Biochemical and structural characterization of Mycoplasma genitalium proteins MG438 and MG200

• Autores: Bárbara Luísa Machado Calisto
• Directores de la Tesis: Ignacio Fita Rodríguez (dir. tes.)
• Lectura: En la Universitat Autònoma de Barcelona ( España ) en 2010
• Idioma: inglés
• Tribunal Calificador de la Tesis: Antonio Planas Sauter (presid.), Pedro M. Alzari (secret.), Josep Vendrell Roca (voc.)
• Enlaces
  • Tesis en acceso abierto en: DDD
• Resumen

  • Mycoplasma genitalium is a self-replicating cell with a small genome size and just a small percentage of protein structures known, whose genome was completely unravelled in 1995. In the present work two M. genitalium proteins, MG438 and MG200, potentially implicated in the microorganism virulence and/or pathogenicity processes, were mainly studied from a structural point of view. The MG438 protein constitutes a type I Restriction-Modification (R-M) S subunit. These subunits are involved in the DNA sequence recognition in type I R-M systems. In M. genitalium, MG438 appears as an orphan S subunit suggesting it can have other functions rather than its regular activity in the methyltransferase (MTase) and restriction endonuclease (REase) complexes from type I R-M systems. The MG438 crystal structure, the first described from a type I R-M S subunit, was determined by the Multiple-wavelength Anomalous Dispersion method and refined at 2.3 Å resolution. The protein consists of two globular domains of about 150 residues each, separated by a pair of 40 residues long antiparallel ¿-helices, which form a left-handed super-coil. The globular domains correspond to the variable Target Recognition Domains (TRDs) while the coiled-coil structure correspond to the central (CR1) and C-terminal (CR2) Conserved Regions, respectively. Moreover, the MG438 structure presents an overall cyclic topology with an intra-subunit two-fold axis that superposes the N- and C-half parts of the molecule, each half containing a TRD and a CR. The straight structural resemblance found to exist between TRDs and the small domain of type II R-M N6-adenine DNA TaqI MTase, together with the presence of the intra-subunit quasi-symmetry in MG438, allowed the proposal of a model for the recognition of the target DNAs by the S subunits. The comparison between the MG438 structure and the almost simultaneously reported structure of an S subunit from the archae Methanococcus jannaschii, S.Mja, highlights the preserved structural features despite the low sequence identity. The comparison also reveals important differences in the TRDs and in their disposition with respect to the CRs. M. genitalium presents a complex cytoskeleton with a differentiated terminal organelle that is involved in cell adherence and motility. The MG200 multi-domain protein, thought to be localized in the terminal organelle, was found to be directly involved in mycoplasma motility. Production of the full-length MG200 protein revealed that it behaves as a tetramer in solution but also has a high tendency to aggregate and an intrinsic heterogeneity which prevents crystallization and, consequently, the X-ray crystal structure determination. The crystal structure of the EAGRb domain from the MG200 protein was solved to 2.9 Å resolution by the Single-wavelength Anomalous Dispersion method. This structure is the first piece of structural information from any terminal organelle protein. The domain presents an essentially new fold containing an accurate intra-domain symmetry, which relates with a sequence repeat. The EAGRb forms a dimer, contained in the crystal asymmetric unit, which is stabilized by a conserved hydrophobic core. Some of the domain features, such as its plasticity and the presence and organization of the intra- and inter-subunits symmetry axes, which result in the unbalance of interactions, strongly suggest a role for the EAGRb in protein-protein interactions. Information on the possible quaternary structures for each of the MG200 protein domains, together with preliminary single particle Electron Microscopy studies, allowed the proposal of a 222 (D2) symmetry for the full-length MG200 protein. The particular properties of the MG200 DnaJ and EAGRb domains could permit the interaction with other components of the terminal organelle and result in the formation of supra-molecular structures as the ones observed in the terminal organelles of M. pneumoniae cells.