Structural analyses of gibberellin-mediated DELLA protein degradation

Structural analyses of gibberellin-mediated DELLA protein degradation | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Structural analyses of gibberellin-mediated DELLA protein degradation Soyaab Islam , KunWoong Park , Eunju Kwon , Dong Young Kim doi: https://doi.org/10.1101/2025.02.08.637281 Soyaab Islam 1 College of Pharmacy, Yeungnam University , Gyeongsan, 38541 Republic of Korea Find this author on Google Scholar Find this author on PubMed Search for this author on this site KunWoong Park 1 College of Pharmacy, Yeungnam University , Gyeongsan, 38541 Republic of Korea 2 Structural Biology Division, Baobab AiBIO Co. Ltd. , Incheon, 21983 Republic of Korea Find this author on Google Scholar Find this author on PubMed Search for this author on this site Eunju Kwon 3 Division of Life Science, Gyeongsang National University , Jinju, 52828 Republic of Korea 4 Research Institute of Molecular Alchemy, Gyeongsang National University , Jinju, 52828 Republic of Korea Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: eunjukwon{at}gnu.ac.kr dyokim{at}ynu.ac.kr Dong Young Kim 1 College of Pharmacy, Yeungnam University , Gyeongsan, 38541 Republic of Korea Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: eunjukwon{at}gnu.ac.kr dyokim{at}ynu.ac.kr Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT Gibberellin promotes plant growth by downregulating growth-repressor DELLA proteins. The gibberellin receptor GID1 binds to DELLA proteins in the presence of gibberellin, triggering their degradation through polyubiquitination by SCF SLY 1 /GID 2 ubiquitin E3 ligase. Despite extensive studies, the molecular mechanisms by which DELLA proteins assemble with SCF SLY 1 /GID 2 to regulate plant growth remain poorly understood. Here, we present two cryo-electron microscopy structures of the Arabidopsis thaliana DELLA protein RGA in complex with GID1A and GID1A-SLY1-ASK2, respectively. Structural analysis revealed that RGA interacts with GID1A and SLY1 through non-overlapping binding surfaces, stabilizing the proteins. This suggests that the SCF SLY 1 -RGA-GID1A complex assembles through stepwise stabilization induced by gibberellin. Furthermore, the structures indicate that RGA does not interact with IDD family transcription factors when bound to SLY1, suggesting that the binding of DELLA proteins to GID1/SLY1 and transcription factors is mutually exclusive. These findings provide insights into how DELLA proteins regulate transcription factor activity in response to gibberellin. INTRODUCTION Gibberellin (GA) is a plant hormone that regulates various aspects of plant growth and development, including seed germination, stem elongation, flowering, and fertility. This hormone belongs to a group of structurally related diterpenoid acids, with a total of 136 natural compounds being identified as GAs in bacteria, fungi, and plants 1 . Among these, four GAs (GA 1 , GA 3 , GA 4 , and GA 7 ) are considered bioactive forms 2 , which are perceived by the GA INSENSITIVE DWARF1 (GID1) 3 – 5 . GID1 is a soluble GA receptor composed of an N-terminal lid and a catalytically inactive hydrolase domain (IHD) 6 , 7 . The catalytically nonfunctional active site of the IHD forms a GA binding pocket, and the N-terminal lid closes the pocket through a disorder-to-order conformational change upon GA binding 6 , 7 . GA-bound GID1 interacts with DELLA proteins (named after the Asp-Glu-Leu-Leu-Ala sequence motif) to promote plant growth. The DELLA proteins serve as coactivators or corepressors for various gene regulatory proteins that govern plant growth and development 8 – 11 . Several transcription factors featuring a basic helix-loop-helix (bHLH) DNA binding motif, such as PHYTOCHROME INTERACTING FACTORs (PIFs) and BRASSINAZOLE RESISTANT 1 (BZR1), bind to DELLA proteins and become inactivated 12 – 16 . Conversely, some INDETERMINATE DOMAIN (IDD) and basic leucine-zipper (bZIP) family proteins are activated upon interaction with DELLA proteins 17 – 21 . Physiologically, DELLA proteins act as repressors of plant growth, a function regulated by GA 8 , 9 , 22 . A depletion of GA, resulting from mutations in GA biosynthesis, leads to failed germination, extreme dwarfism, and unstable flowering development. The phenotypes associated with GA depletion can be rescued by an additional deletion mutation of the DELLA genes, indicating that the primary role of GA is to suppress the activity of DELLA proteins 8 , 9 , 22 . In this context, DELLA mutants with reduced GA sensitivity enable a “green revolution” wherein plants exhibit dwarfism while grain yields increase 23 . DELLA proteins consist of an N-terminal regulatory domain (RD) and a C-terminal GRAS domain (named after the proteins GAI, RGA, and SCARECROW) 8 , 9 . The RD is responsible for the GA-dependent binding of DELLA proteins to GID1. The RD directly binds to the closed lid of GID1 upon GA binding through the DELLA, LExLE, and VHYNP motifs 6 . Consequently, the deletion of these motifs renders plant growth insensitive to GA 10 , 24 . The GRAS domain plays a crucial role in the transcriptional regulation of DELLA proteins; mutations in this domain result in the loss of DELLA protein activity, leading to a tall and slender growth phenotype 9 , 22 , 25 . While monocots such as rice and barley possess a single DELLA gene 9 , 25 , the model dicot Arabidopsis thaliana contains five DELLA genes: REPRESSOR OF GA1-3 ( RGA) , GA-INSENSITIVE ( GAI ), RGA-LIKE1 ( RGL1 ), RGL2 , and RGL3 26 . Although these DELLA genes exhibit both distinct and redundant roles in regulating plant growth and development 27 – 29 , DELLA proteins are physiologically complementary to one another 14 , suggesting that the functional diversification of DELLA