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Regulatory ligand binding in plant chalcone isomerase–like (CHIL) proteins

2023; Elsevier BV; Volume: 299; Issue: 6 Linguagem: Inglês

10.1016/j.jbc.2023.104804

1083-351X

Emma R Wolf-Saxon, Chad C Moorman, Anthony E. Castro, Alfredo Ruiz-Rivera, Jeremy P. Mallari, Jason R. Burke,

Polyamine Metabolism and Applications

Chalcone isomerase–like (CHIL) protein is a noncatalytic protein that enhances flavonoid content in green plants by serving as a metabolite binder and a rectifier of chalcone synthase (CHS). Rectification of CHS catalysis occurs through direct protein–protein interactions between CHIL and CHS, which alter CHS kinetics and product profiles, favoring naringenin chalcone (NC) production. These discoveries raise questions about how CHIL proteins interact structurally with metabolites and how CHIL–ligand interactions affect interactions with CHS. Using differential scanning fluorimetry on a CHIL protein from Vitis vinifera (VvCHIL), we report that positive thermostability effects are induced by the binding of NC, and negative thermostability effects are induced by the binding of naringenin. NC further causes positive changes to CHIL–CHS binding, whereas naringenin causes negative changes to VvCHIL–CHS binding. These results suggest that CHILs may act as sensors for ligand-mediated pathway feedback by influencing CHS function. The protein X-ray crystal structure of VvCHIL compared with the protein X-ray crystal structure of a CHIL from Physcomitrella patens reveals key amino acid differences at a ligand-binding site of VvCHIL that can be substituted to nullify the destabilizing effect caused by naringenin. Together, these results support a role for CHIL proteins as metabolite sensors that modulate the committed step of the flavonoid pathway. Chalcone isomerase–like (CHIL) protein is a noncatalytic protein that enhances flavonoid content in green plants by serving as a metabolite binder and a rectifier of chalcone synthase (CHS). Rectification of CHS catalysis occurs through direct protein–protein interactions between CHIL and CHS, which alter CHS kinetics and product profiles, favoring naringenin chalcone (NC) production. These discoveries raise questions about how CHIL proteins interact structurally with metabolites and how CHIL–ligand interactions affect interactions with CHS. Using differential scanning fluorimetry on a CHIL protein from Vitis vinifera (VvCHIL), we report that positive thermostability effects are induced by the binding of NC, and negative thermostability effects are induced by the binding of naringenin. NC further causes positive changes to CHIL–CHS binding, whereas naringenin causes negative changes to VvCHIL–CHS binding. These results suggest that CHILs may act as sensors for ligand-mediated pathway feedback by influencing CHS function. The protein X-ray crystal structure of VvCHIL compared with the protein X-ray crystal structure of a CHIL from Physcomitrella patens reveals key amino acid differences at a ligand-binding site of VvCHIL that can be substituted to nullify the destabilizing effect caused by naringenin. Together, these results support a role for CHIL proteins as metabolite sensors that modulate the committed step of the flavonoid pathway. Flavonoids are a class of specialized metabolites that are ubiquitous across terrestrial land plants. The biological functions of flavonoids and their derivatives vary widely and include floral pigments, phytoalexins, structured scaffolds, and cellular signals (1Falcone Ferreyra M.L. Rius S.P. Casati P. Flavonoids: biosynthesis, biological functions, and biotechnological applications.Front. Plant Sci. 2012; 3: 222Crossref PubMed Scopus (1127) Google Scholar, 2Weng J.K. Noel J.P. Chemodiversity