Crystal Structural Analysis of Oryza sativa SGT1-TPR Domain

Abstract

SGT1 (the suppressor of the G2 allele of Skp1) functions as an adaptor protein that positively regulates plant defense and developmental processes. It comprises three functional domains: the tetratricopeptide repeat (TPR) domain, Chord SGT1 motif (CS), and SGT1-specific motif (SGS). In this study, we resolved the crystal structure of the Oryza sativa OsSGT1-TPR domain at 1.53 Å resolution. Structural analysis showed that the TPR domain adopts a homo-dimeric architecture stabilized by salt bridges (mediated by K52/R79/R109) and hydrophobic interactions (involving F17). Functional validation through gel filtration chromatography revealed that the disruption of the dimerization interface via F17A/K52A/R79A mutations caused complete dissociation into monomers, establishing the essential role of TPR-mediated oligomerization in maintaining the structural stability of full-length OsSGT1. Yeast two-hybrid assays showed that the dimerization disruption of SGT1 mutants retained the interaction with OsHSP81-2 (an HSP90 ortholog) and OsRAR1, indicating that SGT1 oligomerization serves primarily as a structural stabilizer rather than a prerequisite for partner interaction. Evolutionary analysis through the sequence alignment of plant SGT1 proteins revealed the conservation of the dimerization interface residues. This study provides structural insights into the conserved molecular features of SGT1 proteins and highlights the functional significance of their oligomerization state.

1. Introduction

Plant innate immunity relies on nucleotide-binding leucine-rich repeat (NLR) proteins, which detect pathogen effectors to activate effector-triggered immunity (ETI)—this refers to a robust defense response often accompanied by programmed cell death [1]. The HSP90-RAR1-SGT1 (HRS) chaperone complex has emerged as a central regulatory hub governing NLR protein stability and immune signal transduction [2,3]. SGT1 (a suppressor of the G2 allele of Skp1) functions through two distinct mechanisms: (1) acting as a co-chaperone maintaining NLR conformational stability, as validated by its role in the Tsw-mediated recognition of the Tomato spotted wilt virus NSm effector [4] and the phosphorylation-dependent potentiation of Arabidopsis RPS2 immunity [5] and (2) serving as an evolutionary battleground where pathogen effectors such as Ralstonia solanacearum RipAC and Puccinia striiformis PstSIE1 subvert immunity by competitively binding to SGT1 and disrupting HRS complex integrity [6,7]. Structural studies reveal that HSP90-SGT1-RAR1 forms symmetric complexes that facilitate NLR oligomerization [8], while the dynamic reorganization of SGT1 interactomes shifts from developmental regulation to immune signaling upon activation [9,10].

In rice, the multifunctional protein OsSGT1 plays a pivotal role in immune resistance. It participates in chemically induced resistance against Xanthomonas oryzae pv. Oryzae (Xoo) by regulating salicylic acid (SA) signaling and senescence-associated genes (SAGs) [11], while interacting with OsHsp90 through critical residues (e.g., Y173) to stabilize the chaperone complex [12]. The RNAi-mediated silencing of OsSGT1 severely impairs probenazole-induced resistance to Magnaporthe oryzae [13]. Conversely, the overexpression of OsSGT1-GFP enhances resistance to the specific Xoo strain DY89031 but may reduce resistance to another strain (PXO99), indicating pathogen-specific regulatory effects. Notably, OsSGT1 synergizes with OsRAR1 to regulate innate immunity, and the co-overexpression of both significantly enhances disease resistance [14]. Beyond pathogen defense, OsSGT1 facilitates melatonin-mediated stress adaptation, including drought tolerance and chromium detoxification, by modulating downstream signaling components [15]. Collectively, these findings underscore OsSGT1′s multifaceted role in plant immune networks, positioning it as a prime target for disease-resistant crop breeding [16].

