Crystal structure of the N-terminal domain of the effector protein SidI of Legionella pneumophila reveals a glucosyl transferase domain.

The Gram-negative bacterium Legionella pneumophila is an accidental human pathogen that can cause a life-threatening respiratory infection called Legionellosis. In the course of infection, L. pneumophila injects more than 300 effector proteins into the host cell. The effector proteins modify the intracellular environment in order to create a stable compartment for proliferation within the host cell. The effector protein SidI has been shown to potently inhibit host translation upon translocation. SidI is able to interact with the translation elongation factor eEF1A, which has been hypothesized to be a target of SidI. A postulated glycosyltransferase domain in the C-terminal half may be responsible for the toxic effect of SidI. Here, we present the crystal structure of an N-terminal fragment of SidI containing residues 37-573. The structure is divided into three subdomains, two of which display a novel fold. The third subdomain shows close structural homology to GT-B fold glycosyltransferases. Based on structural analysis we predict that the two previously identified residues R453 and E482 assume roles in the catalytic activity of SidI. Furthermore, we show that the N-terminal fragment of SidI is able to directly interact with a postulated target, the translation elongation factor eEF1A.

Crystal structure of the metaeffector MesI (Lpg2505) from Legionella pneumophila.

Persistence and replication of the gram-negative bacterium Legionella pneumophila in the human host cell depend on so-called effector proteins that target diverse cellular functions and modulate them in favor of the pathogen. We solved the crystal structure of the L. pneumophila effector protein MesI de novo to a resolution of 2.2 Å. The 34 kDa polypeptide chain folds into two distinct α-helical domains. The larger C-terminal domain shows similarity to tetratricopeptide repeat proteins. Using size-exclusion chromatography, we confirmed that MesI binds tightly to full-length SidI and that deletion of either the N- or the C-terminus weakens the interaction. Based on the three-dimensional structure of MesI we suggest a possible binding mode for SidI and identified two homologs of MesI within the proteome of L. pneumophila that do not bind to SidI, but may act as specific inhibitors of other yet to be identified effectors.

Survivin does not influence the anti-apoptotic action of XIAP on caspase-9.

Survivin inhibits apoptosis in numerous tumor cell lines and has emerged as promising target for cancer therapy. The anti-apoptotic effect of survivin was attributed to a direct interaction with XIAP (X-linked inhibitor of apoptosis) and to an indirect effect, where survivin antagonizes the anti-XIAP action of Smac. The direct interaction is thought to lead to synergistic inhibition of caspase-9 and, at the same time, to enhanced stability of XIAP by reducing its auto-ubiquitination. Using recombinant proteins, we have investigated the influence of survivin on the inhibition of caspase-9 by XIAP in vitro. With a fluorescence-based assay for the apoptosome-stimulated activity of caspase-9, we show that survivin has no effect on the inhibition of caspase-9 by XIAP, neither in the presence nor in the absence of Smac. Employing analytical size exclusion chromatography (SEC) and analytical ultracentrifugation, we show that survivin does not physically interact with XIAP. We confirm in vitro that XIAP ubiquitinates itself in the presence of the appropriate recombinant enzymes and Mg2+-ATP and could show that survivin neither influences the kinetics nor the extent of XIAP's self-ubiquitination. Our results call for a revision of the current view of how survivin interferes with the mitochondrial pathway of apoptosis.

Development of an in vitro assay for the detection of polymerization of the pyrin domain of ASC.

Multicomponent protein complexes called inflammasomes play a major role in the innate immune system by activating proinflammatory cytokines and promoting a highly inflammatory form of programmed cell death, called pyroptosis. A hallmark of the function of the nucleotide-binding domain, leucine-rich repeat and NLRP3-mediated inflammasome assembly is the polymerization of ASC into large filaments. The ASC filaments recruit and activate procaspase-1 by induced proximity. We developed an in vitro assay for monitoring the polymerization of the pyrin domain of ASC by microscale thermophoresis. We have validated the assay by analyzing the effects of buffer conditions, mutations of ASC and the use of seeds on the polymerization behavior of ASC.