# Source paper archive - Source title: Core MCC of Arabidopsis thaliana: structural dissection provides new insights into Mad2, BubR1 and CDC20 - Author(s): Eduardo Gamosa, Emanuel Victor Nogueira Gotardo, Paula Guimaraes Custodio, Gabriela Pimentel Serodio, Izabela Silva dos Santos, Marco Antonio Lopes Cruz - Source landing URL: https://www.biorxiv.org/content/10.64898/2026.11.06.764920v1 - PDF URL: https://www.biorxiv.org/content/10.64898/2026.11.06.764920v1.full.pdf - PDF SHA-256: 87d4bd7d9936704ae983e1381ef6e954d0c76a94b6634ad10ff7f2d4a3752df8 - Converter: pymupdf4llm 0.0.27 - Limits: Automated PDF text extraction; reading order, equations, tables and formatting may be incomplete or incorrect. Images are omitted, and no OCR was performed. Markup and links are neutralized for safe downloading. Consult the original PDF for the authoritative version. --- [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 **Core MCC of** _**Arabidopsis thaliana**_ **: structural dissection provides new** 2 **insights into Mad2, BubR1 and CDC20.** 3 4 Eduardo Alves Gamosa de Oliveira [1 ] Emanuel Victor Nogueira Gotardo [1], Paula 5 Guimarães Custodio [1], Gabriela Pimentel Serodio [1,3], Izabela Silva dos Santos [1] 6 and Marco Antonio Lopes Cruz [1,2 ] 7 8 1- Current address: Laboratório de Biotecnologia Vegetal, Instituto de 9 Biodiversidade e Sustentabilidade, Centro de Ciências da Saúde, 10 Universidade Federal do Rio de Janeiro (BIOTEC/NUPEM/CCS/UFRJ) 11 Av. Amaro Reinaldo dos Santos Silva, 764, São José do Barreto, Macaé. 12 CEP 27965-045 13 2- Department of Mechanistic Cell Biology, Max Planck Institute of Molecular 14 Physiology (MPI) - Otto-Hahn-Strasse 11, 44227 Dortmund, Germany. 15 3- Current address: Departamento de Bioquímica, Instituto de Química, 16 Universidade de São Paulo Avenida Professor Lineu Prestes, 748, São 17 Paulo SP 05508-000, Brasil. 18 **E-mail:** marcocruz@macae.ufrj.br 19 20 **Abstract** 21 The spindle assembly checkpoint (SAC) is a mechanism executed mainly 22 by the mitotic checkpoint complex (MCC) to monitor the anchoring of mitotic 23 spindle microtubules to ensure correct chromosome segregation. Although 24 knowledge of protein‒protein interactions in the plant cell cycle has progressed 25 in recent years, information on the SAC mechanism in this context remains [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 26 scarce. Here, we used bigBac, chemical crosslinking, mass spectrometry and 27 structural analysis to identify protein‒protein interactions (PPIs) among 28 AtCDC20, AtBubR1 (BMF2) and AtMad2, which are components of the core MCC 29 in _A. thaliana._ Fifty-three nonredundant crosslinks were identified, with multiple 30 binding sites in different domains and subdomains of these proteins, such as the 31 β-propeller (AtCDC20), tetratricopeptide domain, and KEN boxes, including a 32 new KET box (AtBubR1) and Horma domain (AtMad2). We also identified a 33 hierarchy of interactions between these three proteins, with the potential to 34 regulate signalling through the SAC molecular mechanism. Interactions that 35 characterize the Mad2 tetramer, with the coupling of two dimers, were also 36 identified. No PPIs were identified for AtBub1 (BMF1) or AtBub3:1, revealing a 37 significant difference in MCC formation compared with that in other eukaryotes. 