC:MAX:DNA complex. This heterodimer assembly functions as a central hub in cellular growth control by regulating many biological functions, including proliferation, apoptosis, differentiation and transformation. Overexpression or deregulation of MYC is observed in up to 70% of human aggressive cancer forms, including prostate and breast cancers. By combining SAS with biophysical methods, the work presented in this thesis reveals novel information on the shape and dynamics of biomolecular assemblies critical to health and disease.This thesis comprises five chapters, each dealing with a different aspect of the work in those years. The first chapter introduces the reader to the motivations of this research, and it will give the reader a brief state of the art of the two projects. In the second chapter, I will give you all the theoretical instruments to understand better all the methods used in this thesis, I write first to provide an overview regarding the proteins and their capability to bind other macromolecules. I then will exploit the basics of the small-angle technique, focusing on the neutron contrast variation: the fundamental technique used throughout this thesis and the ab-initio modelling.In the third chapter, Methods, I will discuss the SAS measurements and the requirements for the experiments themselves, the procedure for the data reduction and the data processing and analysis to obtain the structural information.The fourth chapter is a summary of the results of the submitted papers and my contributions:Small-angle X-ray and neutron scattering of MexR and its complex with DNA supports a conformational selection binding modelResolving the DNA interaction of the MexR antibiotic resistance regulatory proteinUpgraded D22 SEC-SANS set-up dedicated to the biology communitySAS studies on the regulation of MYC303:MAX:DNA and MAX:MAX:DNA binding in cancer.