genes may result from their spatial and temporal expression patterns. DELLA proteins are downregulated through polyubiquitination mediated by SLEEPY 1 (SLY1) in Arabidopsis and GID2 in rice. SLY1/GID2 acts as a substrate adaptor that recruits the GID1-GA-DELLA complex to the SCF (an acronym for SKP, CULLIN, F-BOX) ubiquitin E3 ligase for polyubiquitination and subsequent proteasomal degradation of DELLA proteins. Mutations in the SLY1 / GID2 gene block GA-induced proteolysis of DELLA proteins, resulting in a GA-insensitive phenotype 30 – 32 . This underscores the critical role of SCF SLY 1 /GID 2 -mediated degradation of DELLA proteins as a key regulatory pathway in GA-induced plant growth. The physiological functions of DELLA proteins have been extensively studied. However, the molecular mechanisms by which GA regulates the interactions of DELLA proteins with SCF SLY 1 /GID 2 and transcription factors remain poorly understood. This manuscript presents the cryogenic electron microscopy (cryo-EM) structures of the GID1A-GA 3 -RGA and GID1A-GA 3 -RGA-SLY1-ASK2 complexes, which were determined at resolutions of 2.66 Å and 2.80 Å, respectively. These structures provide insights into the mechanisms by which GA suppresses the activity of DELLA proteins and by which SCF SLY 1 /GID 2 mediates their degradation. RESULTS Overall structure of the GID1A-RGA complex To elucidate the interaction between GID1 and DELLA proteins, we purified the GID1A-RGA complex and determined its cryo-EM structure. The RGA protein, when expressed in Escherichia coli , was largely insoluble, with only a small soluble fraction that was unstable and prone to forming aggregates ( Fig. S1A–S1C ). GID1A was also mostly insoluble when expressed in E. coli in the presence of GA 3 , but small soluble fractions were eluted in a homogeneous form through size-exclusion chromatography (SEC) ( Fig. S1D–S1F ). The solubility and homogeneity of RGA and GID1A significantly improved when they were coexpressed in the presence of GA 3 . The GID1A-RGA complex, expressed in the presence of GA 3 , was purified via immobilized metal affinity chromatography (IMAC) and SEC (Fig S1G–S1I) . The SEC-multiangle light scattering (MALS) experiment demonstrated that the purified GID1A-RGA forms a homogeneous heterodimer ( Fig. 1A ). The cryo-EM map of the GID1A-RGA complex was reconstructed at an overall resolution of 2.66 Å, using 251,864 particles selected from the cryo-EM images of the purified GID1A-RGA ( Figs. 1B–D , S2, and S3 ). The atomic model was built by tracing the cryo-EM map and refining it through iterative model correction and structural refinement. The correlation coefficient between the final structure and map (CC mask ) was 0.84, and no outliers were found in the Ramachandran plot. The final structure comprised one GID1A monomer, one RGA monomer, one GA 3 molecule, and 248 water molecules ( Fig. S3D and Table S1 ). Download figure Open in new tab FIGURE 1. Cryo-EM structure of the RGA-GID1A complex. (A) SEC-MALS analysis of purified RGA-GID1A. The absorbance at 280 nm is shown in blue, and the experimental molar masses of the eluate are represented in red. The dotted line indicates the calculated molecular weight of the RGA-GID1A heterodimer (102.6 kDa). (B) Representative two-dimensional classes of RGA-GID1A particles. (C, D) Cryo-EM map of RGA-GID1A shown in two different orientations. GID1A, RGA RD , and RGA GRAS are colored pink, yellow, and cyan, respectively. (E, F) GA 3 -bound GID1A within the cryo-EM structure of RGA-GID1A. The central β-sheet is highlighted in (F). The secondary structures are denoted as h1–h10 for the helices and s1–s8 for the strands. GA 3 is depicted as a green stick model. (G, H) RGA in the cryo-EM structure of RGA-GID1A. The core β-sheet is shown in (H). The secondary structure is denoted as H1–H19 for helices and S1–S9 for strands. The helices in (E) and (G) are color-coded as in the cryo-EM maps shown in (C) and (D). In the cryo-EM structure of the GID1A-RGA complex, GID1A consists of an N-terminal lid (GID1A Lid ; residues 10–50) and an IHD (GID1A IHD ; residues 61–343) ( Fig. 1E ). GID1A IHD exhibited an α/β fold with a parallel β-sheet (topological order: s1, s2, s4, s3, s5, s6, s7, and s8) and peripheral helices (h3–h10) ( Figs. 1E , 1F , and S4A ). GID1A Lid contains two helices (h1 and h2) that cover the GA 3 molecule bound to GID1A IHD , completely enclosing GA 3 within the space between GID1A IHD and GID1A Lid . The overall fold of GID1A is nearly identical to that observed in the crystal structure of the GID1A-GAI RD complex (PDB ID: 2ZSH) 6 ( Fig. S4B ), with an RMSD value of 0.6 Å for 334 Cα atoms. RGA comprises an N-terminal RD (RGA RD ; residues 42–108) and a C-terminal GRAS domain (RGA GRAS ; residues 205–584) ( Fig. 1G ). The segment (residues 109–204) linking RGA RD and RGA GRAS was not visible in the cryo-EM map. The flexible linker among DELLA proteins was highly variable in both length and sequence ( Fig. S5 ), suggesting that this linker may play a role in the specific functions of each DELLA protein. RGA RD consists of four helices (H1–H4) and forms a concave groove that facilitates GID1A binding ( Fig. 1F ). The RGA RD region in the cryo-EM structure did not exhibit significant conformational differences when compared to GAI RD in the crystal structure of the GAI RD -GID1A complex (PDB ID: 2ZSH), with an RMSD value between RGA RD and GAI RD of 1.3 Å for 60 Cα atoms ( Fig. S6A ). RGA GRAS displayed an α/β fold with a twisted core β-sheet and peripheral helices (H8–H14, H18, and H19) ( Fig. 1G ). The core β-sheet consisted of eight β-strands arranged topologically in the order S3, S2, S1, S4, S5, S9, S8, and S7 ( Fig. 1H ). Strand S9 is longer than the other strands and forms additional hydrogen