Although SGT1 functions as a component of the HRS complex to maintain homeostasis between immunity and development, the structural basis underlying this regulation remains poorly characterized. To provide further insight into the SGT1 structure, we determined the crystal structure of the OsSGT1-TPR domain at a 1.53 Å resolution. Structural analyses revealed a conserved dimerization interface within the TPR domain that mediates OsSGT1 homodimerization. This plant-specific dimerization interface exhibits significant divergence from its yeast counterpart. The Y2H assay showed that dimerization-deficient mutants retain their binding capacity to both OsHSP81-2 and OsRAR1, suggesting that TPR-mediated dimerization primarily stabilizes the structural stability of OsSGT1 rather than directly mediating partner interactions.

2. Materials and Methods

2.1. Protein Expression and Purification

The full-length OsSGT1 and TPR domain (encoding residues 1–120) was amplified by PCR using sticky-end cloning, which was ligated into the NcoI/XhoI-digested pETM13 expression vector to generate an N-terminal hexahistidine-tagged fusion construct [17]. Sequence-validated recombinant vectors were transformed into Escherichia coli BL21 (DE3) competent cells for heterologous expression. Transformed cultures were grown at 37 °C in Lysogeny Broth (LB) medium until the mid-log phase (OD600 = 0.6–0.8) was reached, followed by induction with 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 18 °C for 16 h. Cells were harvested by centrifugation and resuspended in lysis buffer (20 mM Tris-HCl, 500 mM NaCl, pH 7.0) and lysed via ultrasonication at an amplitude of 40% (5 cycles: 3 s on and 3 s off). The supernatants of OsSGT1 and TPR domain proteins collected by centrifugation (12,000× g, 20 min, 4 °C) were applied to a Ni-NTA agarose column pre-equilibrated with lysis buffer. His-tagged proteins were eluted with a linear gradient of 50–500 mM imidazole in lysis buffer. Proteins were further purified by size-exclusion chromatography (SEC) on a Superdex 200 Increase column equilibrated with a gel-filtration buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.0). Purified OsSGT1 and TPR were concentrated to 13 mg/mL with Amicon Ultra-15 centrifugal devices (Millipore) and stored at −80 °C for crystallization trials.

2.2. Crystallization and X-Ray Diffraction Analysis

Initial crystallization screening was performed using commercially available sparse-matrix kits (Hampton Research, Aliso Viejo, CA, USA) via the sitting-drop vapor diffusion method at 18 °C with an Oryx4 robotic system (Douglas Instruments Limited, Berkshire, UK). Using the sitting-drop vapor diffusion method, 0.5 μL drops (composed of a 0.25 μL protein solution [13 mg/mL in the gel-filtration buffer] and 0.25 μL reservoir solution) were equilibrated over 35 μL of reservoir solution. Microcrystals of TPR proteins were initially observed in multiple conditions from the index screen. Crystal morphology optimization was achieved by iteratively adjusting precipitant concentrations (PEG 3350: 18–28% w/v) and additive screening (0.1–0.3 M MgCl₂). The final crystallization condition contained 0.2 M of magnesium chloride hexahydrate, 0.1 M of Tris-HCl (pH 8.5), and 25% (w/v) PEG 3350, yielding diffraction-quality single crystals within 7 days. Crystals were briefly soaked in a reservoir solution containing 20% (v/v) glycerol as the cryoprotectant and flash-cooled in liquid nitrogen. X-ray diffraction data were collected at the Shanghai Synchrotron Radiation Facility (beamline BL18U1) (Shanghai Synchrotron Radiation Facility, Shanghai, China) using a wavelength of 0.9792 Å. A Pilatus3 6 M detector recorded diffraction images at 100 K. Crystals were diffracted to the 1.53 Å resolution and belonged to space group P3, with unit cell parameters (a = 93.493 Å, b = 93.493 Å, c = 32.941 Å). The dataset was indexed, integrated, and scaled using HKL-2000 _(version v722) [18]. Data quality was valued with phenix.xtriage (version 1.18.2-3874) [19].

2.3. Structure Determination and Refinement

The initial model of TPR was generated by a molecular replacement using 7OBE [20] as the search model by Phaser (version 1.18.2-3874) [21]. The structure was manually rebuilt using Coot (version 0.8.9.2) and refined with Phenix in an iterative manner [22,23]. Water molecules were built stepwise into the structure during the refinement. Structural figures were generated by PyMOL software (version 2.5). Refinement statistics are detailed in

Table 1. Data collection and refinement statistics.

