 |
PDBsum entry 2fd4
|
|
|
|
References listed in PDB file
|
 |
|
Key reference
|
|
|
Title
|
|
A bacterial inhibitor of host programmed cell death defenses is an e3 ubiquitin ligase.
|
|
|
Authors
|
|
R.Janjusevic,
R.B.Abramovitch,
G.B.Martin,
C.E.Stebbins.
|
|
|
Ref.
|
|
Science, 2006,
311,
222-226.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
The Pseudomonas syringae protein AvrPtoB is translocated into plant cells, where
it inhibits immunity-associated programmed cell death (PCD). The structure of a
C-terminal domain of AvrPtoB that is essential for anti-PCD activity reveals an
unexpected homology to the U-box and RING-finger components of eukaryotic E3
ubiquitin ligases, and we show that AvrPtoB has ubiquitin ligase activity.
Mutation of conserved residues involved in the binding of E2
ubiquitin-conjugating enzymes abolishes this activity in vitro, as well as
anti-PCD activity in tomato leaves, which dramatically decreases virulence.
These results show that Pseudomonas syringae uses a mimic of host E3 ubiquitin
ligases to inactivate plant defenses.
|
|
|
|
|
|
|
|
Figure 2.
Fig. 2. AvrPtoB mimics host RING-finger and U-box proteins. (A)
Structural alignment of the AvrPtoB CTD (the core fold) with the
RING-finger and U-box structures of Rbx1 [Protein Data Bank
(PDB) ID 1LDJ [PDB]
, Rag1 (PDB ID 1RMD [PDB]
), PrP19 (PDB ID 1N87 [PDB]
), and AtPUB14 (PDB ID 1T1H [PDB]
). (B) Visualization of the E2-binding site residues of Rbx1
with homologous regions in AvrPtoB and AtPUB14 [the polypeptide
backbone from the alignment in panel (A)]. The corresponding
residue numbers are indicated next to the amino acid. (C)
Structure-based sequence alignment of AvrPtoB, Rbx1, and AtPUB14
focusing on the conserved, core fold. Secondary structure of
AvrPtoB is indicated in blue above the sequence. Black
highlights the three putative E2-binding residues shown in panel
(B). The location of the AvrPtoB disordered insertion is
indicated.
|
|
Figure 3.
Fig. 3. The AvrPtoB CTD has eukaryotic-like ubiquitin ligase
activity. (A) Autoubiquitination activity of AvrPtoB full-length
protein (FL) and a CTD GST-AvrPtoB(436-553) fusion that was used
for crystallization [labeled GST-(436-553]. In vitro
ubiquitination assays were performed in the presence of ATP with
the indicated combinations of proteins as described in (9).
Proteins were resolved by SDS-polyacrylamide electrophoresis
(SDS-PAGE) and were subjected to immunoblot analysis with
indicated antibodies. Polyubiquitinated forms of AvrPtoB FL and
AvrPtoB(436-553) were detected with antibodies against Ub or
against Ub and GST, respectively. Antibodies against His[6] were
used for detection of 2XHis[6]-tagged ubiquitin-conjugating
proteins (E2, human UbcH5c or its Arabidopsis homolog AtUBC8;
colored in red; position indicated with arrows). FL mutants
F479A, F525A, and P533A (33) are the E2-binding site mutants of
full-length AvrPtoB. E1, ubiquitin-activating enzyme; Ub,
ubiquitin. One asterisk indicates position of
GST-AvrPtoB(436-553); two asterisks indicate the position of
GST. SYPRO FL: gel was stained with SYPRO Ruby protein gel
stain, and the position of wt FL AvrPtoB and each E2-binding
site mutant is indicated by a black arrow. (B) Time-dependent in
vitro autoubiquitination of GST-AvrPtoB(436-553). Reaction mix
containing E1, E2 (Arabidopsis His[6]-UbcH8),
GST-AvrPtoB(436-553), and ubiquitin was incubated for indicated
times at 30°C as described (9), visualized by SDS-PAGE. The
ubiquitinated proteins, GST-AvrPtoB(436-553), and His[6]-UbcH8
were detected in immunoblot analysis with the indicated
antibodies as in Fig. 3A.
|
|
|
|
|
|
The above figures are
reprinted
by permission from the AAAs:
Science
(2006,
311,
222-226)
copyright 2006.
|
|
|
|
|
|
|