bonds with strand S6, aligning in the same direction as strand S8 and extending beyond the core β-sheet. In addition to the α/β fold, RGA GRAS features a helical bundle composed of helices H5–H8, H15, and H16. Overall, RGA GRAS exhibited a rod-shaped structure that included both the α/β fold and the helical bundle. A structural homology search via the DALI server 33 indicated that RGA GRAS shares a fold with GRAS family proteins. Notably, Arabidopsis SCARECROW-LIKE 3 (SCL3; PDB ID: 6KPD) was superimposed onto RGA GRAS , yielding the lowest RMSD value of 1.7 Å for 353 Cα atoms ( Fig. S6B ). Some GRAS domains form homodimers or heterodimers through their helical bundles, corresponding to the LHRI region 34 , 35 . It has been suggested that RGA also forms a homodimer through its LHRI region (helices H5–H7) 15 . However, RGA GRAS in the cryo-EM structure of GID1A-RGA did not form a homodimer despite the solvent-exposed surface of the LHRI region ( Fig. S6C and S6D ). Consistent with this observation, larger particles containing RGA homodimers were not detected in the two-dimensional (2D) classification of the cryo-EM particles ( Fig. S2 ). The surfaces of GID1A and RGA RD at their binding interface are partially positively and negatively charged, respectively. Conversely, those of GID1A and RGA GRAS at their binding interface are relatively hydrophobic ( Fig. S6E and S6F ). The binding of GA 3 in the GID1A pocket GID1A IHD possesses a GA-binding pocket formed by the helices h3 and h8, the preceding loops (l2 and l3), and the loop l4 located between strand s8 and helix h10 ( Fig. 2A and S4B ). GA 3 directly forms hydrogen bonds with residues S116, Y127, and F238 of GID1A IHD , and hydrophobic interaction with Y247 through its methyl group within the pocket ( Fig. 2A ). The GA-binding pocket was slightly larger than GA 3 , allowing water molecules to occupy the space and mediate interactions between GA 3 and the pocket ( Fig. 2A ). In the crystal structure of the GAI RD -GID1A complex 6 , four water molecules bridged the interactions between GID1A and GA 3 . In the cryo-EM structure of the RGA-GID1A complex, three water molecules directly mediate the interactions between GID1A and GA 3, similar to the interactions observed in the crystal structure of GAI RD -GID1A ( Fig. 2A ). The GA-binding pocket was directly covered by helix h1 of GID1A Lid ( Fig. 2B ). GA 3 bound F27 in helix h1 of GID1A Lid through hydrophobic interaction. As with the GA 3 -binding pocket, water molecules also mediate additional interactions between GA 3 and GID1A Lid ( Fig. 2B and 2C ). Download figure Open in new tab FIGURE 2. The GA 3 -binding pocket of GID1A. (A) GA 3 in the binding pocket. (B) GA 3 covered by GID1A Lid . (A) and (B) are views from opposite directions centered on GA 3 . GA 3 is shown as a yellow stick model, and GID1A is represented as a pink surface model. Residues interacting with GA 3 are depicted as blue stick models and surface models. Water molecules are shown as red spheres, and the hydrogen bonding network around GA 3 is indicated by dotted lines. The yellow surface indicates the RGA. (C) Interactions between GID1A and GA 3 . The GID1A residues involved in the hydrogen bonding network and hydrophobic interactions are highlighted in blue and green, respectively. Interactions between GID1A and RGA The RGA interacted with GA 3 -bound GID1A over an extensive binding interface that encompassed both RGA RD and RGA GRAS ( Fig. 3A ). The RGA surface area was buried by 11.4% within the GID1A-binding interface (2,498 Å 2 in 21,833 Å 2 ), resulting in a reduction in ΔG by −33.2 kcal/mol. Among the four helices of RGA RD , helix H1, which contains the DELLA motif, contacts the surface around helix h3 of GID1A mainly through hydrophobic interactions. Additionally, a water molecule bridged interactions between RGA RD -L46 and GID1A-R133/L323 through hydrogen bonding ( Fig. 3B ). Helix H2 of RGA RD contacted the N-terminal loop and helix h1 of GID1A, which encapsulates GA 3 . RGA RD -E67 formed ionic bonds with GID1A-R13, and RGA RD -E70 formed hydrogen bonds with GID1A-K28, which is the next residue of F27 that directly contacts GA 3 ( Fig. 3C ). Residues participating in hydrophobic interactions were clustered near the ionic and hydrogen bonds. Helix H3 of RGA RD contacted the C-terminus of helix h1. The loop L1 between helices H3 and H4 formed a slight contact with helix h2 ( Fig. 3D ). Helix H4, which contains the VHYNP motif, contacts helix h1 of GID1A through hydrophobic interactions. These observations indicate that RGA RD is responsible for GID1A Lid binding. The surface of RGA RD is buried by 26.9% within the binding interface (1,310 Å 2 in 4,869 Å 2 ), contributing to a ΔG reduction of −22.8 kcal/mol. Download figure Open in new tab FIGURE 3. Interactions between GID1A and RGA. (A) The binding interface between GID1A and RGA. RGA is shown as a low-transparency surface model. RGA and GID1A at the binding interface are depicted as cartoon models. Residues of GID1A, RGA RD , and RGA GRAS , involved in interactions at the binding interface, are highlighted in purple, yellow, and cyan, respectively. Water molecules are shown as red spheres. (B–G) Detailed interactions between GID1A and RGA. The GID1A, RGA RD , and RGA GRAS models are presented in pink, yellow, and cyan, respectively. The blue and red dotted lines indicate hydrogen bonds and ionic interactions, respectively. (B, C) Interactions between GID1A and RGA RD . (D) Interactions of GID1A with RGA RD and RGA GRAS at the junctional area. (E–G) Interactions between GID1A and RGA GRAS . RGA GRAS interacts with GID1A through multiple binding motifs, forming a large binding interface ( Fig. 3A ). First, helix H19 and strand S7 of RGA GRAS contacted residues surrounding helix