Beamline	RRSF BL18U1
Wavelength	0.9792
Resolution range	22.46–1.53 (1.59–1.53) a
Space group	P 3
Unit cell	93.493 93.493 32.941 90 90 120
Total reflections	478,266
Unique reflections	48,592 (4851)
Multiplicity	18.9 (17.0)
Completeness (%)	99.95 (100.00)
Mean I/sigma(I)	48.41 (3.40)
Wilson B-factor	20.8
Rmerge b	0.075 (0.780)
Rmeas	0.091 (0.748)
Rp.i.m	0.021 (0.180)
CC1/2	0.998 (0.879)
Reflections used in refinement	48,589 (4851)
Reflections used for Rfree	2001 (198)
Rwork c	0.176 (0.209)
Rfree	0.190 (0.213)
Number of non-hydrogen atoms	2039
Macromolecules	1777
Solvent	262
No. of protein residues	231
R.m.s. deviations bond lengths (Å) (bonds)	0.006
R.m.s. deviations bond angles (°) (angles)	0.84
Ramachandran plot (%) d
Ramachandran favored	97.36
Ramachandran allowed	2.64
Ramachandran outliers	0.00
Rotamer outliers	0.00
Clashscore	3.43
Average B-factor	28.1
Macromolecules	26.75
Solvent	37.57
Number of TLS groups	8

a: The numbers in parenthesis are for the highest resolution data shell. b: R_merge =∑_hkl∑_I (|I_i (hkl)– < I (hkl) > |)/∑_hkl∑_iI_i (hkl). c: R_work = ∑_hkl (||F_obs|–|F_calc||)/∑I_hkl|F_obs|. d: As evaluated by MolProbity.

. The atomic coordinate of the structure was deposited in the PDB (accession code 9UIB).

2.4. Yeast Two-Hybrid Assay

The coding sequences of OsSGT1 and OsTPR were inserted into the GAL4 DNA-Binding Domain (BD) vector pGBKT7 to generate bait proteins, while the coding sequences of OsHSP81-2 and OsRAR1 were cloned into the GAL4 Activation Domain (AD) vector pGADT7 to create prey proteins. The prey vectors were co-transformed with their corresponding bait vectors into the Saccharomyces cerevisiae strain AH109. Yeast cells expressing both plasmids were selected by growth on a synthetically defined (SD) medium lacking tryptophan and leucine (SD/-Trp/-Leu). Protein–protein interactions were verified by plating these transformants onto the SD medium with a further deficiency in histidine and adenine (SD/-Trp/-Leu/-His-Ade) at 30 °C for 3–5 days.

2.5. Circular Dichroism Spectroscopy

The purified wild-type and mutant proteins were analyzed by circular dichroism (CD) spectroscopy using a Chirascan-plus spectropolarimeter (Applied Photophysics Limited, Surrey, UK) at room temperature, with a protein concentration of approximately 0.2 mg/mL. CD spectra were collected in the wavelength range of 180–280 nm, and the final data were obtained by subtracting the blank spectrum of the corresponding buffer.

3. Results and Discussion

The recombinant protein TPR domain or full-length OsSGT1 proteins both formed homodimers, which were consistent with size-exclusion chromatography results (Figure 1A). We only achieved the crystals of the TPR domain and determined the crystal structure of the TPR domain at a 1.53 Å resolution, belonging to space group P3 (Figure 1B). All residues were matched into the electron density map, and detailed statistics on data collection and refinement are summarized in Table 1. The overall structure of the TPR domain (Protein Data Bank (PDB): 9UIB) adopts a canonical fold consisting of seven α-helices (α1–α7) interconnected by short loops (Figure 1C). Within the asymmetric unit, two TPR molecules were assembled into a homodimer via interactions of the loop region and α1 helix of one subunit with the concave surface of its adjacent counterpart (Figure 1C). This dimeric arrangement stabilizes the interface observed in the crystal lattice. Comparing this to the structure of the Saccharomyces cerevisiae SGT1-TPR domain, OsTPR was superimposed by the ScSGT1-TPR domain (PDB: 5AN3) with an RMSD of 2.6 Å for all main chain atoms, indicating conserved structural features (Figure 2). However, the dimerization interface of OsTPR was positioned opposite to that of ScTPR, indicating divergent dimerization mechanisms between plants and yeast (Figure 2).

