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Structural basis for protein glutamylation by the Legionella pseudokinase SidJ

ABSTRACT: Legionella pneumophila (LP) avoids phagocytosis by secreting nearly 300 effector proteins into the host cytosol. SidE family of effectors (SdeA, SdeB, SdeC and SidE) employ phosphoribosyl ubiquitination to target multiple host Rab GTPases and innate immune factors. To suppress the deleterious toxicity of SidE enzymes in a timely manner, LP employs a metaeffector named SidJ. Upon activation by host Calmodulin (CaM), SidJ executes an ATP-dependent glutamylation to modify the catalytic residue Glu860 in the mono-ADP-ribosyl transferase (mART) domain of SdeA. SidJ is a unique glutamylase that adopts a kinase-like fold but contains two nucleotide-binding pockets. There is a lack of consensus about the substrate recognition and catalytic mechanism of SidJ. Here, we determined the cryo-EM structure of SidJ in complex with its substrate SdeA in two different states of catalysis. Our structures reveal that both phosphodiesterase (PDE) and mART domains of SdeA make extensive contacts with SidJ. In the pre-glutamylation state structure of the SidJ-SdeA complex, adenylylated E860 of SdeA is inserted into the non-canonical (migrated) nucleotide-binding pocket of SidJ. Structure-based mutational analysis indicates that SidJ employs its migrated pocket for the glutamylation of SdeA. Finally, using mass spectrometry, we identified several transient autoAMPylation sites close to both the catalytic pockets of SidJ. Our data provide unique insights into the substrate recognition and the mechanism of protein glutamylation by the pseudokinase SidJ. Legionella pneumophila (LP) employs the metaeffector SidJ to suppress the toxicity of SdeA and other LP SidE effector family members by catalysing the glutamylation of the catalytic Glu residue. Here, the authors present the cryo-EM structures of SidJ in complex with SdeA in two different states, which together with mutagenesis analysis provide insights into the substrate recognition and the mechanism of protein glutamylation by SidJ.

SUBMITTER: Adams M

PROVIDER: S-EPMC8548325 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Effector proteins translocated by the Dot/Icm type IV secretion system determine the virulence of Legionella pneumophila (L. pneumophila). Among these effectors, members of the SidE family (SidEs) regulate several cellular processes through a unique phosphoribosyl ubiquitination mechanism mediated by another effector, SidJ. Host-cell calmodulin (CaM) activates SidJ to glutamylate the SidEs of ubiquitin (Ub) ligases and to make a balanced Ub ligase activity. Given the central role of SidJ in this regulatory process, studying the nature of evolution of sidJ is important to understand the virulence of L. pneumophila and the interaction between the bacteria and its hosts. By studying sidJ from a large number of L. pneumophila strains and using various molecular evolution algorithms, we demonstrated that intragenic recombination drove the evolution of sidJ and contributed to sidJ diversification. Additionally, we showed that four codons of sidJ which are located in the N-terminal (NTD) (codons 58 and 200) and C-terminal (CTD) (codons 868 and 869) domains, but not in the kinase domain (KD) had been subjected to strong positive selection pressure, and variable mutation profiles of these codons were identified. Protein structural modeling of SidJ provided possible explanations for these mutations. Codons 868 and 869 mutations might engage in regulating the interactions of SidJ with CaM through hydrogen bonds and affect the CaM docking to SidJ. Mutation in codon 58 of SidJ might affect the distribution of main-chain atoms that are associated with the interaction with CaM. In contrast, mutations in codon 200 might influence the α-helix stability in the NTD. These mutations might be important to balance Ub ligase activity for different L. pneumophila hosts. This study first reported that intragenic recombination and positive Darwinian selection both shaped the genetic plasticity of sidJ, contributing to a deeper understanding of the adaptive mechanisms of this intracellular bacterium to different hosts.

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Structural basis for protein glutamylation by the Legionella pseudokinase SidJ

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