h2 of GID1A ( Fig. 3D and 3E ). RGA GRAS -E560 formed an ionic bond with GID1A-R51, and L535 and K541 of RGA GRAS established hydrogen bonds with GID1A-Y48 and GID1A-H44, respectively. Notably, GID1A-Y48 interacted with both RGA GRAS and RGA RD ( Fig. 3D ). Second, loop L2 (residues 205–211) of RGA GRAS , which serves as a linker connecting RGA RD and RGA GRAS , contacts the surface loops and strand s1 of GID1A. The residues S205, T206, R207, V209, and L211 in loop L2 of RGA GRAS formed hydrogen bonds with the GID1A residues N58, N56, N56/A57/N58/P92, D67, and L69, respectively ( Fig. 3F ). Third, helix H15 of RGA GRAS contacted the loop between strands s1 and s2. RGA GRAS -D478 and E481 formed ionic bonds with GID1A-R72 ( Fig. 3G ). Additionally, several hydrophobic interactions further contributed to the interaction between RGA GRAS and GID1A. These hydrophobic interactions were predominantly found around hydrogen and ionic bonds, and helix H5 also bound to GID1A-L95 through hydrophobic interactions. The RGA GRAS surface was buried by 7.2% within the binding interface (1,258 Å 2 in 17,497 Å 2 ), with a ΔG reduction of −10.7 kcal/mol. Furthermore, seven water molecules bridged the interactions between RGA GRAS and GID1A ( Fig. 3B , 3D–F ). Despite the extensive binding area of RGA GRAS , GID1A, when coexpressed with RGA GRAS in the presence of GA 3 , precipitated during purification under high-salt buffer conditions ( Fig. S7A–I ). This observation suggests that RGA RD is a prerequisite for the interactions and stability between RGA GRAS and GID1A. Overall structure of the GID1A-RGA-SLY1-ASK2 complex DELLA proteins are polyubiquitinated by SCF SLY 1 /GID 2 ubiquitin E3 ligase in the presence of GA, leading to their proteasomal degradation 30 – 32 . To elucidate the mechanisms by which the DELLA protein binds to SCF SLY 1 /GID 2 , we purified the Arabidopsis thaliana GID1A-RGA-SLY1-ASK2 complex and determined its cryo-EM structure. The GID1A-RGA-SLY1-ASK2 complex was purified via a copurification strategy. When SLY1 and ASK2 were coexpressed, only ASK2 was successfully purified, whereas SLY1 precipitated during purification due to its low stability ( Fig. S7K–L ). However, similar to the copurification of GID1A and RGA ( Fig. S1 ), the stability of SLY1 was significantly enhanced when SLY1-ASK2 was co-purified with GID1A-RGA in the presence of GA 3 ( Fig. S7M–O ). To purify the GID1A-RGA-SLY1-ASK2 complex, GID1A-RGA was expressed in E. coli in the presence of GA 3 , and the SLY1-ASK2 complex was expressed in E. coli without GA 3 . The cell lysates containing the recombinant GID1A-RGA and SLY1-ASK2 were mixed in balanced quantities. Purification of the GID1A-RGA-SLY1-ASK2 complex was performed via IMAC and SEC under high-salt buffer conditions with 0.5 M NaCl ( Fig. S7M–O ). The SEC-MALS experiment demonstrated that the purified GID1A-RGA-SLY1-ASK2 complex forms a homogeneous heterotetramer ( Fig. 4A ). These results indicate that the protein assembly of the GID1A-RGA-SLY1-ASK2 complex stabilizes the individual proteins involved. To determine the cryo-EM structure of the GID1A-RGA-SLY1-ASK2 complex, its cryo-EM map was reconstructed at 2.80 Å resolution, with 65,737 particles selected from cryo-EM images of the purified complex. As the cryo-EM map did not clearly show the density of SLY1-ASK2, an additional map focusing on SLY1-ASK2 was constructed at an overall resolution of 3.08 Å. The complete map covering all regions of the GID1A-RGA-SLY1-ASK2 complex was obtained by combining these two maps ( Figs. 4B–D , S8, and S9 ). The final model structure of the GID1A-RGA-SLY1-ASK2 complex exhibited a CC mask of 0.78, with no outliers in the Ramachandran plot ( Table S1 ). Download figure Open in new tab FIGURE 4. Cryo-EM structure of the GID1A-RGA-SLY1-ASK2 complex. (A) SEC-MALS analysis of purified RGA-GID1A-SLY1-ASK2. The absorbance at 280 nm is shown in blue, and the experimental molar masses of the eluate are depicted in red. The dotted line indicates the calculated molecular weight of the RGA-GID1A-SLY1-ASK2 heterotetramer (138.6 kDa). (B) Representative two-dimensional classes of RGA-GID1A-SLY1-ASK2 particles. (C, D) Cryo-EM map showing two different orientations. GID1A, RGA RD , RGA GRAS , SLY1, and ASK2 are colored pink, yellow, cyan, purple, and orange, respectively. (E) Cartoon model in the same orientation as (D). The color scheme matches the cryo-EM map in (C) and (D). The secondary structures are labeled A1–A9 for the helices of SLY1, α1–α8 for the helices of ASK2, and β1–β2 for the strands of ASK2. The GID1A-RGA in the cryo-EM structure of GID1A-RGA-SLY1-ASK2 was nearly identical to that in the cryo-EM structure of GID1A-RGA. Both GID1A-RGA structures were superimposed, with an RMSD value of 0.6 Å for 773 Cα atoms ( Fig. S10A ). No significant conformational differences were detected between the two GID1A-RGA complexes, except for a local conformational change in the loop between strand S2 and helix H11 of RGA (residues 362–367). Although this loop is situated near the SLY1-binding interface of RGA, no direct interaction between the loop and SLY1 was observed ( Fig. 4D ). SLY1 formed a helical structure comprising nine helices (Α1–A9) ( Figs. 4E and S10B ). The three N-terminal helices A2–A4 form an F-box motif responsible for SKP1 binding, and the C-terminal long helix A9 is oriented toward the RGA surface. Structural homology searches revealed that SLY1 shares an overall fold with human F-box-only protein 31 (FBXO31; PDB ID: 5VZT), except for helix A9. Superimposing SLY1 with FBXO31 yielded an RMSD value of 2.5 Å for 90 Cα atoms ( Fig. S10C ). In the cryo-EM structure, SLY1 binds to ASK2 and RGA through its N-terminal F-box motif and C-terminal helix A9, respectively, thereby linking RGA to the