Protein polyglutamylation catalyzed by the bacterial calmodulin-dependent pseudokinase SidJ
- Weill Institute for Cell and Molecular Biology, Cornell University, United States
- Cornell University, United States
Figures
Figure 1 with 1 supplement
SidJ binds CaM through its C-terminal IQ motif.
(A) Multiple sequence alignment of IQ motifs (‘IQXXXR’), which mediate the binding of CaM, from the indicated proteins. Protein names are followed by a two-letter representation of the species and …
Figure 1—figure supplement 1
Size exclusion chromatography analysis of the SidJ–CaM complex.
(A–E) Size exclusion chromatogram profile of purified recombinant protein (top) and the peak fractions visualized by SDS-PAGE followed by Coomassie staining (bottom). (A) SidJ; (B) CaM; (C) SidJ IQ …
Figure 2 with 1 supplement
Overall structure of the SidJ–CaM complex.
(A) Schematic diagram of SidJ domain architecture. SidJ is comprised of a N-terminal regulatory domain (NRD) in red, a base domain (BD) in yellow, a kinase-like catalytic domain in blue, and a …
Figure 2—figure supplement 1
CaM EF-hand coordinated with one Ca2+.
CaM is represented as a pink cartoon with the residues that coordinate with the Ca2+ ion shown as sticks. The green mesh represents the Fo–Fc difference map contoured to 3σ. Note that the conserved …
Figure 3 with 3 supplements
The core of SidJ adopts a protein kinase fold.
(A) Cartoon diagram of the kinase-like domain of SidJ. Secondary structure elements that are conserved in protein kinases are colored in blue. Ca2+ ions are shown as purple spheres while the …
Figure 3—figure supplement 1
Multiple sequence alignment of SidJ kinase-like domain homologs.
The NCBI BLAST server was used to identify proteins that are homologous to SidJ. Sequences corresponding to the kinase-like domain of SidJ (315–756) were aligned by Clustal Omega and colored using …
Figure 3—figure supplement 2
Multiple sequence alignment of representative protein kinases.
The secondary structure of PKA is labeled above the sequence, and PKA residue numbers are marked on the top of the alignment. Identical residues are highlighted in red and similar residues in …
Figure 3—figure supplement 3
SidJ lacks canonical kinase activity but exhibits auto-AMPylation activity.
(A) Two concentrations of SidJ (0.1 μM and 1 μM) were incubated with CaM, MgCl2 and [γ-32P]ATP without substrate, with MBP, and with SdeA 1–910 for 30 min at 37°C. Proteins were separated by 12% …
Figure 4 with 2 supplements
SidJ catalyzes the polyglutamylation of SdeA.
Reconstructed ion chromatograms for the SdeA peptide (residues 855–877) from (A) cells grown in light medium and co-transfected with GFP-SdeA and mCherry vector control or (B) cells grown in heavy …
Figure 4—figure supplement 1
MS/MS analysis of the SdeA peptide modified by SidJ.
(A) The MS2 spectrum of the SdeA peptide (residues 855–877, prepared from light medium) displays two signature ions, y3 and y10, which correspond to the two y ions generated from two labile prolines …
Figure 4—figure supplement 2
The structural context of the SdeA peptide modified by SidJ.
Left: overall structure of SdeA in complex with Ub and NADH (PDB ID: 5YIJ). Right: enlarged view of the mART active site of SdeA. The peptide (residues 855–877) shown in cyan was polyglutamylated at …
Figure 5
SidJ suppresses the PR-ubiquitination activity of SdeA.
(A) SdeA Core was first incubated with SidJ for 30 min at 37°C with MgCl2, ATP, and CaM, and in the presence or absence of glutamate. Then, the SdeA-mediated ADP-ribosylation of Ub was initiated by …
Figure 6 with 1 supplement
Molecular determinants of SidJ-mediated polyglutamylation.
(A) Overall structure of the SidJ kinase-like domain. (B) Enlarged view of the kinase catalytic site of SidJ. Key catalytic residues are displayed in sticks. Pyrophosphate is represented by sticks …
Figure 6—figure supplement 1
Inhibition of SdeA-catalyzed Ub ADP-ribosylation by SidJ mutants.
(A) SdeA core was first treated by SidJ or its kinase active-site mutants for 30 min at 37°C. After the treatment, the SdeA mediated ADP-ribosylation of Ub was initiated by the addition of Ub and …
Figure 7 with 5 supplements
Activation of SidJ by CaM.
