Abstract:The kinase domain transfers phosphate from ATP to substrates. However, the Legionella effector SidJ adopts a kinase fold yet catalyzes calmodulin (CaM)-dependent glutamylation to inactivate the SidE ubiquitin ligases. The structural and mechanistic basis in which the kinase domain catalyzes protein glutamylation is unknown. Here we present cryo-EM reconstructions of SidJ:CaM:SidE reaction intermediate complexes. We show that the kinase-like active site of SidJ adenylates an active site Glu in SidE resultin… Show more
“…SidJ R500A mutant protein exhibits more autoAMPylation and acyl adenylylate formation compared to WT SidJ (Figure S6). Interestingly, a recent pre-print by Osinski et al, shows that SidJ R500 co-ordinates free L-Glutamate along with R522 which plays a crucial part in SidJ-enabled nucleophilic attack by L-Glutamate 30 . This explains why SidJ R500A mutant shows increased autoAMPylation while being defective in glutamylation of SdeA.…”
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 serine ubiquitination to target an array of 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.
“…SidJ R500A mutant protein exhibits more autoAMPylation and acyl adenylylate formation compared to WT SidJ (Figure S6). Interestingly, a recent pre-print by Osinski et al, shows that SidJ R500 co-ordinates free L-Glutamate along with R522 which plays a crucial part in SidJ-enabled nucleophilic attack by L-Glutamate 30 . This explains why SidJ R500A mutant shows increased autoAMPylation while being defective in glutamylation of SdeA.…”
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 serine ubiquitination to target an array of 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.
“…This second step likely involves another essential region in SidJ, named the “migrated” nucleotide-binding site ( 139 ), which may help in the optimal positioning of the acyl-adenylated SdeA and the free glutamate to take the reaction to completion. A recent cryo-EM reconstruction of the SidJ:CaM:SdeA intermediate complex revealed that while the pseudokinase active site is responsible for the acyl-adenylation reaction, it is the migrated nucleotide binding pocket that carries out the glutamylation reaction, with Arg522 SidJ playing the crucial role of positioning the donor Glu to attack the acyl-adenylate intermediate and subsequent formation of the Glu-Glu isopeptide bond on SdeA ( 143 ). Figure 8 Regulation of the activity of SidE members by SidJ and SdeD.…”
Section: Side Proteins Sidj and Sded: Atypical Ubiquitination Of Rab-gtpasesmentioning
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