in Selaginella: a reference system for parallel and convergent metabolic evolution in terrestrial plants.Front. Plant Sci. 2013; 4: 119Crossref PubMed Scopus (54) Google Scholar). In animals, dietary flavonoids are medically important antioxidants that also have beneficial procognitive and anti-inflammatory functions (3Jaeger B.N. Parylak S.L. Gage F.H. Mechanisms of dietary flavonoid action in neuronal function and neuroinflammation.Mol. Aspects Med. 2018; 61: 50-62Crossref PubMed Scopus (52) Google Scholar). The flavonoid pathway in green plants begins with chalcone synthase (CHS), a type II polyketide synthase that catalyzes the condensation of one p-coumaroyl-CoA and three malonyl-CoAs into 2ʹ,4,4ʹ,6ʹ-tetrahydroxychalcone, also known as naringenin chalcone (NC). Downstream, chalcone isomerase (CHI) cyclizes NC to form 2S-naringenin (4Bednar R.A. Hadcock J.R. Purification and characterization of chalcone isomerase from soybeans.J. Biol. Chem. 1998; 263: 9582-9588Abstract Full Text PDF Google Scholar, 5Yonekura-Sakakibara K. Higashi Y. Nakabayashi R. The origin and evolution of plant flavonoid metabolism.Front. Plant Sci. 2019; 10: 943Crossref PubMed Scopus (199) Google Scholar). The chalcone isomerase–like (CHIL) protein family is noncatalytic, yet it shares a "CHI fold" with CHI (6Ralston L. Subramanian S. Matsuno M. Yu O. Partial reconstruction of flavonoid and isoflavonoid biosynthesis in yeast using soybean type I and type II chalcone isomerases.Plant Physiol. 2005; 137: 1375-1388Crossref PubMed Scopus (197) Google Scholar, 7Ngaki M.N. Louie G.V. Philippe R.N. Manning G. Pojer F. Bowman M.E. et al.Evolution of the chalcone-isomerase fold from fatty-acid binding to stereospecific catalysis.Nature. 2012; 485: 530-533Crossref PubMed Scopus (143) Google Scholar). Genetic knockouts of CHIL reduce flavanol production in Japanese morning glory (Ipomoea nil) and Arabidopsis thaliana (8Morita Y. Takagi K. Fukuchi-Mizutani M. Ishiguro K. Tanaka Y. Nitasaka E. et al.A chalcone isomerase-like protein enhances flavonoid production and flower pigmentation.Plant J. Cell Mol. Biol. 2014; 78: 294-304Crossref PubMed Scopus (87) Google Scholar, 9Jiang W. Yin Q. Wu R. Zheng G. Liu J. Dixon R.A. et al.Role of a chalcone isomerase-like protein in flavonoid biosynthesis in Arabidopsis thaliana.J. Exp. Bot. 2015; 22: 7165-7179Crossref Scopus (100) Google Scholar). Overexpression of CHIL partially rescues the loss-of-function phenotype in A. thaliana and enhances flavanol production in Dracaena cambodiana (9Jiang W. Yin Q. Wu R. Zheng G. Liu J. Dixon R.A. et al.Role of a chalcone isomerase-like protein in flavonoid biosynthesis in Arabidopsis thaliana.J. Exp. Bot. 2015; 22: 7165-7179Crossref Scopus (100) Google Scholar, 10Zhu J. Zhao W. Li R. Guo D. Li H. Wang Y. et al.Identification and characterization of chalcone isomerase genes involved in flavonoid production in Dracaena cambodiana.Front. Plant Sci. 2021; 12616396Google Scholar). CHIL-mediated flavanol production is upregulated via CHS binding, causing CHS to reduce production of a polyketide side product, p-coumaroyltriacetic acid lactone (CTAL) in favor of NC (11Waki T. Mameda R. Nakano T. Yamada S. Terashita M. Ito K. et al.A conserved strategy of chalcone isomerase-like protein to rectify promiscuous chalcone synthase specificity.Nat. Commun. 