Architektur und Funktion eubakterieller Dodecine - eine neuartige Proteinfamilie Kofaktor-speichernder Proteine

Dodecins are small flavin binding proteins, which were discovered during an inverse structural genomics project in the archaebacterium H. salinarum. The monomeric subunits, which are only 7.4 kDa in size consist of a simple beta-alpha-beta-beta-fold and adopt a hollow spheric dodecameric complex with cubic 23-point symmetry. The flavins are bound as dimers along the twofold symmetry axes of the dodecameric complex and are sandwiched between two neighbouring tryptophan residues resulting in an aromatic tetrade, which is unique so far. This work is the basis for the understanding of flavin binding in eubacterial dodecins. Solving the 2.6 and 1.7 Å crystal structures of the heterologously expressed dodecins from T. thermophilus and M. tuberculosis showed a different flavin-binding mode for eubacterial dodecins than published for the H. salinarum dodecin. Single point mutations of residues, which differ in the eubacterial dodecins and the haloarchaeal type showed the same binding mode as observed for the wild type T. thermophilus dodecin, as was shown by the 2.6 and 1.3 Å crystal structures of the T. thermophilus dodecin R45A and R65A mutants. Spectroscopic investigation of the T. thermophilus dodecin and its W38Y and W38F mutants which were performed in order to investigate the function of the aromatic tetrade give clear indication of electronic interaction between the flavin and its neighbouring amino acid, which results in case of the WT dodecin in complete deactivation of the flavin activated state in only 70 fs. Beside flavin dimers eubacterial dodecins also contain endogenously bound coenzyme A, as was shown by X-ray crystallography and mass spectrometry. The CoA molecules are bound non-covalently as trimers in the threefold symmetric face II in such a way that the free thiol group is surrounded by the protein. Such a trimeric assembly of CoA molecules has never been reported in literature before. Also for the first time a dodecin dataset with atomic resolution (1.08 Å) was collected, that can serve as a basis for further calculations of dodecin-flavin interactions. Based on the obtained knowledge the function of dodecin as bifunctional cofactor storage protein can be postulated; this protein binds CoA and flavins in a non-reactive state und reduces cellular photosensitive effects resulting from these cofactors.