reSeArCH Letter
glutamylase that catalyses the ligation of glutamate moieties to E860
of SdeA.
We further investigated the mechanism of the CaM-dependent
glutamylase activity of SidJ by structural analysis. A truncated SidJ that
lacks the first 99 residues (SidJ(∆N99)) showed activity that was indis-
tinguishable from that of the full-length protein. Biophysical analysis
indicated that it formed a stable heterodimer with CaM at a ratio of
1:1 (Extended Data Fig. 4). We solved a structure at 2.71 Å resolution
of the SidJ(∆N99)–CaM complex, using a 2.95 Å-resolution structure
of the SidJ(Se-Met)–CaM derivative as the search model (Extended
Data Table 1). In our structure, two SidJ–CaM heterodimeric com-
plexes are found in one asymmetric unit (Extended Data Fig. 5 a).
Analysis of the intersubunit contacts in the asymmetric unit suggests
that the interface between the two SidJ molecules in the structure
results from crystal packing. In the complex, SidJ(∆N99) folds into
three distinct domains that we designated as the N-terminal domain,
the central domain and the C-terminal domain (Fig. 4a). CaM docks
onto the carboxyl end that contains the IQ motif (Fig. 4b). The inter-
face area between SidJ and CaM is about 1,574 Å^2 , which accounts for
17.6% of the surface of CaM.
SidJ(∆N99) interacts extensively with CaM through hydrogen
bonds and salt bridges. Specifically, Q830 and Q842 of SidJ engage
in hydrogen-bonding interactions with E85 and S102 of the CaM
C-lobe, respectively. Other hydrogen bonds include S808(SidJ)
and E812(SidJ):R38(CaM), R804(SidJ):S39(CaM), R660(SidJ) and
R796(SidJ):E15(CaM) (Fig. 4b). Mutations in these residues reduced
the binding affinity of SidJ for CaM (Fig. 4c).
In order to determine the role of ATP in the activity of SidJ, we
crystallized the SidJ(∆N99)–CaM complex in the presence of ATP
and obtained a structure at 3.11 Å resolution (Extended Data Table 1).
We observed an AMP moiety bound in a pocket formed in the
central domain. This domain—along with approximately one hundred
additional residues—has been designated as the kinase domain in a
recent study^16 , in which the same pocket is shown to be occupied by
pyrophosphate and Mg^2 + ions. The AMP moiety—which is probably
a product of ATP breakdown, induced by SidJ—is coordinated by
R352, K367, Y532, N534, R536 and D545 (Fig. 4d). Substitution of
R352, K367, N534, R536 or D545 by alanine abolished the activity of
SidJ, whereas a mutation in the distal Y443 had no effect (Fig. 4e). The
binding of AMP does not cause obvious conformational changes in the
SidJ(∆N99)–CaM complex (Extended Data Fig. 5b). In our structures,
we observed CaM in a relatively closed conformation^17 with one Ca^2 +
coordinated in the EF1 site of CaM (Extended Data Fig. 6a). However,
the B-factor of Ca^2 + was relatively high, indicating partial occupancy
of the ion; this is consistent with the partial disorder found in the CaM
polypeptide in the crystals. CaM remained active even after dialysisN-terminal domain Central domain C-terminal domain1 312 624 873Construct for crystallization
CDCTD
CaMNTD180°E85S102R660Q830Q842R796E15E12S39R38S808E812R804a100150100(^15032) P-AMP–
SidJ
f
Ub-4×Flag–
Rab33b
150
IB: Flag
IB: SdeA
4 ×Flag–
Rab33b
37
100 IB: SidJ
kDa h
g
IB: Flag (Rab33b-Ub)
IB: SidJ
37
100
25
150 IB: SdeA
kDa
IB: Flag
(^37) (Rab33b-Ub)
100
150
kDa
IB: SidJ
IB: SdeA
b
e
c
SidJ Kd (nM)
Wild type 6.51
R660A 98.28
R796A 634.66
R804A 6,120.72
E812A 78.03
Q842A 5,121.22
Binding af nity
100 873
N lobe C lobe
kDa
IB: Flag
(Rab33b-Ub)
N
C C
N
d
Y532
R352 R536
K367
D545
N534
AMP
Wild typeD545AR352AK367AY443AN534AR536A
Wild typeD545AR352AK367AY443AN534AR536A –ATP+ATP+ATP-
α-S
+ApCpp
–ATP+ATP+ATP-
γ-S
+AMP-PNP
+ADP+AMP+Ade
Fig. 4 | Structural analysis of the mechanism of SidJ-catalysed
glutamylation. a, The domain organization of SidJ. SidJ consists of the
N-terminal domain (orange), the central domain (CD; yellow) and the
C-terminal domain (CTD; green). b, Ribbon diagram representation of
the SidJ–CaM complex. The top panels show the N-terminal domain
(NTD; orange), the central domain (CD; yellow) and the C-terminal
domain (CTD; green) of SidJ, and CaM (red). The N and C termini of
SidJ are labelled with letters. The missing residues are shown as dashed
lines. The bottom panels depict interactions between SidJ and the N-lobe
and C-lobe of CaM. Residues important for binding are shown as sticks
and hydrogen bonds are indicated by dashed lines. c, Binding of CaM to
SidJ and its mutants. The binding affinity was evaluated using microscale
thermophoresis. Kd was calculated by the NanoTemper Analysis 2.2.4
software. Data shown are one representative from three experiments
with similar results. d, Ribbon representation of the SidJ–CaM–AMP
complex. Key SidJ residues involved in AMP binding are shown as yellow
sticks, AMP is shown as magenta sticks. Hydrogen bonds are shown
as dashed lines. The electron density of a simulated annealing Fo − Fc
omit map for AMP is shown, contoured at 3.0σ. e, Mutational analysis
of residues that are important for the binding of AMP. Each SidJ mutant
was incubated with SdeA, ATP, l -glutamate and CaM for 2 h before
measuring the ubiquitin ligase activity of SdeA. f, g, Activation of SidJ
by ATP analogues. The indicated compounds were incubated with SdeA,
GST–SidJ, l -glutamate and CaM for 2 h at 37 °C before monitoring the
activity of SdeA in the ubiquitination of Rab33b. Note that analogues
that cannot be hydrolysed at the α site cannot activate SidJ. h, The role of
residues important for AMP binding in SidJ self-AMPylation. Each SidJ
mutant was incubated with^32 P-α-ATP, Mg^2 + and CaM for 2 h at 37 °C
and the incorporation of^32 P-α-ATP was detected by autoradiography. In
c, e–h, data shown are one representative from at least three independent
experiments with similar results.
390 | NAtUre | VOL 572 | 15 AUGUSt 2019