SCF complex as a substrate adaptor for the ubiquitin E3 ligase. ASK2 formed a helical structure consisting of seven helices (α1–α7) and contains an additional two-stranded short β-sheet at its N-terminus ( Fig. 4E ). ASK2 exhibited a fold similar to that of the F-box binding protein SKP1 of the SCF complex and it was superimposed with Arabidopsis thaliana ASK1, with the lowest RMSD value (1.2 Å for 132 Cα atoms). SLY1 as a substrate adaptor for RGA ubiquitination In the cryo-EM structure of the GID1A-GA 3 -RGA-SLY1-ASK2 complex, RGA GRAS forms a surface groove near RGA RD that binds SLY1. Although RGA RD and GID1A are located close to SLY1, no direct interaction was observed between SLY1 and either RGA RD or GID1A ( Fig. 4B ). Helix A9 of SLY1 directly binds to helices H9–H11 of RGA GRAS . Specifically, residues K126, S130, Y137, and S141 of helix A9 in SLY1 formed hydrogen bonds with residues Q311, A338/Q341, P337, Q332, and K375 of RGA GRAS , respectively ( Fig. 5A ). Additionally, the RGA-binding interface of SLY1 exhibited high hydrophobicity ( Fig. 5B and 5C ). Twenty-two hydrophobic interactions were identified around the hydrogen bonds between RGA GRAS and SLY1, suggesting that the hydrophobic surface of SLY1 is stabilized through binding to RGA. This is consistent with the observation that SLY1 precipitated during the purification of the SLY1-ASK2 complex but was stabilized and coeluted during copurification with ASK2 and GID1A-RGA ( Fig. S7 ). Moreover, SLY1 was stabilized and coeluted with ASK2 and RGA GRAS in SEC during purification when SLY1-ASK2 was copurified with RGA GRAS ( Fig. S7P–R ). These findings suggest that SLY1 stability is enhanced through its interactions with RGA GRAS and ASK2. Download figure Open in new tab FIGURE 5. Interactions of SLY1 with RGA GRAS and ASK2. (A) Cartoon model depicting the binding interface between SLY1 and RGA GRAS . The residues involved in the interactions are shown as purple and cyan stick models for SLY1 and RGA GRAS , respectively. (B, C) Surface charge distribution at the binding interface of RGA GRAS (B) and SLY1 (C). The black-lined boxes highlight the binding interface. The electrostatic potential from red (−10 kcal/mol· e ) to blue (+10 kcal/mol· e ) is mapped onto the solvent-accessible surfaces. (D, E) Structural comparison between RGA GRAS -SLY1 (D) and SHR-JKD (E). RGA GRAS and SHR are shown in the same orientation after superimposition. (D) RGA GRAS is represented as a cyan surface model, and SLY1 is displayed as a purple cartoon model. The SLY1-binding surface of RGA GRAS is colored yellow. (E) SHR is depicted as a blue surface model, and JKD is depicted as a teal cartoon model with the JKD-binding surface of SHR colored red. (F) Steric clash between RGA RD and JKD in the superimposed structures of the RGA and SHR-JKD. The surface of JKD clashing with RGA RD is colored red. (G) Cartoon model illustrating the binding interface between SLY1 and ASK2. (H, I) Surface charge distribution at the binding interface of SLY1 (H) and ASK2 (I). The electrostatic potential, ranging from red (−10 kcal/mol· e ) to blue (+10 kcal/mol· e ), is plotted on the solvent-accessible surfaces. The GRAS domain of Arabidopsis SHORT ROOT (SHR GRAS ) binds to the zinc-finger transcription factor JACKDAW (JKD), a member of the IDD family, through a hydrophobic surface groove 34 . When the SHR GRAS -JKD structure was superimposed onto that of RGA, the JKD-binding surface of SHR GRAS overlapped with the SLY1-binding surface of RGA GRAS . Moreover, JKD exhibited steric clashes with RGA RD ( Fig. 5D – 5F ). This indicates that DELLA proteins bind the IDD family transcription factors in a similar manner to JKD binding to SHR GRAS and that RGA RD likely prevents transcription factor binding when RGA forms a complex with GA-bound GID1A. SLY1 bound to ASK2 on the opposite surface from its RGA-binding interface through its F-box motif. This was primarily mediated by helix A3 of SLY1. Specifically, residues D48, K50, T51, A53, S56, C57, and V58 in helix A3 of SLY1 formed ionic or hydrogen bonds with ASK2 residues K137, A169, K137, N166, N166, R145 and R145, respectively ( Fig. 5G ). Additionally, SLY1-H63 in helix A4 and SLY1-R101 in helix A7 formed additional ionic bonds with ASK2-E165 ( Fig. 5G ). Like the RGA-binding surface, the ASK2-binding surface of SLY1 also showed high hydrophobicity ( Fig. 5H and 5I ). Consistent with this hydrophobicity, only ASK2 was successfully purified, but SLY1 was precipitated during purification when SLY1 and ASK2 were coexpressed ( Fig. S7J–L ). DISCUSSION DELLA proteins are key regulators of plant growth and development 8 – 11 , 23 . Their activity is modulated by GA-induced GID1 binding, followed by the polyubiquitination of DELLA proteins 30 – 32 . In this study, we determined two cryo-EM structures of RGA complexes. The atomic models of these complexes were constructed for both the main and side chains by tracing the cryo-EM map. Notably, water molecules and GA 3 were identified in the cryo-EM map of RGA-GID1A, despite the relatively small size of the protein complex for cryo-EM structure analysis. The atomic model in the cryo-EM structure of RGA-GID1A is approximately 86 kDa, excluding the flexible long linker. These cryo-EM structures elucidate the assembly mechanism of the ubiquitin E3 ligase responsible for the degradation of DELLA proteins. GID1A conceals GA 3 and interacts with RGA, which in turn binds to GA 3 -bound GID1A and SLY1 through distinct binding interfaces. Additionally, SLY1 possesses separate binding interfaces for RGA and ASK2 ( Fig. 6A ). Thus, these findings reveal that the GID1A-RGA-SLY1-ASK2 complex assembles in a chain-like manner via distinct, nonoverlapping binding interfaces. Download figure Open in new tab FIGURE 