Ab Initio Modelling of the Structure of ToxA-like and MAX Fungal Effector Proteins

Abstract

Pathogenic fungal diseases in crops are mediated by the release of effector proteins that facilitate infection. Characterising the structure of these fungal effectors is vital to understanding their virulence mechanisms and interactions with their hosts, which is crucial in the breeding of plant cultivars for disease resistance. Several effectors have been identified and validated experimentally; however, their lack of sequence conservation often impedes the identification and prediction of their structure using sequence similarity approaches. Structural similarity has, nonetheless, been observed within fungal effector protein families, creating interest in validating the use of computational methods to predict their tertiary structure from their sequence. We used Rosetta ab initio modelling to predict the structures of members of the ToxA-like and MAX effector families for which experimental structures are known to validate this method. An optimised approach was then used to predict the structures of phenotypically validated effectors lacking known structures. Rosetta was found to successfully predict the structure of fungal effectors in the ToxA-like and MAX families, as well as phenotypically validated but structurally unconfirmed effector sequences. Interestingly, potential new effector structural families were identified on the basis of comparisons with structural homologues and the identification of associated protein domains.

Keywords: MAX effector family; MaxCluster; Rosetta; TM-score; ToxA-like effector family; ab initio modelling; fungal effector protein.

Figure 1

Structures of effector proteins from the ToxA-like family: (a) ToxA (PDB ID: 1ZLD), (b) AvrL567 (PDB ID: 2QVT) and (c) Avr2 (Fol) (PDB ID: 5OD4). The β-strands are shown in purple and the α-helix is shown in cyan. The structural topology of the secondary structures is shown below each corresponding structure. Figure was used under the terms of the Creative Commons Attribution License (CC-BY 4.0) [24].

Figure 2

Structures of effector proteins from the MAX family: Avr1CO39 (PDB ID: 5ZNG), AvrPia (PDB ID: 2N37), AvrPib (PDB ID: 5Z1V), AvrPikD (PDB ID: 6FUB), AvrPiz-t (PDB ID: 2LW6) and ToxB (PDB ID: 2MM0). The β-strands are shown in purple, and the α-helix is shown in cyan. The structural topology of the secondary structures is shown below each protein.

Figure 3

Scatter plots of the distribution of Rosetta energy scores (in Rosetta energy units (REU)) versus RMSD (left panel) and TM-score (right panel) of ToxA-like effector templates ToxA (A), Avr2 (Fol) (B), and AvrL567 (C). RMSD was calculated as the average deviation between the Cα of all residues in each model and the native structure (in Ångstrom). TM-score values above 0.5 indicate similarity in fold between the ab initio model and the native structure. Plots were derived based on structural models obtained with the highest nstruct of 50,000 models per run, and only models with negative REU values were included in the analysis.

Figure 4

Scatter plots of the distribution of Rosetta energy scores (in Rosetta energy units (REU)) versus RMSD (left panel) and TM-score (right panel) of MAX effector templates ToxB (A), AvrPib (B), AvrPik (C), AvrCO39 (D), AvrPia (E), and AvrPiz-t (F). RMSD was calculated as the average deviation between the Cα of all residues in each model and the native structure (in Ångstrom). TM-score values above 0.5 indicate similarity in fold between the ab initio model and native structure. Plots were derived based on structural models obtained with the highest nstruct of 50,000 models per run, and only models with negative REU values were included in the analysis.

Figure 5

Best structural models for ToxA-like effector templates ToxA (A), Avr2 (Fol) (B), and AvrL567 (C) predicted with Rosetta ab initio modelling. All models are shown in cyan in ribbon representation and each model was superimposed onto their respective XRD or NMR template structure (shown in gold). RMSD and TM-score values are shown on the right-hand side.

Figure 6

Best structural models for MAX effector templates ToxB (A), AvrPib (B), AvrPik (C), Avr1CO39 (D), AvrPia (E) and AvrPiz-t (F) predicted with Rosetta ab initio modelling. All models are shown in blue in ribbon representation, and each model was superimposed onto their respective XRD or NMR template structure (shown in gold). RMSD and TM-score values are shown on the right-hand side.

Figure 7

Comparison of Rosetta ab initio models of ToxA-like family (shown in cyan) ToxA (A), Avr2 (Fol) (B) and AvrL567 (C) with models generated with Robetta (shown in dark green) and QUARK (shown in light green). Each model was superimposed onto its respective XRD or NMR structure (shown in gold) and the corresponding RMSD and TM-score values are shown to the right-hand side of each model.

Figure 8

Comparison of Rosetta ab initio models of MAX family (shown in blue) ToxB (A), AvrPib (B), AvrPitz (C), Avr1CO39 (D), AvrPia (E) and AvrPik (F) with models generated with Robetta (shown in dark blue), and QUARK (shown in purple). Each model was superimposed onto its respective XRD or NMR structure (in gold) and the corresponding RMSD and TM-score values are shown to the right-hand side of each model.

Figure 9

Rosetta ab initio models of structurally unconfirmed effector proteins obtained from PHI-base: Avr2 (F. fulva) (A), AvrStb6 (B), BAS2 (C), MiSSP7 (D), BAS4 (E), PstSCR1 (F), ECP1 (G), AVRA1 (H), Iug6 (I) and PstPEC6 (J). All models are represented with a rainbow colour scheme, from dark blue at the N-terminus to dark red at the C-terminus.

Figure 10

Superimposition of the final models of AvrStb6 (A) and PstSCR1 (B) with the structures of XcpV of T2SS and TplE1 of T6SS, respectively. Rosetta models are coloured in green and homologous structures in cyan.