2020; 11: 870Crossref PubMed Scopus (50) Google Scholar, 12Ni R. Zhu T.T. Zhang X.S. Wang P.Y. Sun C.J. Qiao Y.N. et al.Identification and evolutionary analysis of chalcone isomerase-fold proteins in ferns.J. Exp. Bot. 2020; 71: 290-304Crossref PubMed Scopus (33) Google Scholar, 13Liu Y. Wu L. Deng Z. Yu Y. Two putative parallel pathways for naringenin biosynthesis in Epimedium wushanense.RSC Adv. 2021; 11: 13919-13927Crossref PubMed Google Scholar, 14Xu H. Lan Y. Xing J. Li Y. Liu L. Wang Y. AfCHIL, a type IV chalcone isomerase, enhances the biosynthesis of naringenin in metabolic engineering.Front. Plant Sci. 2022; 13891066Google Scholar). By rectifying the catalytic activity of CHS away from CTAL and toward NC, CHIL serves as a positive regulator of metabolic flux in the flavonoid pathway. Protein–enzyme binding between CHIL and CHS, as well as CHIL-induced CHS rectification, is conserved across diverse lineages of green plants (11Waki T. Mameda R. Nakano T. Yamada S. Terashita M. Ito K. et al.A conserved strategy of chalcone isomerase-like protein to rectify promiscuous chalcone synthase specificity.Nat. Commun. 2020; 11: 870Crossref PubMed Scopus (50) Google Scholar). In Humulus lupulus, CHIL attenuates the production of two prenylated chalconoids, xanthohumol and demethylxanthohumol, into flavanols through direct binding and stabilization of the open-ring form of the metabolites (15Ban Z. Qin H. Mitchell A.J. Liu B. Zhang F. Weng J.K. et al.Noncatalytic chalcone isomerase-fold proteins in Humulus lupulus are auxiliary components in prenylated flavonoid biosynthesis.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E5223-E5232Crossref PubMed Scopus (64) Google Scholar). CHIL is the subject of two recent reviews with perspectives that frame these discoveries in the context of the flavonoid pathway, metabolon research, and the idea that CHIL may act as a sensor to regulate flux (16Waki T. Takahashi S. Nakayama T. Managing enzyme promiscuity in plant specialized metabolism: a lesson from flavonoid biosynthesis: mission of a "body double" protein clarified.Bioessays. 2021; 43e2000164Crossref PubMed Scopus (6) Google Scholar, 17Dastmalchi M. Elusive partners: a review of the auxiliary proteins guiding metabolic flux in flavonoid biosynthesis.Plant J. Cell Mol. Biol. 2021; 108: 314-329Crossref PubMed Scopus (9) Google Scholar). Despite recent findings, it is yet unknown how CHIL proteins interact structurally with metabolites. Additional research is needed to reveal the structural bases underlying the recently described functions of CHIL proteins. Here, we present evidence of ligand selectivity in CHIL proteins and ligand-mediated regulation of CHIL–CHS complexes. Two CHILs are studied that represent distantly related species in the lineage of green plants: the lone CHIL from grape seed, Vitis vinifera (VvCHIL), and one of two known CHILs from the bryophyte, Physcomitrella patens (PpCHIL-A). Ligand-induced changes to protein Tms, or thermal shifts, reveal characteristics of CHIL binding to various metabolites possessing either chalcone, flavanone, or flavone-based scaffolds. Chalcones induce positive thermal shifts that are thermostabilizing for PpCHIL-A and VvCHIL. In addition, NC binding to CHIL increases the formation of CHIL–CHS complexes. Naringenin and quercetin decrease the thermostability of VvCHIL, and naringenin reduces protein–protein binding between VvCHIL and CHS. The effect of ligand-induced protein fold destabilization is similar to the studied example of Dwarf 14 (D14), a strigolactone receptor that undergoes a conformational change to facilitate signal transduction (18Hamiaux C. Drummond R.S. Janssen B.J. Ledger S.E. Cooney J.M. Newcomb R.D. et al.DAD2 is an α/β hydrolase likely to be involved in the perception of the plant branching hormone, strigolactone.Curr. Biol. 2012; 22: 2032-2036Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). These results suggest that CHILs may amplify or attenuate flux through the flavonoid pathway via metabolite-sensing mechanisms that regulate CHIL–CHS complexes. To examine the structural bases for ligand-binding differences, high-resolution protein X-ray crystal structures of VvCHIL and PpCHIL-A are presented. A comparison of these structures reveals key amino acid differences at a protein surface pocket that canonically serves as an active site in related CHIs and a ligand binding site in fatty acid–binding proteins (FAPs) and fungal heme-binding proteins (7Ngaki M.N. Louie G.V. Philippe R.N. Manning G. Pojer F. Bowman M.E. et al.Evolution of the chalcone-isomerase fold from fatty-acid binding to stereospecific catalysis.Nature. 