6. GA-induced assembly of the SCF SLY 1 -RGA-GID1A complex. (A) Schematic representation of the interactions between GID1A, RGA, SLY1, and ASK2. Each protein is depicted as a bar, with the lower and upper halves illustrating known structural motifs and the secondary structures at the binding interfaces. Red, blue, and gray dots indicate residues involved in ionic bonds, hydrogen bonds, and hydrophobic interactions, respectively. (B) Model of GA-induced stepwise assembly for RGA degradation and transcription factor regulation. F-box proteins serve as substrate-binding components of SCF ubiquitin E3 ligases. The substrate binding of F-box proteins is regulated primarily by post-translational modifications of the substrate degron, such as phosphorylation and glycosylation 36 . In contrast, the substrate binding of SLY1 is regulated in response to the GA hormone 30 – 32 . Our cryo-EM structures elucidate the mechanism by which DELLA proteins are degraded in response to this hormone. In the cryo-EM structure of the GID1A-RGA-SLY1-ASK2 complex, SLY1 interacts with RGA GRAS but not with RGA RD or GID1A. Consistent with this, SLY1 is copurified with RGA GRAS and ASK2 when RGA RD and GID1A are absent, indicating that RGA GRAS is sufficient for SLY1 binding. Therefore, the interaction between SLY1 and RGA GRAS is likely regulated through RGA RD , as GA-bound GID1A is essential for RGA ubiquitination. The RD of DELLA proteins is stabilized upon GID1 binding through a disorder-to-order transition 6 . Consistently, RGA GRAS is purified in a homogeneous form, whereas the full-length RGA tends to form aggregates. Given that RGA is costabilized by forming a heterodimer with GID1A in the presence of GA, RGA likely stabilizes through a conformational rearrangement of RGA RD upon GID1 binding, thereby becoming accessible to SLY1. This suggests that DELLA proteins expose the SLY1-binding site upon GID1 binding, rendering them susceptible to ubiquitination ( Fig. 6B ). SLY1 is stabilized through its interaction with RGA. It was observed that SLY1 precipitated in the absence of RGA-GID1A, but it stabilized when copurified with RGA-GID1A and ASK2. Consistent with this observation, the C-terminal helix A9 of SLY1, which functions as a substrate-binding motif, exhibits hydrophobicity. Thus, the hydrophobic C-terminal helix of SLY1 appears to be stabilized by binding to RGA through hydrophobic interactions. Homogeneous forms of RGA and SLY1 were purified exclusively through a co-purification strategy. In summary, the results of the cryo-EM structure and protein-protein interaction analyses suggest that the SCF SLY 1 -RGA-GID1A complex is assembled in a stepwise manner, stabilizing the proteins in response to GA signals ( Fig. 6B ). In this model, GA induces the assembly of SCF SLY 1 for the degradation of DELLA proteins. GID1 Lid binds GA and is costabilized with DELLA RD , facilitating further interactions between GID1 IHD and DELLA GRAS . The interaction between GID1 and DELLA proteins exposes the SLY1-binding surface and potentially restricts the access of transcription factors to DELLA proteins. SLY1 binds to the exposed hydrophobic groove of DELLA GRAS and assembles the SCF complex for DELLA ubiquitination ( Fig. 6B ). Some bHLH transcription factors are inactivated by binding to DELLA proteins 12 – 16 , whereas some IDD and bZIP family proteins are activated by binding them 17 – 21 . Given that the DNA-binding motifs of transcription factors are not conserved, different types of transcription factors are likely to interact with distinct surfaces of DELLA proteins. These transcription factors appear to bind to the GRAS domain of DELLA proteins 34 , 35 ; however, the specific binding sites and regulatory mechanisms of these interactions remain unclear. Our cryo-EM structures provide insights into how DELLA proteins regulate transcription factors. A structural comparison between GID1A-RGA-SLY1 and SHR GRAS -JDK 34 revealed that the zinc-finger motif of IDD family proteins accommodates the SLY1-binding groove of RGA GRAS but sterically clashes with RGA RD . Given that the RD of DELLA proteins transitions from a random coil to an ordered conformation upon the binding of GID1A-GA 6 , it is evident that DELLA RD inhibits the binding of IDD family proteins to DELLA GRAS through its interaction with GID1-GA and the resulting conformational rearrangement. In this context, the direct interaction between GID1 and DELLA GRAS may also restrict the binding of other transcription factors to RGA GRAS , as GID1A covers a substantial surface area of RGA GRAS . Assuming that the binding sites of GID1 and transcription factors on DELLA GRAS partially overlap, GID1 could competitively prevent transcription factors from binding to DELLA proteins ( Fig. 6B ). METHODS Plasmid preparation The DNA sequences encoding full-length RGA (residues 1–587; UniProt ID Q9SLH3), SLY1 (residues 1–151; UniProt ID Q9STX3), and ASK2 (residues 1–171; UniProt ID Q9FHW7) were amplified from the cDNA of A. thaliana (Gelvin Arabidopsis CYFP-cDNA stock number CD4-58, The Arabidopsis Information Resource) 37 via polymerase chain reaction. The gene encoding GID1A (residues 1–345; UniProt ID Q9MAA7) was synthesized following codon optimization on the basis of the codon usage preferences of E. coli (Bioneer, Daejeon, South Korea). The genes for RGA and ASK2 were inserted into the pET-His-SUMO vector, and SLY1 was inserted into the pRSF-His-SUMO vector 38 . Both vectors express a target protein with the N-terminal 6×His-SUMO tag removed by tobacco etch virus (TEV) protease. GID1A was inserted into the pCDF-His vector 38 , which expresses a target protein with the N-terminal 6×His removed by TEV protease. Purification of the GID1A-RGA complex Plasmids for the expression of RGA