(A) The structure of the SidJ–CaM complex showing the C-lobe of CaM (pink) ‘gripping’ the IQ-motif helix (cyan) of SidJ. (B) A 120° rotated view of the complex in panel (A) showing that the N-lobe …
Figure 7—figure supplement 1
Electrostatic surface potential analysis of the interaction between SidJ and CaM.
(A) The SidJ–CaM complex with SidJ depicted in ribbon and CaM shown with a surface representation colored by electrostatic surface potential, with red being negatively charged with −5 eV and blue …
Figure 7—figure supplement 2
The C-lobe of CaM in the SidJ-CaM complex adopts a semi-open conformation.
(A) A structural comparison of the CaM C-lobe in the SidJ–CaM complex (pink) with apo-CaM (blue) (PDB ID: 1CFC). (B) Structural overlay of the CaM C-lobe in the SidJ-CaM complex (pink) with CaM …
Figure 7—figure supplement 3
The N-lobe of the CaM in the SidJ–CaM complex adopts a closed conformation.
(A) A structural comparison of the CaM N-lobe in the SidJ–CaM complex (pink) with that in apo-CaM (blue) (PDB ID: 1CFC). (B) A structural comparison of the CaM N-lobe in the SidJ–CaM complex (pink) …
Figure 7—figure supplement 4
Reaction time course of SidJ in varied Ca2+ concentrations.
(A) In vitro glutamylation time course of SdeA by SidJ with a concentration of 20 nM SidJ–CaM in buffer containing 1 mM EGTA. Reaction time points were collected and time is labeled above the graph …
Figure 7—figure supplement 5
CaM adopts a unique conformation in the SidJ–CaM complex.
(A) Structural comparison of CaM (pink) bound to SidJ IQ helix with that (green) bound to myosin V IQ1 (PDB ID: 2IX7). IQ helixes are displayed as cylinders colored in a spectrum from blue to red …
Figure 8
Hypothetic reaction model for SidJ-mediated polyglutamylation of SdeA.
SidJ has a kinase-like catalytic cleft, a regulatory nucleotide-binding pocket and a C-terminal CaM-binding IQ helix. Binding of a nucleotide to the allosteric regulatory site and to CaM with the IQ …
Author response image 1
A pilot enzymatic assay of SidJ with a variable concentration of CaM and Ca2+.
(A) In vitro glutamylation of SdeA with 0.5 μM SidJ and five-fold serial dilutions of CaM starting at a concentration of 10 uM. CaM concentration is labeled in nM concentrations. SidJ and CaM were …
Tables
Table 1
Data collection, phasing, and structural refinement statistics.
https://doi.org/10.7554/eLife.51162.006| SeMet SidJ–CaM | Native SidJ–CaM (PDB ID: 6PLM) | |
|---|---|---|
| Synchrotron beam lines | NSLS II 17-ID-1 (AMX) | NSLS II 17-ID-1 (AMX) |
| Wavelength (Å) | 0.97949 | 0.97949 |
| Space group | P21 | P21 |
| Cell dimensions | ||
| a, b, c (Å) | 105.08, 104.08, 109.65 | 105.35, 103.79, 110.19 |
| α, β, γ (o) | 90, 104.49, 90 | 90, 104.69, 90 |
| Maximum resolution (Å) | 2.85 | 2.59 |
| Observed reflections | 371,678 | 482,266 |
| Unique reflections | 69,809 | 69,809 |
| Completeness (%) | 99.5 | 97.7 |
| <I > /<σ>a | 43.20 (15.30) | 38.20 (13.20) |
| Rsyma,b (%) | 0.024 (0.068) | 0.043 (0.091) |
| Phasing methods | SAD | Native |
| Heavy atom type | Se | – |
| Number of heavy atoms/ASU | 12 | – |
| Resolution (Å)a | – | 29.32 (2.59) |
| Rcrys/Rfree (%)a,c | – | 17.6/24.1 |
| Rms bond length (Å) | – | 0.0142 |
| Rms bond angles (°) | – | 1.8174 |
| Most favored/allowed (%) | – | 96.65/3.35 |
| Generous/disallowed (%) | – | 0 |
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a Values in parentheses are for the highest-resolution shell.
bRsym = ΣhΣi|II(h) −<I(h)|/ΣhΣiII(h).
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cRcrys = Σ(|Fobs|−k|Fcal|)/Σ|Fobs|. Rfree was calculated for 5% of reflections randomly excluded from the refinement.
Additional files
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Source data 1
Source data.
- https://doi.org/10.7554/eLife.51162.024
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Supplementary file 1
Key Resources Table.
- https://doi.org/10.7554/eLife.51162.025
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Transparent reporting form
- https://doi.org/10.7554/eLife.51162.026