2012; 485: 530-533Crossref PubMed Scopus (143) Google Scholar, 19Jez J.M. Bowman M.E. Dixon R.A. Noel J.P. Structure and mechanism of the evolutionarily unique plant enzyme chalcone isomerase.Nat. Struct. Biol. 2000; 7: 786-791Crossref PubMed Scopus (279) Google Scholar, 20Schmitz J.M. Wolters J.F. Murray N.H. Guerra R.M. Bingman C.A. Hittinger C.T. Pagliarini D.J. Aim18p and Aim46p are chalcone isomerase domain-containing mitochondrial hemoproteins in Saccharomyces cerevisiae.J Biol Chem. 2023; 299: 102981Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar). Site-directed mutagenesis is used to exchange a key amino acid between VvCHIL and PpCHIL-A, resulting in a variant of VvCHIL that is no longer destabilized by naringenin binding, as well as a variant of PpCHIL-A possessing enhanced naringenin binding. Interestingly, mutations to the canonical active-site/ligand-binding pocket have very little effect on NC binding for VvCHIL, suggesting distinct binding sites for NC and naringenin. Together, these findings reveal how divergent structural attributes of CHIL proteins direct ligand selectivity and protein conformational stability to affect CHIL–CHS interactions. The ability of CHILs to act as receptors that sense and respond to downstream metabolite concentrations reveals a logic that is universal to committed steps of metabolic pathways. Differential scanning fluorimetry (DSF) is a sensitive and label-free method for measuring the Tm of a protein, defined as the temperature at which 50% of a protein sample is unfolded (21Niesen F.H. Berglund H. Vedadi M. The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability.Nat. Protoc. 2007; 2: 2212-2221Crossref PubMed Scopus (1735) Google Scholar). Positive Tm changes have been used to measure binding between a CHIL protein and chalcones (15Ban Z. Qin H. Mitchell A.J. Liu B. Zhang F. Weng J.K. et al.Noncatalytic chalcone isomerase-fold proteins in Humulus lupulus are auxiliary components in prenylated flavonoid biosynthesis.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E5223-E5232Crossref PubMed Scopus (64) Google Scholar). To elucidate a broader scope of CHIL–chalcone interactions, DSF was used to determine the thermal shifts that occur to VvCHIL and PpCHIL-A upon binding naturally occurring chalcones containing different substitution patterns (Fig. 1). Titration of NC into PpCHIL-A produces a positive thermal shift, indicating a protein fold–stabilizing effect with a maximum thermal shift (ΔTmmax) of 1.7 ± 0.2 °C. A single-site binding equation was used to fit the apparent equilibrium binding dissociation constant (apparent Kd) of 58 ± 16 μM (Fig 2A). Because the apparent Kd values are derived by DSF, they do not account for the temperature dependence of the binding constant (22Vivoli M. Novak H.R. Littlechild J.A. Harmer N.J. Determination of protein-ligand interactions using differential scanning fluorimetry.JoVE. 2014; 9151809Google Scholar). When the experiment is conducted using VvCHIL, NC binds with an apparent Kd of 24 ± 5 μM, producing a ΔTmmax of 3.5 ± 0.2 °C (Fig. 2B). The apparent Kd values for NC binding to VvCHIL and PpCHIL-A are similar to the reported Kd of 43 μM and ΔTmmax of 6.4 °C for HlCHIL1 from H. lupulus, measured by DSF (15Ban Z. Qin H. Mitchell A.J. Liu B. Zhang F. Weng J.K. et al.Noncatalytic chalcone isomerase-fold proteins in Humulus lupulus are auxiliary components in prenylated flavonoid biosynthesis.