 |
PDBsum entry 1lvc
|
|
|
|
|
 |
Contents |
 |
|
|
|
|
|
|
|
|
|
|
|
|
* Residue conservation analysis
|
|
|
|
|
References listed in PDB file
|
|
|
Key reference
|
|
|
Title
|
|
Physiological calcium concentrations regulate calmodulin binding and catalysis of adenylyl cyclase exotoxins.
|
|
|
Authors
|
|
Y.Shen,
Y.S.Lee,
S.Soelaiman,
P.Bergson,
D.Lu,
A.Chen,
K.Beckingham,
Z.Grabarek,
M.Mrksich,
W.J.Tang.
|
|
|
Ref.
|
|
EMBO J, 2002,
21,
6721-6732.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
Edema factor (EF) and CyaA are calmodulin (CaM)-activated adenylyl cyclase
exotoxins involved in the pathogenesis of anthrax and whooping cough,
respectively. Using spectroscopic, enzyme kinetic and surface plasmon resonance
spectroscopy analyses, we show that low Ca(2+) concentrations increase the
affinity of CaM for EF and CyaA causing their activation, but higher Ca(2+)
concentrations directly inhibit catalysis. Both events occur in a
physiologically relevant range of Ca(2+) concentrations. Despite the similarity
in Ca(2+) sensitivity, EF and CyaA have substantial differences in CaM binding
and activation. CyaA has 100-fold higher affinity for CaM than EF. CaM has N-
and C-terminal globular domains, each binding two Ca(2+) ions. CyaA can be fully
activated by CaM mutants with one defective C-terminal Ca(2+)-binding site or by
either terminal domain of CaM while EF cannot. EF consists of a catalytic core
and a helical domain, and both are required for CaM activation of EF. Mutations
that decrease the interaction of the helical domain with the catalytic core
create an enzyme with higher sensitivity to Ca(2+)-CaM activation. However, CyaA
is fully activated by CaM without the domain corresponding to the helical domain
of EF.
|
|
|
|
|
|
|
|
Figure 4.
Figure 4 The activation of EF and CyaA-N by wild-type CaM and
two series of CaM mutants. EF (1 nM) and CyaA-N (0.7 nM) were
used for an adenylyl cyclase activity assay in the presence of
0.1  M
free Ca^2+ with CaM mutants CaM 41/75 and 85/112. Each mutant
has two cysteine mutations to lock either the N- or the
C-terminal domain of CaM in the closed conformation (A and B).
The same concentrations of EF and CyaA-N were used for an
adenylyl cyclase activity assay in the presence of 1 M
free Ca^2+ with CaM mutants B1Q, B2Q, B3Q and B4Q. Each mutant
has a mutation inactivating one of four calcium-binding sites (C
and D). Means  SE
are representative of at least two experiments.
|
|
Figure 5.
Figure 5 Effect of calcium and magnesium ions on adenylyl
cyclase activity of EF and CyaA-N (A and B) and an EF mutant,
EF-H577N (C and D). Adenylyl cyclase activity assays were
performed with 10 M
CaM at 0.1 M
Ca^2+ (filled circles), 0.3 M
Ca^2+ (open circles) and 1.0 M
Ca^2+ (filled triangles) in the presence of 1 nM EF (A), and 0.7
nM CyaA-N (B). To analyze the mutant form of EF, adenylyl
cyclase activities were measured with 10 M
CaM and 1 nM EF or 66 nM EF-H577N with either 0.3 M
Ca^2+ (C) or 10 mM Mg^2+ (D). Both were buffered by 10 mM EGTA.
Maximal activities for EF in the magnesium titration are 1616
s^-1 (0.1 M
Ca^2+), 1074 s^-1 (0.3 M
Ca^2+), and 1009 s^-1 (1.0 M
Ca^2+) (A) and those for CyaA-N are 2106 s^-1 (0.1 M
Ca^2+), 1674 s^-1 (0.3 M
Ca^2+) and 1385 s^-1 (1.0 M)
(B). Maximal activities for EF and EF-H577N were 1208 s^-1 and 4
s^-1 (C) and those for EF and EF-H577N were 2726 s^-1 and 6 s^-1
(D), respectively. Means SE
are representative of at least two experiments.
|
|
|
|
|
|
The above figures are
reprinted
from an Open Access publication published by Macmillan Publishers Ltd:
EMBO J
(2002,
21,
6721-6732)
copyright 2002.
|
|
|
Secondary reference #1
|
|
|
Title
|
|
Structural basis for the activation of anthrax adenylyl cyclase exotoxin by calmodulin.
|
|
|
Authors
|
|
C.L.Drum,
S.Z.Yan,
J.Bard,
Y.Q.Shen,
D.Lu,
S.Soelaiman,
Z.Grabarek,
A.Bohm,
W.J.Tang.
|
|
|
Ref.
|
|
Nature, 2002,
415,
396-402.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Figure 3.
Figure 3: CPK representation of EF structures. a, EF alone;
b, CaM -EF. CaM-contacting residues are in red, and the atoms
for switch A, B and C are in cyan, orange and yellow,
respectively.
|
|
Figure 5.
Figure 5: The active site of EF and its comparison with mAC.
a, Ball-and-stick representation of the EF active site. b,
Proposed mechanism of catalysis of EF. For clarity, only
residues that are directly involved in 3' O- to P  nucleophilic
attack are shown. c, Ball-and-stick representation of the mAC
active site. d, Secondary structures of the EF catalytic core
and the mAC catalytic core with the same view as in a and c. The
nucleotide (3'dATP in EF -CaM and ATP- S
in mAC) and the metal ion are in black and grey, respectively.
The O4', C4' and P of
ATP- S
are shown in a similar position to those of 3'dATP in a for
comparison. C1a and C2a of mAC are in green and yellow,
respectively.
|
|
|
|
|
|
The above figures are
reproduced from the cited reference
with permission from Macmillan Publishers Ltd
|
|
|
|
|
|
|
