Strukturelle Studien zur Wechselwirkung humaner Rho-Proteine mit Effektoren und Regulatoren

Die vorliegende Arbeit beschäftigt sich mit der Charakterisierung des Aktivierungs-mechanismus der kleinen GTPase Cdc42 durch den Austauschfaktor SopE von $\textit {Salmonella typhimurium}$ sowie mit der Regulation der p21-aktivierten Proteinkinase $\alpha$PAK durch Cdc42. SopE ist essentiell für die Invasivität von $\it {Salmonella}$. Die Röntgenkristallstruktur der katalytischen Domäne von SopE im Komplex mit seinem Rho-Zielprotein Cdc42 zeigt, dass SopE keine Strukturähnlichkeiten mit Dbl-homologen eukaryontischen Austauschfaktoren aufweist. Der Mechanismus der Katalyse scheint dagegen ähnlich zu sein. Die p21-aktivierten Kinasen (PAKs) sind an Prozessen der intrazellulären Signalweiterleitung beteiligt. Sie werden durch die Rho-Proteine Cdc42·GTP und Rac1-GTP aktiviert, indem inhibitorische Wechselwirkungen, die zwischen dem regulatorischen Bereich und der katalytischen Domäne von PAK bestehen, unterbrochen werden

Structural basis for the reversible activation of a Rho protein by the bacterial toxin SopE

The bacterial enteropathogen Salmonella typhimurium employs a type III secretion system to inject bacterial toxins into the host cell cytosol. These toxins transiently activate Rho family GTP-binding protein-dependent signaling cascades to induce cytoskeletal rearrangements. One of these translocated Salmonella toxins, SopE, can activate Cdc42 in a Dbl-like fashion despite its lack of sequence similarity to Dbl-like proteins, the Rho-specific eukaryotic guanine nucleotide exchange factors. To elucidate the mechanism of SopE-mediated guanine nucleotide exchange, we have analyzed the structure of the complex between a catalytic fragment of SopE and Cdc42. SopE binds to and locks the switch I and switch II regions of Cdc42 in a conformation that promotes guanine nucleotide release. This conformation is strikingly similar to that of Rac1 in complex with the eukaryotic Dbl-like exchange factor Tiam1. However, the catalytic domain of SopE has an entirely different architecture from that of Tiam1 and interacts with the switch regions via different amino acids. Therefore, SopE represents the first example of a non-Dbl-like protein capable of inducing guanine nucleotide exchange in Rho family proteins

Human nonsense-mediated mRNA decay factor UPF2 interacts directly with eRF3 and the SURF complex

Nonsense-mediated mRNA decay (NMD) is an mRNA degradation pathway that regulates gene expression and mRNA quality. A complex network of macromolecular interactions regulates NMD initiation, which is only partially understood. According to prevailing models, NMD begins by the assembly of the SURF (SMG1-UPF1-eRF1-eRF3) complex at the ribosome, followed by UPF1 activation by additional factors such as UPF2 and UPF3. Elucidating the interactions between NMD factors is essential to comprehend NMD, and here we demonstrate biochemically and structurally the interaction between human UPF2 and eukaryotic release factor 3 (eRF3). In addition, we find that UPF2 associates with SURF and ribosomes in cells, in an UPF3-independent manner. Binding assays using a collection of UPF2 truncated variants reveal that eRF3 binds to the C-terminal part of UPF2. This region of UPF2 is partially coincident with the UPF3-binding site as revealed by electron microscopy of the UPF2-eRF3 complex. Accordingly, we find that the interaction of UPF2 with UPF3b interferes with the assembly of the UPF2-eRF3 complex, and that UPF2 binds UPF3b more strongly than eRF3. Together, our results highlight the role of UPF2 as a platform for the transient interactions of several NMD factors, including several components of SURF

Co-expression of protein complexes in prokaryotic and eukaryotic hosts: Experimental procedures, database tracking and case studies

Structure determination and functional characterization of macromolecular complexes requires the purification of the different subunits in large quantities and their assembly into a functional entity. Although isolation and structure determination of endogenous complexes has been reported, much progress has to be made to make this technology easily accessible. Co-expression of subunits within hosts such as Escherichia coli and insect cells has become more and more amenable, even at the level of high-throughput projects. As part of SPINE (Structural Proteomics In Europe), several laboratories have investigated the use co-expression techniques for their projects, trying to extend from the common binary expression to the more complicated multi-expression systems. A new system for multi-expression in E. coli and a database system dedicated to handle co-expression data are described. Results are also reported from various case studies investigating different methods for performing co-expression in E. coli and insect cells

The cryo-EM structure of the UPF-EJC complex shows UPF1 poised toward the RNA 3´ end.

8 páginas, 7 figuras -- PAGS nros. 498-505Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance pathway that degrades aberrant mRNAs containing premature termination codons (PTCs). NMD is triggered upon the assembly of the UPF surveillance complex near a PTC. In humans, UPF assembly is prompted by the exon junction complex (EJC). We investigated the molecular architecture of the human UPF complex bound to the EJC by cryo-EM and using positional restraints from additional EM, MS and biochemical interaction data. The heptameric assembly is built around UPF2, a scaffold protein with a ring structure that closes around the CH domain of UPF1, keeping the helicase region in an accessible and unwinding-competent state. UPF2 also positions UPF3 to interact with the EJC. The geometry is such that this transient complex poises UPF1 to elicit helicase activity toward the 3′ end of the mRNPThis work was funded by the Spanish Government (SAF2008-00451 and SAF2011-22988 to O.L.) and the Red Temática de Investigación Cooperativa en Cáncer from the Instituto de Salud Carlos III (RD06/0020/1001 to O.L. and contract to R.M.). O.L. is additionally supported by the Human Frontiers Science Program (RGP39/2008 to O.L.), the Fundación Ramón Areces and the Government from the Autonomous Region of Madrid (S2010-BMD-2316). This work was also supported by the Max Planck Gesellschaft, the Sonderforschungsbereich SFB646, the Gottfried Wilhelm Leibniz Program of the Deutsche Forschungsgemeinschaft and the Center for Integrated Protein Science Munich (E.C.)Peer reviewe

Structure and E3-ligase activity of the Ring–Ring complex of Polycomb proteins Bmi1 and Ring1b

Polycomb group proteins Ring1b and Bmi1 (B-cell-specific Moloney murine leukaemia virus integration site 1) are critical components of the chromatin modulating PRC1 complex. Histone H2A ubiquitination by the PRC1 complex strongly depends on the Ring1b protein. Here we show that the E3-ligase activity of Ring1b on histone H2A is enhanced by Bmi1 in vitro. The N-terminal Ring-domains are sufficient for this activity and Ring1a can replace Ring1b. E2 enzymes UbcH5a, b, c or UbcH6 support this activity with varying processivity and selectivity. All four E2s promote autoubiquitination of Ring1b without affecting E3-ligase activity. We solved the crystal structure of the Ring–Ring heterodimeric complex of Ring1b and Bmi1. In the structure the arrangement of the Ring-domains is similar to another H2A E3 ligase, the BRCA1/BARD1 complex, but complex formation depends on an N-terminal arm of Ring1b that embraces the Bmi1 Ring-domain. Mutation of a critical residue in the E2/E3 interface shows that catalytic activity resides in Ring1b and not in Bmi1. These data provide a foundation for understanding the critical enzymatic activity at the core of the PRC1 polycomb complex, which is implicated in stem cell maintenance and cancer