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E5223-E5232Crossref PubMed Scopus (64) Google Scholar). HlCHIL1 (A0A2U7XUH7) is a relatively distant ortholog of VvCHIL and PpCHIL-A and is more closely related to the family of FAPs, perhaps possessing functions different from other CHILs (15Ban Z. Qin H. Mitchell A.J. Liu B. Zhang F. Weng J.K. et al.Noncatalytic chalcone isomerase-fold proteins in Humulus lupulus are auxiliary components in prenylated flavonoid biosynthesis.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E5223-E5232Crossref PubMed Scopus (64) Google Scholar) (Fig. S1). To determine which functional groups of NC are directly involved in binding to CHIL proteins, other naturally occurring chalcones were tested. The trihydroxy-substituted chalcones, 2ʹ,4,4ʹ-trihydroxychalcone (isoliquiritigenin) and 2,2ʹ,4ʹ-trihydroxychalcone, both possess a dihydroxy-substituted A ring, which differs from the trihydroxy-substituted A ring of NC (Fig. 1). In addition, 2,2ʹ,4ʹ-trihydroxychalcone has a hydroxyl at C2 on the B ring instead of at C4, as in NC, naringenin, and 2ʹ,4,4ʹ-trihydroxychalcone (Fig. 1). The results of the DSF experiments show that 2ʹ,4,4ʹ-trihydroxychalcone binds PpCHIL-A with an apparent Kd of 41 ± 10 μM and ΔTmmax of 2.1 ± 0.2 °C and VvCHIL with an apparent Kd of 127 ± 51 μM, with a ΔTmmax of 3.8 ± 0.1 °C (Fig. 2, C and D). The ligand, 2,2ʹ,4ʹ-trihydroxychalcone, binds PpCHIL-A with an apparent Kd of 85 ± 23 μM and ΔTmmax of 2.7 ± 0.2 °C and VvCHIL with an apparent Kd of 286 ± 23 μM and ΔTmmax of 3.3 ± 0.7 °C (Fig. 2, E and F). The most significant outcome of this comparative set of experiments is that VvCHIL is selective for NC binding relative to other chalcones, whereas PpCHIL-A is not. This is supported by the finding that VvCHIL has approximately 5-fold and 11-fold smaller apparent Kd values for NC binding relative to 2ʹ,4,4ʹ-trihydroxychalcone and 2,2ʹ,4ʹ-trihydroxychalcone, respectively. These differences reveal components of a preferred binding epitope on chalcone ligands, which includes the 6ʹOH on the A ring and a hydroxyl at the C4 versus C2 position on the B ring. PpCHIL-A has lower selectivity for different chalcones such that it does not bind with an appreciable difference to NC or 2ʹ,4,4ʹ-trihydroxychalcone and has only a twofold greater apparent Kd for 2,2ʹ,4ʹ-trihydroxychalcone. Together, these results suggest that VvCHIL and PpCHIL-A may possess different structural elements for binding to chalcones and other related metabolites. Naringenin is the product of NC cyclization by CHI and possesses a flavanone scaffold that is conformationally restricted relative to chalcones (Fig. 1). The protein X-ray crystal structure of naringenin bound to type II CHI from Medicago sativa (MsCHI-II) reveals the location and orientation through which naringenin binds to the enzyme active site (19Jez J.M. Bowman M.E. Dixon R.A. Noel J.P. Structure and mechanism of the evolutionarily unique plant enzyme chalcone isomerase.Nat. Struct. Biol. 2000; 7: 786-791Crossref PubMed Scopus (279) Google Scholar). When MsCHI-II is heated in the presence of increasing concentrations of naringenin, the increase in Tm produces a ΔTmmax of 2.9 ± 0.2 °C and an apparent Kd of 1019 ± 115 μM (Fig. 3A). Ligand concentrations used are reported in Table S1. For PpCHIL-A, protein thermostability also increases with increasing naringenin, producing a ΔTmmax of 3.6 ± 0.3 °C and an apparent Kd of 762 ± 39 μM (Fig. 3B). These experiments reveal that PpCHIL-A is similar to MsCHI-II in the thermal shift produced by naringenin, suggesting that PpCHIL-A and MsCHI-II may have similar modes of binding to