and GID1A (pET-His-SUMO-RGA and pCDF-His-GID1A) were co-introduced into the E. coli strain BL21-Star (DE3) (Thermo Fisher Scientific, Waltham, MA, USA). The transformed cells were cultured in Luria-Bertani (LB) medium at 37 °C. When the optical density at 600 nm of the culture reached 0.6–0.7, the media was cooled at 4 °C for 1 h. Subsequently, 0.4 mM isopropyl β-thiogalactopyranoside was added to induce protein expression, along with 0.4 mM gibberellic acid (GA 3 ), to promote stable expression of the RGA-GID1A complex. After being cultured at 12 °C for 36 h, the cells were harvested via centrifugation at 3,000 × g for 10 min, resuspended in buffer A (20 mM HEPES pH 7.5, 0.2 M NaCl, 5% (v/v) glycerol, 0.5 mM TCEP, and 0.4 mM GA 3 ), and lysed via sonication. The cell lysates were treated with DNase I (Roche, Mannheim, Germany) and RNase A (Roche) (10 μg/mL each) for 30 min on ice. Insoluble debris was removed by centrifugation at 20,000 × g for 30 min at 4 °C. The GID1A-RGA complex was purified using immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC). The clarified cell lysate was applied to a 5 mL HisTrap nickel-chelating column (Cytiva, Marlborough, MA, USA). The column was washed with 80 mM imidazole to remove nonspecifically bound proteins from the resin. Proteins bound to the resin were eluted using a linear imidazole gradient ranging from 0.08 to 1.0 M on an AKTA-purifier FPLC (Cytiva). Eluate fractions containing 6×His-SUMO-RGA and 6×His-GID1A were pooled and treated with TEV protease at 4 °C overnight. After confirming the complete cleavage of the N-terminal tags, 6×His-SUMO and 6×His, the protein solution was concentrated and loaded onto a PD-10 desalting column (Cytiva) equilibrated with buffer A to remove imidazole from the protein solution. To remove the N-terminal tag, the protein fractions eluted from the desalting column were passed through HisPur Ni-NTA resin using a gravity-flow column (Thermo Fisher Scientific, Waltham, MA, USA). The eluates were loaded onto a Superdex 200 preparative grade column (Cytiva) that had been preequilibrated with buffer B (20 mM HEPES pH 7.5, 0.2 M NaCl, and 0.5 mM TCEP). The eluate fractions corresponding to the monodisperse UV peak were pooled after confirmation via SDS-PAGE and subsequently concentrated to 5 mg/mL. Purification of the GID1A-RGA-SLY1-ASK2 complex Plasmids for the expression of SLY1 and ASK2 (pRSF-His-SUMO-SLY1 and pET-His-SUMO-ASK2) were co-introduced into the E. coli strain BL21-star (DE3). The SLY1-ASK2 complex was expressed using the same procedures as the RGA-GID1A complex, with the exception that GA 3 was not added to the LB medium during protein expression. The cells expressing RGA-GID1A and SLY1-ASK2 were cultured and subsequently harvested. The harvested cell pellets were combined and resuspended in buffer C (20 mM HEPES pH 7.5, 0.5 M NaCl, 5% (v/v) glycerol, 0.5 mM TCEP, and 0.4 mM GA 3 ). The GID1A-RGA-SLY1-ASK2 complex was purified following the same procedures used for the RGA-GID1A complex. The final SEC was conducted using a Superdex 200 preparative grade column (Cytiva) preequilibrated with buffer D (20 mM HEPES pH 7.5, 0.5 M NaCl, and 0.5 mM TCEP). The fractions containing the GID1A-RGA-SLY1-ASK2 complex were pooled and concentrated to a final concentration of 3 mg/mL. Cryo-EM data collection Purified complexes of GID1A-RGA and GID1A-RGA-SLY1-ASK2 (3 µL each) were applied to glow-discharged Cu 300 mesh QUANTIFOIL R1.2/1.3 holey carbon grids (SPI Supplies, West Chester, PA, USA). The grid was blotted using a Vitrobot Mark IV (Thermo Fisher Scientific) with humidity-saturated filter paper (Ted Pella, Redding, CA, USA). After blotting for 3 s at a blot force of 5, the grid was rapidly plunged into liquid ethane. Micrographs were collected on a 300 kV Krios G4 cryo-transmission electron microscope equipped with a Falcon 4 detector and a Selectris-X energy filter (Thermo Fisher Scientific). The energy filter slit was adjusted to 10 eV. In total, 2,362 micrographs for the RGA-GID1A complex and 1,925 micrographs for the GID1A-RGA-SLY1-ASK2 complex were collected with a pixel size of 0.7451 Å at a magnification of 165,000x, for a total dose of 60 electrons per Å 2 . The nominal defocus value ranged from −0.5 to −1.9 µm ( Table S1 ). Data processing and structure determination Image processing was conducted using cryoSPARC (v.4.4.0) software 39 . The raw movies were motion-corrected using Patch Motion Correction, and the defocus value for each micrograph was estimated using Patch CTF Estimation. To construct the cryo-EM map of the GID1A-RGA complex, particles were identified using Template Picker with templates obtained from a previous pilot data collection. A total of 2,804,928 particles were picked from 2,362 micrographs and extracted with a box size of 400 pixels for 2D classification. After multiple rounds of 2D classification, a set of 273,109 particles was obtained. Particles were subsequently repicked from the micrographs using the TOPAZ program in the cryoSPARC platform 40 , yielding a total of 676,577 extracted particles. The two sets of particles were combined, and duplicates were removed. The particles were then subjected to additional rounds of 2D classification to eliminate poor-quality particles, resulting in a final selection of 507,751 particles. An initial ab initio model was reconstructed from these particles and classified into four classes. Of these, 265,845 particles belonging to the major class were subjected to motion correction using reference-based motion correction. Finally, the cryo-EM map of GID1A-RGA was reconstructed from the motion-corrected 251,864 particles using heterorefinement, with an estimated overall resolution of 2.66 Å ( Figs. S2 and S3 ). To reconstruct the cryo-EM map of GID1A-RGA-SLY1-ASK2, a