PDBsum entry 2fd4

Ligase

PDB id: 2fd4

Contents

Protein chain

105 a.a.

Waters ×51

PDB id:

2fd4

Name:

Ligase

Title:

Crystal structure of avrptob (436-553)

Structure:

Avirulence protein avrptob. Chain: a. Fragment: c-terminal domain, residues 436-553. Engineered: yes

Source:

Pseudomonas syringae. Organism_taxid: 317. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Biol. unit:

Dimer (from PQS)

Resolution:

1.80Å R-factor: 0.243 R-free: 0.267

Authors:

R.Janjusevic,C.E.Stebbins

Key ref:

R.Janjusevic et al. (2006). A bacterial inhibitor of host programmed cell death defenses is an E3 ubiquitin ligase. Science, 311, 222-226. PubMed id: 16373536 DOI: 10.1126/science.1120131

Date:

13-Dec-05 Release date: 27-Dec-05

Protein chain

Q8RSY1  (HPAB2_PSESM) -  Effector protein HopAB2 from Pseudomonas syringae pv. tomato (strain ATCC BAA-871 / DC3000)

Seq: 553 a.a.

Struc: 105 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.2.3.2.- - ?????

DOI no: 10.1126/science.1120131

Science 311:222-226 (2006)

PubMed id: 16373536

A bacterial inhibitor of host programmed cell death defenses is an E3 ubiquitin ligase.

R.Janjusevic, R.B.Abramovitch, G.B.Martin, C.E.Stebbins.

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.

Selected figure(s)

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.

Figures were selected by the author.