title: Interaction map of the Trypanosoma cruzi ribosomal P protein complex (stalk) and the elongation factor 2.
abstract: The large subunit of the eukaryotic ribosome possesses a long and protruding stalk formed by the ribosomal P proteins. This structure is involved in the translation step of protein synthesis through interaction with the elongation factor 2 (EF-2). The Trypanosoma cruzi stalk complex is composed of four proteins of about 11 kDa, TcP1α, TcP1β, TcP2α, TcP2β and a fifth TcP0 of about 34 kDa. In a previous work, a yeast two-hybrid (Y2H) protein-protein interaction map of T. cruzi ribosomal P proteins was generated. In order to gain new insight into the assembly of the stalk, a complete interaction map was generated by surface plasmon resonance (SPR) and the kinetics of each interaction was calculated. All previously detected interactions were confirmed and new interacting pairs were found, such as TcP1β-TcP2α and TcP1β-TcP2β. Moreover P2 but not P1 proteins were able to homo-oligomerize. In addition, the region comprising amino acids 210-270 on TcP0 was identified as the region interacting with P1/P2 proteins, using Y2H and SPR. The interaction domains on TcP2β were also mapped by SPR identifying two distinct regions. The assembly order of the pentameric complex was assessed by SPR showing the existence of a hierarchy in the association of the different P proteins forming the stalk. Finally, the TcEF-2 gene was identified, cloned, expressed and refolded. Using SPR analysis we showed that TcEF-2 bound with similar affinity to the four P1/P2 ribosomal P proteins of T. cruzi but with reduced affinity to TcP0.
authors.affiliation: Laboratorio de Biología Molecular de la Enfermedad de Chagas (LaBMECh), Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI), National Research Council (CONICET), Buenos Aires, Argentina.
authors.forename: Cristian R
authors.initials: CR
authors.lastname: Smulski
authors.forename: Silvia A
authors.initials: SA
authors.lastname: Longhi
authors.forename: Maximiliano Juri
authors.initials: MJ
authors.lastname: Ayub
authors.forename: Martin M
authors.initials: MM
authors.lastname: Edreira
authors.forename: Leandro
authors.initials: L
authors.lastname: Simonetti
authors.forename: Karina A
authors.initials: KA
authors.lastname: Gómez
authors.forename: Joaquín N
authors.initials: JN
authors.lastname: Basile
authors.forename: Olivier
authors.initials: O
authors.lastname: Chaloin
authors.forename: Johan
authors.initials: J
authors.lastname: Hoebeke
authors.forename: Mariano J
authors.initials: MJ
authors.lastname: Levin
mesh_terms: D000595:Amino Acid Sequence; D017125:Genes, Protozoan; D007700:Kinetics; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism*; D020652:Peptide Elongation Factor 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000737:chemistry / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D020349:Surface Plasmon Resonance; D014349:Trypanosoma cruzi / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: D046912:Multiprotein Complexes; D020652:Peptide Elongation Factor 2; D015800:Protozoan Proteins; D012269:Ribosomal Proteins