naringenin. For VvCHIL, naringenin binding produces the opposite effect on the thermal shift relative to PpCHIL-A and MsCHI-II, resulting in reduced protein Tms at high naringenin concentrations (Fig. 3C). The loss of DSF signal at high naringenin makes the VvCHIL data unsuitable for quantitative analysis of ligand binding because the condition of saturation of ligand binding cannot be determined. The first derivative of the change in fluorescence of SYPRO orange dye was used to calculate the Tm under various conditions. From these data, it is apparent that the negative thermal shift of VvCHIL in the presence of high naringenin is different from the positive and dose-dependent thermal shifts of naringenin with MsCHI-II or PpCHIL-A (Fig. 3, D–F). Notably, the negative thermal shift of VvCHIL is comparatively switch like, capturing two distinct states (Fig. 3F). This suggests that a less-stable conformational ground state of the VvCHIL protein fold is favored upon naringenin binding. Quercetin is relatively abundant in shoots and leaves of V. vinifera when compared with other flavonoids, such as naringenin (23Goufo P. Singh R.K. Cortez I. A reference list of phenolic compounds (including stilbenes) in grapevine (Vitis vinifera L.) roots, woods, canes, stems, and leaves.Antioxidants (Basel, Switzerland). 2020; 9: 398PubMed Google Scholar, 24Rätsep R. Karp K. Maante-Kuljus M. Aluvee A. Kaldmäe H. Bhat R. Recovery of polyphenols from vineyard pruning wastes-shoots and cane of hybrid grapevine (vitis sp.) cultivars.Antioxidants (Basel, Switzerland). 2021; 10: 1059PubMed Google Scholar). In the flavonoid pathway, quercetin is made three steps downstream of naringenin. Quercetin differs from naringenin by additional hydroxyl groups on the B and C rings and an α,β-unsaturated carbonyl within the C ring (Fig. 1). Because of the relative abundance of quercetin in plants, we sought to determine whether quercetin also causes a fold-destabilizing effect when binding VvCHIL. Titrating quercetin into VvCHIL induces a negative thermal shift to the protein above 750 μM (Fig. 3G). Unfortunately, the DSF signal for the PpCHIL-A protein is very low relative to other proteins studied (Fig. 3E), and the intrinsic fluorescence of quercetin obstructs the weak fluorescence signal from the SYPRO orange dye in the DSF assay for this protein. Therefore, the effects of quercetin on PpCHIL-A by DSF were not measured. The intrinsic fluorescence of quercetin has been used to measure protein–ligand binding directly (25Gutzeit H.O. Henker Y. Kind B. Franz A. Specific interactions of quercetin and other flavonoids with target proteins are revealed by elicited fluorescence.Biochem. Biophys. Res. Commun. 2004; 318: 490-495Crossref PubMed Scopus (65) Google Scholar). Naringenin does not have similar fluorescent properties that would make it suitable for this assay (25Gutzeit H.O. Henker Y. Kind B. Franz A. Specific interactions of quercetin and other flavonoids with target proteins are revealed by elicited fluorescence.Biochem. Biophys. Res. Commun. 2004; 318: 490-495Crossref PubMed Scopus (65) Google Scholar). Binding-induced fluorescence enhancements of quercetin in the presence of increasing concentrations of CHIL were measured to determine Kd values of 347 ± 35 μM for VvCHIL and 240 ± 121 μM for PpCHIL-A (Fig. 3, H and I). The destabilization effect of quercetin on VvCHIL, as measured by DSF, is observed for quercetin concentrations between 750 μM and 1 mM (Fig. 3G). It is notable that the reduction in thermostability correlates closely with quercetin concentrations relevant for binding to VvCHIL, as measured by fluorescence (Fig. 3H); for example, based on the Kd value for VvCHIL, the fraction of VvCHIL bound to 750 μM quercetin is 0.68. Together, these