The structural analysis of the TPR domain revealed that its dimerization was driven by salt bridges and hydrophobic interactions (Figure 3A). Specifically, the core residues K14, K52, R79, and R109 in chain A stabilized the interaction interface by forming hydrogen bonds and salt bridges with side chains of residues S11, D20, and D19 in adjacent subunits, respectively (Figure 3A). Furthermore, residue F17 participated in hydrophobic interactions with V18 to further stabilize the dimer interface. To validate the self-interaction interface, we compared the oligomerization states of wild-type and mutant proteins (the TPR domain or full-length OsSGT1) using size-exclusion chromatography. The gel filtration assay revealed that the wild-type TPR domain (−35 kDa) and full-length OsSGT1 (−84 kDa) eluted as stable dimers, while mutants (K52A, R79A, R109A, and F17A) migrated as monomers with apparent molecular masses of 16 kDa (isolated TPR mutants) and 42 kDa (full-length SGT1 mutants) (Figure 3B). These results demonstrate that the disruption of interfacial residues (K52A, R79A, R109A, or F17A), but not K14A, abolishes SGT1 dimerization in vitro, which is consistent with structural models of the TPR domain. Circular dichroism (CD) analysis in the far UV region of the spectra showed characteristic α-helical signatures (minima at −222 nm) for both wild-type and mutant TPR proteins, indicating that the mutations did not significantly perturb the overall structural stability (Figure S1). Sequence alignment confirmed that these critical residues (K52, R79, R109, F17A) are highly conserved across plant species (Figure 4), underscoring the functional importance of the TPR domain in mediating SGT1 oligomerization.

In plants, HSP90s, RAR1, and SGT1 proteins directly interact to form the ternary HRS complex, which stabilizes NLR proteins and regulates NLR-mediated resistance to diverse pathogens [10,24,25,26,27]. To further confirm whether the formation of this complex depends on SGT1 dimerization, we performed Y2H assays to analyze the interactions between OsSGT1 (wild-type or dimerization-disrupted mutants) and OsHSP81-2 or RAR1, respectively. When wild-type (WT) or mutant OsSGT1 proteins were used as bait, and OsHSP81-2 or RAR1 were used as prey, no obvious autoactivation was observed on TDO or QDO media (Figure 5). Both WT and mutants interacted with OsHSP81-2 and RAR1, indicating that SGT1 oligomerization is not required for HRS complex assembly (Figure 5).

The HRS chaperone complex is evolutionarily conserved across eukaryotes and plays diverse biological functions, including the assembly of kinetochore in eukaryotes and NLR-triggered plant immunity [3,25,28,29,30]. Previous structural and functional studies have shown that the TPR domain of ScSGT1 directly binds to the BTB domain of Skp1, thereby coupling ScSGT1 to both Hsp90-dependent signaling pathways and Skp1/Cullin/F-box (SCF) ubiquitin ligase-mediated protein degradation pathways [31,32]. Notably, the TPR domain is critical for the dimerization of full-length Sgt1 in yeast [32,33]. In this study, we provided the structural characterization of the rice OsSGT1-TPR domain and elucidated its role in mediating self-association in solution. Mutations targeting the oligomerization region abolished OsSGT1 dimerization. In addition, structural analysis revealed that the dimer interface is evolutionarily conserved in plants but different in yeast. The self-association interface of OsSGT1 overlaps spatially with the Skp1-binding interface of ScSGT1 (Figure S2). This structural divergence leads to the inability of OsSGT1 to bind Skp1 (Figure S3). However, dimerization-deficient mutants remain bound to OsHSP81-2 (HSP90 ortholog) and OsRAR1, indicating that TPR-mediated homodimerization is dispensable for partner recognition but contributes to scaffold complex stability. These findings indicate that the evolutionary conservation of this dimer interface in plant lineages reflects its critical role in stabilizing immune signaling complexes.