Molecular Characterization of LubX: Functional Divergence of the U-Box Fold by Legionella pneumophila

Abstract

Summary LubX, is part of Legionella pneumophila’s large arsenal of effectors that are translocated into the host cytosol during infection. Despite such unique features as the presence of two U-box motifs and its targeting of another effector SidH, the molecular basis of LubX’s activity remains poorly understood. Here we show that the N-terminal of LubX is able to activate an extended number of ubiquitin conjugating (E2) enzymes including UBE2W, UBEL6 and all tested members of UBE2D and UBE2E families. Crystal structures of LubX alone and in complex with UBE2D2 revealed drastic molecular diversification between the two U-box domains, with only the N-terminal U-box retaining E2 recognition features typical for its eukaryotic counterparts. Extensive mutagenesis followed by functional screening in a yeast model system captured functionally important LubX residues including Arg121 critical for interactions with SidH. Combined, this data provides a new molecular insight into function of this unique pathogenic factor.

The Legionella pneumophila effector Ceg4 is a phosphotyrosine phosphatase that attenuates activation of eukaryotic MAPK pathways

“…In these assays, Ceg4 demonstrated the highest activity toward phosphotyrosine ( Figure 2C, see also Supplemental Figure S1E). To determine if Ceg4 is active against protein substrates, we screened for its ability to remove the phosphate group from a selection of 53 phosphopeptides, chosen for their importance to signaling in Saccharomyces cerevisiae, which has been successfully used as a model eukaryotic system in characterization of bacterial effector functions including those from Legionella (30,59). This set included pSer-, pThr-and pTyrcontaining sequences.…”

“…Effectors with this PTM activity have been identified in the arsenals of many bacterial pathogens, including E. coli (26,27), Shigella flexneri (24,28), Salmonella spp. (26,29) and Legionella pneumophila (30,31).The most common PTM in eukaryotic cells is phosphorylation, in which a phosphate group from a donor molecule such as ATP onto hydroxyl functional groups on residues (serine, threonine or tyrosine) of the targeted protein (32,33). This PTM is catalyzed by kinases and the human genome encodes several hundred protein kinases divided into tyrosine and serine/threonine specific enzymes (34).…”

“…Effectors with this PTM activity have been identified in the arsenals of many bacterial pathogens, including E. coli (26,27), Shigella flexneri (24,28), Salmonella spp. (26,29) and Legionella pneumophila (30,31).…”

Exploitation of the host cell ubiquitin machinery by microbial effector proteins

“…LubX possesses two U-box domains (Quaile et al, 2015;Kubori et al, 2008), but only the N-terminal one interacts with E2s and exhibits E3 ligase activity. Interestingly, the ubiquitylation activity of LubX is not directed against a host factor, but against another L. pneumophila effector protein, SidH (Kubori et al, 2010).…”

Ubiquitination Accomplished: E1 and E2 Enzymes Were Not Necessary

“…Of the 300 effector proteins L. pneumophila secretes within host cells, many are employed to override substrate selectivity of host SCF E3 complexes (Bruckert and Abu Kwaik, 2016;Quaile et al, 2015). Well-documented examples include LubX, a U-Box containing protein, and F-box proteins AnkB and LegU1.…”