Structural and funcionalstudies of nse2, the sumo e3 ligase of the smc 5/6 complex

• Autores: Jara Lascorz Lozano
• Directores de la Tesis: David Reverter Cendrós (dir. tes.), Nathalia Varejao Nogueira (dir. tes.)
• Lectura: En la Universitat Autònoma de Barcelona ( España ) en 2022
• Idioma: inglés
• Tribunal Calificador de la Tesis: Josep Vendrell Roca (presid.), Jordi Torres-Rosell (secret.), Luisa María Lois Rojas (voc.)
• Programa de doctorado: Programa de Doctorado en Bioquímica, Biología Molecular y Biomedicina por la Universidad Autónoma de Barcelona
• Materias:
  • Química
    • Bioquímica
      • Biología molecular
• Enlaces
  • Tesis en acceso abierto en: TESEO
• Resumen

  • Protein post-translational modification (PTM) by small ubiquitin-like modifier (SUMO) is a widespread manner of regulation of many cellular functions, including as example transcription or DNA damage repair. SUMO belongs to the ubiquitin-like modifiers (UbL) superfamily of proteins, which are constituted by homologous small protein domains that can be attached to protein targets by the formation of an isopeptidic bond between lysine residues in the substrate and the C-terminal tail of the UbL modifier. This reaction requires the participation of an ATP-dependent enzymatic cascade composed by an: E1, E2 and E3 enzymes.

    Structural Maintenance of Chromosomes (SMC) complexes are ring-shaped heterodimers formed by two SMC proteins and a distinct number of satellite proteins.

    Eukaryotes contain three SMC complexes: cohesin, which maintains the connection between sister chromatids; condensin, which compacts chromosomes; and the Smc5/6 complex, which promotes chromosome disjunction, among other functions. Nse2 is a subunit of the Smc5/6 complex that possesses SUMO E3 ligase activity by the presence of a SP-RING domain that activates the E2~SUMO thioester discharge on the substrates.

    In the present thesis, we first show the design, subcomplex purification, and initial crystallization attempts of truncated constructs of the human Smc5/6 complex. Second, we reveal the enhancement of the SUMO E3 ligase activity of Nse2 upon DNA binding to a positively charged patch in the ARM domain of Smc5, revealing a potential mechanism to restrict SUMOylation to the vicinity of those Smc5/6-Nse2 molecules engaged on DNA.

    Third, we present the crystal structure of the Nse2/Smc5 in complex with the E2-SUMO charged thioester, disclosing the structural details of this multiple E2-E3 protein interface. We finally reveal the role of two SIM (SUMO-Interacting Motif)-like motifs in Nse2, which are restructured upon binding the donor and E2-backside SUMO during the E3-dependent discharge reaction, revealing the enzymatic mechanism of the Nse2 SUMO E3 ligase.