total of 2,183,664 particles were autopicked from 1,925 micrographs using a Blob Picker, with extraction performed at a box size of 400 pixels. After multiple rounds of 2D classification, a set of 756,176 particles was obtained. The particles were then repicked from the micrographs using the TOPAZ program in the cryoSPARC platform 40 , resulting in 1,428,933 extracted particles. These two sets were combined, and duplicates were removed. The particles were then subjected to additional rounds of 2D classification to eliminate poor-quality particles and duplicates, leading to the selection of 682,192 particles. An initial ab initio model was reconstructed from the combined particles and classified into three classes. Of these, 277,898 particles from the major class were motion-corrected using reference-based motion correction. The cryo-EM map of GID1A-RGA-SLY1-ASK2 was reconstructed from the motion-corrected 277,150 particles using nonuniform refinement and classified into 20 clusters using 3D variability analysis. The final cryo-EM map was reconstructed using nonuniform refinement with 65,737 particles selected from a 3D variability, achieving an overall resolution of 2.80 Å. To enhance the map quality of SLY1 and ASK2, a cryo-EM map focusing on SLY1-ASK2 was reconstructed at a resolution of 3.07 Å using local refinement ( Figs. S8 and S9 ). The final map was reconstructed by combining the cryo-EM map of GID1A-RGA-SLY1-ASK2 with the focused map of SLY1-ASK2 using the Phenix.combine_focused_maps 41 . All reported resolutions were estimated using the gold-standard Fourier shell correlation (FSC) = 0.143 criterion ( Figs. S3 and S9 ). The cryo-EM map processing statistics are summarized in Table S1, and the overall workflow of cryo-EM data processing is illustrated in Figs. S2 and S8. Atomic model building and structure analysis The AlphaFold 42 models of GID1A, RGA, SLY1, and ASK2 were used as templates for model building. Each model was fitted into the cryo-EM map using ChimeraX 43 , rebuilt by tracing the cryo-EM map in COOT 36 and refined using Phenix.refinement 41 , 44 . Water molecules were added using Phenix.douse 41 and subsequently confirmed manually. The statistics for Cryo-EM data collection and refinements are summarized in Table S1. Molecular interactions were analyzed using PISA 45 and DIMPLOT 46 . Figures were generated using ChimeraX 43 , PyMOL 47 , and ALSCRIPT 48 . The atomic models and cryo-EM maps have been deposited in the Protein Data Bank with the PDB accession numbers: xxxx for GID1A-RGA and xxxx for GID1A-RGA-SLY1-ASK2. SEC-MALS The molar masses of the GID1A-RGA and GID1A-RGA-SLY1-ASK2 complexes were measured using an SEC-MALS instrument (Wyatt Technology, Santa Barbara, CA, USA). Purified GID1A-RGA (100 µL, 1.5 mg/mL) and GID1A-RGA-SLY1-ASK2 (100 µL, 1 mg/mL) complexes were injected into a Superdex 200 Increase 10/300 GL column (Cytiva) equilibrated with buffer B (20 mM HEPES pH 7.5, 0.5 M NaCl, and 0.2 mM TCEP). The eluate was then applied to inline DAWN Heleos II MALS and Optilab T-Rex differential refractive index detectors (Wyatt Technology). Data analysis was performed using the ASTRA 6 software package (Wyatt Technology), and data were visualized using SigmaPlot 14.0 (Grafiti LLC, Palo Alto, CA, USA). DATA AND CODE AVAILABILITY The final coordinates and cryo-EM maps that support the findings of this study have been deposited in the Worldwide Protein Data Bank ( www.wwpdb.org ) with the following PDB IDs (9LUM, 9LUN, 9LUO, and 9LUP) and EMDB IDs (EMD-63398, EMD-63399, EMD-63400, and EMD-63401). CONFLICT OF INTEREST The authors declare no conflict of interest. AUTHOR CONTRIBUTIONS S.I. and K.P. performed the experiments. S.I., K.P., E.K., and D.Y.K. carried out the data analysis. E.K. and D.Y.K. wrote the manuscript with contributions from all other authors. ACKNOWLEDGMENTS We would like to express our gratitude to the staff at the Korea Basic Science Institute for their assistance with SEC-MALS analyses. REFERENCES 1. ↵ Hedden , P . The Current Status of Research on Gibberellin Biosynthesis . Plant Cell Physiol 61 , 1832 – 1849 ( 2020 ). OpenUrl CrossRef PubMed 2. ↵ Sponsel , V.M . Signal Achievements in Gibberellin Research: The Second Half-Century . Annual Plant Reviews 49 , 1 – 36 ( 2016 ). OpenUrl 3. ↵ Ueguchi-Tanaka , M. et al. GIBBERELLIN INSENSITIVE DWARF1 encodes a soluble receptor for gibberellin . Nature 437 , 693 – 8 ( 2005 ). OpenUrl CrossRef PubMed Web of Science 4. Nakajima , M. et al. Identification and characterization of Arabidopsis gibberellin receptors . 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Share Structural analyses of gibberellin-mediated DELLA protein degradation Soyaab Islam , KunWoong Park , Eunju Kwon , Dong Young Kim bioRxiv 2025.02.08.637281; doi: https://doi.org/10.1101/2025.02.08.637281 Share This Article: Copy Citation Tools Structural analyses of gibberellin-mediated DELLA protein degradation Soyaab Islam , KunWoong Park , Eunju Kwon , Dong Young Kim bioRxiv 2025.02.08.637281; doi: https://doi.org/10.1101/2025.02.08.637281 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Plant Biology Subject Areas All Articles Animal Behavior and Cognition (7622) Biochemistry (17648) Bioengineering (13870) Bioinformatics (41880) Biophysics (21423) Cancer Biology (18553) Cell Biology (25458) Clinical Trials (138) Developmental Biology (13364) Ecology (19866) Epidemiology (2067) Evolutionary Biology (24290) Genetics (15589) Genomics (22475) Immunology (17711) Microbiology (40327) Molecular Biology (17145) Neuroscience (88472) Paleontology (666) Pathology (2826) Pharmacology and Toxicology (4815) Physiology (7635) Plant Biology (15114) Scientific Communication and Education (2044) Synthetic Biology (4286) Systems Biology (9815) Zoology (2268)