Structural basis for the interaction between the potato virus X resistance protein (Rx) and its cofactor Ran GTPase-activating protein 2 (RanGAP2)

Abstract

The potato (Solanum tuberosum) disease resistance protein Rx has a modular arrangement that contains coiled-coil (CC), nucleotide-binding (NB), and leucine-rich repeat (LRR) domains and mediates resistance to potato virus X. The Rx N-terminal CC domain undergoes an intramolecular interaction with the Rx NB-LRR region and an intermolecular interaction with the Rx cofactor RanGAP2 (Ran GTPase-activating protein 2). Here, we report the crystal structure of the Rx CC domain in complex with the Trp-Pro-Pro (WPP) domain of RanGAP2. The structure reveals that the Rx CC domain forms a heterodimer with RanGAP2, in striking contrast to the homodimeric structure of the CC domain of the barley disease resistance protein MLA10. Structure-based mutagenesis identified residues from both the Rx CC domain and the RanGAP2 WPP domain that are crucial for their interaction and function in vitro and in vivo. Our results reveal the molecular mechanism underlying the interaction of Rx with RanGAP2 and identify the distinct surfaces of the Rx CC domain that are involved in intramolecular and intermolecular interactions.

Keywords: Coiled-coil Domain; Plant Molecular Biology; Plant Resistance Protein; Protein Structure; Protein/Protein Interactions; RanGAP; Rx; Structural Biology; X-ray Crystallography.

FIGURE 1.

Characterization of the interaction between Rx CC and RanGAP2 WPP. A, schematic depiction of the domain organization of the Rx and RanGAP2 proteins, including the CC, NB, LRR, WPP, and acidic tail domains. B, Rx CC interacts with RanGAP2 fragments in a pulldown assay. GST-fused RanGAP2 fragments were individually coexpressed with Rx CC (residues 1–122) and purified on glutathione-Sepharose 4B resin. Proteins were visualized by SDS-PAGE and Coomassie Blue staining. C, gel-filtration analysis of the interaction of Rx CC with RanGAP2 WPP. Aliquots of gel-filtration fractions were visualized by SDS-PAGE and Coomassie Blue staining. Numbers indicate the elution fractions. MW, molecular weight standards shown in kilodaltons. D, measurement of binding affinity between Rx CC and RanGAP2 WPP by ITC. Upper panel, raw ITC data. Twenty injections of Rx CC-containing solution were added to the RanGAP2 WPP proteins in the ITC cell. The area of each injection peak corresponds to the total heat released for that injection. Lower panel, the binding isotherm for the Rx CC/RanGAP2 WPP interaction. The integrated heat was plotted against the molar ratio of Rx CC that was added to RanGAP2 WPP in the cell. Data fitting revealed a dissociation constant of 0.039 μm.

FIGURE 2.

The Rx CC domain has a four-helix bundle structure. A, ribbon representation of the Rx CC domain structure. Helices α1, α2, α3, and α4 are indicated. N and C represent the N and C termini, respectively. B, sequence of the Rx CC domain, with the helices shown as cylinders on top. The conserved EDVID motif is boxed.

FIGURE 3.

The RanGAP2 WPP domain forms a three-helix bundle. A, ribbon representation of the crystal structure of the RanGAP2 WPP domain. Helices αA, αB, and αC and the long loop linking αA and αB (loop D) are indicated. N and C represent the N and C termini, respectively. B, structure-based sequence alignment of the WPP domains from potato RanGAP2 and its homolog RanGAP1. Identical amino acids are boxed in red and in yellow. The locations of the three α-helices are indicated on top.

FIGURE 4.

The Rx CC and RanGAP2 WPP domains interact primarily through hydrophobic interactions. A, ribbon representation of the overall structure of the Rx CC-RanGAP2 WPP complex. Rx CC and RanGAP2 WPP are colored cyan and green, respectively. B, illustration of the overall structure of the complex with the same orientation as in A with RanGAP2 WPP shown as a surface representation. The positive, negative, and hydrophobic surfaces on RanGAP2 WPP are colored blue, red, and white, respectively. The Rx CC EDVID motif is colored yellow. C, illustration of the overall structure of the complex with the same orientation as in A with Rx CC shown as a surface representation. The positive, negative, and hydrophobic surfaces are colored blue, red, and white, respectively. D, detailed interactions between Rx CC and RanGAP2 WPP. The side chains of Rx CC and RanGAP2 WPP involved in the interaction are shown in pink and slate, respectively. The side chains of the WPP motif are shown in orange. Relevant amino acid residues are numbered. The hydrogen bond is represented by a dashed red line.

FIGURE 5.

Mutagenesis of residues involved in the interaction between the Rx CC and RanGAP2 WPP domains. A, effects of substitutions in Rx CC on interaction with RanGAP2 WPP. Each GST-tagged Rx CC mutant protein was coexpressed with untagged RanGAP2 WPP and purified on glutathione-Sepharose 4B resin. The eluted proteins were visualized by SDS-PAGE and Coomassie Blue staining. MW, molecular weight; WT, wild-type. B, effects of substitutions in RanGAP2 WPP on interaction with Rx CC.

FIGURE 6.

Functional analysis of Rx and RanGAP2 point mutants in vivo. A, impact of the W90D mutation on Rx-mediated resistance. The Rx NB-LRR fragment was coexpressed in N. benthamiana leaves by Agrobacterium-mediated transient expression together with the wild-type (WT) or the W90D mutant Rx CC domain in the presence of either the PVX CP (upper panel) or PVX-GFP (middle and lower panels). Leaves co-infiltrated with the CP were photographed after 3 days under white light (upper panel), and those co-infiltrated with PVX-GFP were photographed after 6 days under white light (middle panel) or UV light (lower panel). B, impact of point mutations on CC/RanGAP2 binding. Rx CC-HA (WT or W90D) was transiently expressed in N. benthamiana leaves along with RanGAP2-FLAG (WT or A89D). Protein extracts were subjected to anti-HA and anti-FLAG immunoprecipitation (IP), followed by immunoblotting (IB) as indicated. C, NB-LRR binding by the Rx CC W90D mutant. Rx CC-HA (WT or W90D) was agro-expressed in N. benthamiana leaves together with NB-LRR-Myc. Protein extracts were subjected to anti-HA and anti-Myc immunoprecipitation, followed by immunoblotting as indicated.

FIGURE 7.

Further structural analysis of the Rx CC domain. A, superimposition of the Rx CC (cyan) and MLA10 CC (pink) domains. The side chains of the EDVID motif of Rx CC and MLA10 CC are colored slate and yellow, respectively. B, surface representation of the Rx CC domain. The surface formed by the side chains of the EDVID motif is colored yellow. The surface that interacts with RanGAP2 WPP is colored slate. The side chains of RanGAP2 involved in the interaction with Rx and the WPP motif are colored pink and orange, respectively.