measurements verify that binding occurs between quercetin and the CHIL proteins and reveal that quercetin destabilizes VvCHIL; however, the negative thermal shift to VvCHIL caused by quercetin is relatively small when compared with the negative thermal shift caused by naringenin (Fig. 3, C and G). From these findings, it is not straightforward to say whether quercetin binds to CHILs better or worse than chalcones. Because of differences in the binding assays needed for the different ligands, the equilibrium binding constants measured (i.e., apparent Kd versus Kd) do not have a known quantitative relationship. Functional CHS–CHIL binding interactions are conserved throughout green plants and can be constituted using CHS and CHIL proteins from nondistant species (11Waki T. Mameda R. Nakano T. Yamada S. Terashita M. Ito K. et al.A conserved strategy of chalcone isomerase-like protein to rectify promiscuous chalcone synthase specificity.Nat. Commun. 2020; 11: 870Crossref PubMed Scopus (50) Google Scholar). A protein pull-down method was devised using strep-tagged CHS; however, in our experiment, strep-tagged VvCHS failed to bind to the StrepTactin beads, so CHS from A. thaliana (strep-AtCHS) was used to capture untagged VvCHIL. Results of a binary protein-binding experiment show that strep-AtCHS binds to StrepTactin resin, whereas untagged VvCHIL does not (Fig. 4A). When strep-AtCHS and untagged VvCHIL are mixed together in the presence of StrepTactin resin, VvCHIL is observed in the bound fraction because of a direct binding interaction with strep-AtCHS (Fig. 4B). When the pull-down assay is conducted in the presence of increasing naringenin concentrations, VvCHIL–AtCHS binding is increasingly disrupted (Fig. 4B). Native PAGE shows that VvCHIL remains folded in the presence of all ligand concentrations tested, suggesting that the disruption to AtCHS–VvCHIL binding interaction may be due to a conformational change in the VvCHIL protein, rather than unfolding of the protein (Fig. 4B). In contrast to the effect seen with naringenin, the presence of NC enhances binding between strep-AtCHS and VvCHIL (Fig. 4C). The results of the protein pull-down experiments were quantified using densitometry (Figs. S2 and S3). It is notable that the band intensity of strep-AtCHS in the bound fraction decreases with increasing naringenin (Fig. 4B) and increasing NC (Fig. 4C). The effect of reduced strep-AtCHS binding to the StrepTactin resin in the presence of hydrophobic metabolites may be due to inhibited binding of the hydrophobic HPQ motif on the strep-tag to streptavidin-linked beads. Because the amount of untagged VvCHIL in the bound fraction is dependent upon the amount of strep-AtCHS in the bound fraction, bound VvCHIL was normalized. By taking the ratio of band intensities for VvCHIL to AtCHS for each of the StrepTactin-bound fractions on the SDS-PAGE gels, quantification shows that NC increases binding between VvCHIL and AtCHS, whereas naringenin has the opposite effect at 1.2 and 4.3 mM concentrations, decreasing the amount of VvCHIL that AtCHS can bind and pull down (Fig. 4D). Bound VvCHIL is present in bound fractions of non-native SDS-PAGE and native PAGE gels and in similar ratios to strep-AtCHS (Figs. 4B and S4). This analysis shows that VvCHIL protein in streptavidin-bound fractions is not significantly aggregated. When the experiment is conducted using a strep-tagged CHS from P. patens (strep-PpCHS) to capture untagged PpCHIL-A, PpCHIL-A is observed in the bound fractions for conditions without ligand present and with naringenin present (Fig. 4, E and F). When NC is present, bound PpCHIL-A is strongly enriched in the strep-PpCHS–bound fraction relative to the other conditions tested (Fig. 4, E and F). Together, these findings