Structural and Functional Studies on Acyl-CoA Carboxylases of Mycobacterium tuberculosis Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences Madhankumar Anandhakrishnan 2013 Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences Presented by B.Tech., Madhankumar Anandhakrishnan born in Madurai, India Oral examination: Structural and Functional Studies on Acyl-CoA Carboxylases of Mycobacterium tuberculosis Referees: Dr. Rob Meijers Prof. Dr. Robert B. Russell Contents Table of Contents Summary…………………………………………………………………………….IV Zusammenfassung………………………………………………………………….VI Publications……………………………………………………………………….VIII Abbreviations…………………………………………………………………….....IX 1! Introduction ........................................................................................................... 1! 1.1! Mycobacterium tuberculosis ....................................................................................... 1! 1.1.1! Transmission and pathogenesis............................................................................. 2! 1.1.2! The fight against TB ............................................................................................. 4! 1.1.2.1! Host defence mechanisms ............................................................................. 4! 1.1.2.2! Anti-tuberculosis chemotherapy.................................................................... 6! 1.1.3! The cell wall of Mycobacterium tuberculosis..................................................... 10! 1.1.3.1! Biochemical nature of the cell wall ............................................................. 10! 1.1.3.2! Fatty acid biosynthesis................................................................................. 12! 1.2! Acyl-CoA carboxylase.............................................................................................. 14! 1.2.1! Biotin-dependent carboxylases ........................................................................... 14! 1.2.1.1! Functional components................................................................................ 14! 1.2.1.2! Classification of biotin-dependent carboxylases ......................................... 15! 1.2.2! Actinobacterial acyl-CoA carboxylases.............................................................. 17! 1.2.2.1! Physiological roles of actinobacterial YCCs............................................... 17! 1.2.2.2! Phylogeny of the !-subunits ........................................................................ 18! 1.2.2.3! Quaternary structure of acyl-CoA carboxylases.......................................... 21! 1.2.2.4! Acyl-CoA carboxylases of Streptomyces coelicolor................................... 21! 1.2.2.5! Acyl-CoA carboxylases of Mycobacterium tuberculosis............................ 23! 1.3! Aims of this study ..................................................................................................... 26! 2! Materials and Methods ....................................................................................... 27! 2.1! Materials ................................................................................................................... 27! 2.1.1! Chemicals............................................................................................................ 27! 2.1.2! Fine biochemicals ............................................................................................... 27! 2.1.3! Genomic DNA and bacterial strains ................................................................... 27! 2.1.4! Laboratory consumables ..................................................................................... 28! 2.1.5! Molecular cloning kits ........................................................................................ 29! 2.1.6! Chromatography materials.................................................................................. 29! 2.2! Methods..................................................................................................................... 29! 2.2.1! Preparation of competent cells............................................................................ 29! I Contents 2.2.1.1! Preparation of chemically competent E. coli cells ...................................... 29! 2.2.1.2! Preparation of electrocompetent M. smegmatis cells .................................. 30! 2.2.2! Molecular cloning ............................................................................................... 30! 2.2.2.1! Ligase-dependent cloning............................................................................ 30! 2.2.2.2! Ligase-independent cloning......................................................................... 33! 2.2.3! Heterologous overexpression of proteins............................................................ 35! 2.2.3.1! Large-scale expression of proteins in E. coli............................................... 35! 2.2.3.2! Large-scale expression of proteins in M. smegmatis................................... 35! 2.2.4! Purification of proteins ....................................................................................... 36! 2.2.4.1! IMAC purification of polyhistidine-tagged proteins................................... 37! 2.2.4.2! Size-exclusion chromatography .................................................................. 37! 2.2.4.3! Evaluation of protein biochemical purity .................................................... 38! 2.2.4.4! Protein quantification and concentration..................................................... 39! 2.2.4.5! Verification of protein identity.................................................................... 39! 2.2.5! Reconstitution of protein complexes .................................................................. 40! 2.2.5.1! In vitro reconstitution by purification and co-elution.................................. 40! 2.2.5.2! In vivo reconstitution by co-expression....................................................... 41! 2.2.6! Kinetic characterisation ...................................................................................... 42! 2.2.6.1! Michaelis-Menten kinetics .......................................................................... 42! 2.2.6.2! Assay for carboxylase activity..................................................................... 42! 2.2.7! Biophysical characterisation ............................................................................... 43! 2.2.7.1! Static light scattering ................................................................................... 43! 2.2.7.2! Thermofluor................................................................................................. 44! 2.2.7.3! Circular dichroism spectroscopy ................................................................. 44! 2.2.8! Protein crystallisation.......................................................................................... 45! 2.2.8.1! Principles ..................................................................................................... 45! 2.2.8.2! Screening and optimization ......................................................................... 48! 2.2.8.3! Reductive methylation................................................................................. 48! 2.2.9! X-ray crystallography ......................................................................................... 49! 2.2.9.1! Theory.......................................................................................................... 49! 2.2.9.2! Data collection............................................................................................. 53! 2.2.9.3! Structure solution and refinement................................................................ 53! 2.2.10! Small angle X-ray scattering............................................................................. 54! 3! Results and Discussion ........................................................................................ 56! 3.1! Characterisation of components of the propionyl-CoA carboxylase (PCC) of Mycobacterium tuberculosis............................................................................................... 56! II Contents 3.1.1! Results................................................................................................................. 57! 3.1.1.1! AccA3 and AccD5: formation of a binary complex.................................... 57! 3.1.1.2! AccA3-AccD5 is an active carboxylase ...................................................... 58! 3.1.1.3! Crystallisation.............................................................................................. 59! 3.1.1.4! X-ray data collection, structure solution and refinement ............................ 59! 3.1.1.5! Crystal structure of AccD5.......................................................................... 61! 3.1.1.6! Electron microscopy of AccA3-AccD5....................................................... 66! 3.1.1.7! Overexpression in E. coli............................................................................. 67! 3.1.1.8! Co-expression .............................................................................................. 69! 3.1.1.9! Attempts to improve AccA3 behaviour......................................................