Crystal Structure of the Complex between Pseudomonas Effector AvrPtoB and the Tomato Pto Kinase Reveals Both a Shared and a Unique Interface Compared with AvrPto-Pto

Abstract

Resistance to bacterial speck disease in tomato (Solanum lycopersicum) is activated upon recognition by the host Pto kinase of either one of two sequence-unrelated effector proteins, AvrPto or AvrPtoB, from Pseudomonas syringae pv tomato (Pst). Pto induces Pst immunity by acting in concert with the Prf protein. The recently reported structure of the AvrPto-Pto complex revealed that interaction of AvrPto with Pto appears to relieve an inhibitory effect of Pto, allowing Pto to activate Prf. Here, we present the crystal structure of the Pto binding domain of AvrPtoB (residues 121 to 205) at a resolution of 1.9Å and of the AvrPtoB121-205–Pto complex at a resolution of 3.3 Å. AvrPtoB121-205 exhibits a tertiary fold that is completely different from that of AvrPto, and its conformation remains largely unchanged upon binding to Pto. In common with AvrPto-Pto, the AvrPtoB-Pto complex relies on two interfaces. One of these interfaces is similar in both complexes, although the primary amino acid sequences from the two effector proteins are very different. Amino acid substitutions in Pto at the other interface disrupt the interaction of AvrPtoB-Pto but not that of AvrPto-Pto. Interestingly, substitutions in Pto affecting this unique interface also cause Pto to induce Prf-dependent host cell death independently of either effector protein.

Hyaloperonospora arabidopsidis ATR1 effector is a repeat protein with distributed recognition surfaces

“…Oomycete and fungal effectors have been shown to evolve under strong positive selection that drives rapid divergence, making it difficult to detect effector homologs outside their genus using amino acid sequence comparisons or secondary-structure prediction tools. The 3D structure of the Hpa effector ATR1 differs from that of other effector proteins, including AvrL567-A and AvrL567-D, two effectors from Melampsora lini (flax rust) differentially recognized by a cognate R protein L (20), and Pseudomonas syringae effectors AvrPto and AvrB (21,22).…”

Structural Basis for the Interaction between the Potato Virus X Resistance Protein (Rx) and Its Cofactor Ran GTPase-activating Protein 2 (RanGAP2)

“…Protein Expression and Purification-The expression and purification methods used in this study have been described elsewhere in detail (26,27). In summary, the Rx CC domain (residues 1-122) and the RanGAP2 WPP domain (residues 1-112), as well as mutant versions thereof, were subcloned into pGEX-6P-1 (GE Healthcare) and pET-30a (Novagen), respectively, and coexpressed in Escherichia coli BL21(DE3) cells by overnight induction at 16°C.…”

“…Native crystals of the CC-WPP complex were grown in 39% (v/v) Tacsimate (pH 7.0). Selenomethionine-substituted protein was produced using previously established methods (26,27) and was crystallized similarly as described above. The crystals grew to their maximal size within ϳ3 days.…”

Protein Flexibility and Coiled-Coil Propensity: New Insights Into Type III and Other Bacterial Secretion Systems

“…The S. flexneri effector IpaH is a E3 ubiquitin ligase with a C-terminal which consists entirely of -helical bundles and carries the catalytic activity for ubiquitin transfer (Singer et al, 2008). Helical bundle structures are also adopted by AvrPto (Wulf et al, 2004) and AvrPtoB (Dong et al, 2009), although both interact with their target protein kinase Pto via -strand addition (Xing et al, 2007). AvrPto (PDB ids: 1R5E, 2QKW) displays considerable structural plasticity which is consistent with its predicted increased flexibility (Table 1).…”