suggest a role for CHIL proteins as receptors with the capacity to upregulate the formation of CHIL–CHS complexes in the presence of NC, or alternatively in the case of VvCHIL, disrupt existing CHIL–CHS complexes in the presence of high naringenin. The significance of this is underscored by the work showing that CHIL proteins rectify CHS catalysis, thus affecting flux through the flavonoid pathway (11Waki T. Mameda R. Nakano T. Yamada S. Terashita M. Ito K. et al.A conserved strategy of chalcone isomerase-like protein to rectify promiscuous chalcone synthase specificity.Nat. Commun. 2020; 11: 870Crossref PubMed Scopus (50) Google Scholar). These results suggest a model in which CHIL proteins can act as metabolic sensors that provide feedback to CHIL-assisted CHS catalysis at the rate-determining step of the flavonoid pathway. On native PAGE gels, VvCHIL displays two high–molecular weight bands indicative of oligomeric states and a single low–molecular weight band indicative of a monomer (Fig. 4A). Similar high–molecular weight bands are not observed on native PAGE gels for PpCHIL-A or MsCHI-II (Fig. 5A). Size-exclusion chromatography (SEC) of VvCHIL and PpCHIL-A reveals that VvCHIL elutes as two peaks and PpCHIL-A elutes as a single peak (Fig. 5B). When compared with a set of standard reference proteins, the single peak for PpCHIL-A elutes at a volume that calculates to a molecular weight of 36 kDa. The VvCHIL peaks calculate to 26 kDa (major peak) and 68 kDa (minor peak), indicating that a minor amount of VvCHIL is stable as a multimer, perhaps as a trimer in this experiment. For comparison, molecular weights of the monomeric forms of these proteins, calculated from their amino acid sequences, are 23 kDa for PpCHIL-A and 22 kDa for VvCHIL. Differences in SEC-based experimental molecular weight values relative to reference molecular weight values can be due to differences in the hydrodynamic radii of the protein molecules as they elute through a SEC column. To enable a meaningful structural comparison of CHIL proteins, purified proteins were crystallized (Table 1). The protein X-ray crystal structures of PpCHIL-A and VvCHIL reveal CHI-type α/β folds with high structural homology to each other. The RMS value between the α-carbons within the PpCHIL-A and VvCHIL structures, a measure of structural identity, is 0.831 Å.Table 1X-ray crystallography data collection and refinement statisticsCrystal (PDB ID)PpCHIL-A (8DLD)VvCHIL (8DLC)Data collection Space groupC121P3221 Cell dimensionsa, b, c (Å)62.55, 49.88, 76.8249.4199, 49.4199, 170.851α, β, γ (°)90, 110.87, 9090, 90, 120 Resolution (Å)37.94–1.8542.80–1.90 Rmerge0.102 (0.667)0.190 (1.6717) Rpim0.049 (0.326)0.050 (0.439) CC ½0.994 (0.812)0.998 (0.815) I/σI8.1 (2.1)10.7 (2.2) Completeness (%)97.7100.0 Multiplicity5.315.2Refinement Resolution (Å)37.3–1.85 (1.916–1.85)42.8–1.9 (1.968–1.9) No. of unique reflections18,703 (1837)19,936 (1887) Rwork/Rfree0.1744 (0.3002)/0.2203 (0.3598)0.1895 (0.4350)/0.2325 (0.4552) No. of atoms17941807Protein16351665Water159142 B-factors (Å2)Average37.0330.14Protein36.5429.45Water42.9138.13 Rms deviationsBond lengths (Å)0.0110.011Bond angles (°)1.091.11 Ramachandran plot (%)Favored99.5198.05Allowed0.491.95Outliers00 PDB accession codes8DLD8DLCParentheses are for the highest resolution shell. Open table in a new tab Parentheses are for the highest resolution shell. To date, there is no available structural evidence of CHIL proteins bound to ligands. Currently, the only other structure of a CHIL protein is from A. thaliana (AtCHIL) (7Ngaki M.N. Louie G.V. Philippe R.N. Manning G. Pojer F. Bowman M.E. et al.Evolution of the chalcone-isomerase fold from fatty-acid binding to stereospecific catalysis.Nature. 2012; 485: 530-533Crossref PubMed Scopus (1