38 Our findings provide a structural basis for core MCC formation in _A. thaliana_ and 39 offer new insights into the proteins CDC20, BubR1 and Mad2 in the context of 40 the SAC. 41 42 **Introduction** 43 Almost seventy years ago, Alma Howard and Stephen Pelc first described 44 the timeframe of cellular life according to four periods of cell division in root beans 45 ( Howard & Pelc, 1986), paving the way for understanding the eukaryotic cell 46 cycle. However, this area of inquiry in plants is lagging behind the corresponding 47 research in animals and yeast. Therefore, understanding the structural 48 organization and dynamics of protein homo and heterocomplexes related to cell 49 cycle control is important for overcoming this gap. Unlike what occurs in animals, 50 environmental factors such as light intensity, temperature, water availability, and [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 51 nutrient supply can cause significant changes in the cell cycle and development 52 of plants ( Stals, H., & Inzé, 2001) . However, the basic molecular machinery 53 controlling the cell cycle is largely conserved in eukaryotes (Kops et al., 2020; 54 Dewitte & Murray, 2003; De Veylder et al., 2007). Understanding the molecular 55 mechanisms that control the cell cycle during development could contribute to the 56 development of resilient varieties for food production and thus mitigate the effects 57 of ongoing climate change (Carneiro et al., 2021). The spindle assembly 58 checkpoint (SAC) is a regulatory molecular mechanism executed mainly by the 59 mitotic checkpoint complex (MCC) to monitor the anchoring of mitotic spindle 60 microtubules to ensure correct chromosome segregation, and the core MCC is 61 formed by three essential SAC components: CDC20, BubR1 and Mad2 62 ( Musacchio 2015). Although our knowledge of protein‒protein interactions in the 63 plant cell cycle has progressed in recent decades ( Van Leene et al., 2011), 64 information on the SAC mechanism in this context remains scarce. The cell 65 division cycle 20 protein (CDC20) has a β-propeller domain formed by seven 66 WD40 repeats (blades), each with four antiparallel β-strands ( Kapanidou et al., 67 2017). Budding uninhibited by benzimidazole-r elate d 1 (BubR 1) has a 68 tetratricopeptide (TPR) domain ( D'Arcy et al., 2010) and conserved KEN boxes 69 ( Sczaniecka et al., 2008) . Mitotic arrest deficient 2 (Mad2) is a HORMA protein 70 ( Rosenberg et al., 2015) with the capacity to assume two conformations, namely, 71 open (O-Mad2) and closed (C-Mad2), after interacting with Mad1 and CDC20 72 ( Luo et al., 2001; Sironi et al., 2001). Because many studies of SAC interactions 73 involve peptides or truncated proteins, studies involving full-length proteins can 74 provide new insight into this molecular mechanism because the PPI might have 75 remained hidden under these experimental conditions. Chemical crosslinking and [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 76 mass spectrometry (crosslinking-MS) enables the identification of protein‒protein 77 interactions (PPIs) and heterocomplex characterization ( O’Reilly and Rappsilber, 78 2018; Rozbesky ́ et al., 2018; Graziadei et al., 2022; Botticelli et al., 2024) . These 79 techniques combined with baculovirus expression systems ( Pan et al., 2018) and 80 bigBac approaches ( Weissmann et al., 2016) have become robust strategies for 81 PPI identification. Thus, here, we combined these strategies to identify and 82 structurally characterize the interactions between the proteins that form the MCC 83 core in _Arabidopsis thaliana_, AtCDC20 AtBubR1 (BMF2) and AtMad2 (Fig. 1). 84 85 **Results** 86 _**In vitro**_ **formation and components of the core MCC of** _**Arabidopsis thaliana**_ 87 Insect cell culture combined with a baculovirus expression system allows 88 us to produce high-quality cells ( Trowitzsch et al., 2010). Therefore, using bigBac 89 technology ( Weissmann et al., 2016), six pLIB vectors with full-length proteins 90 were constructed, pLIB MBP AtCDC20, pLIB 6his AtBubR1 (BMF2), pLIB 6his 91 AtMad2, 6his AtBub1, 6his AtBub3;1 and pLIB 8TEV AtCDC20 (Fig. 2a), for 92 which the pBIG1a MBP core MCC bigBac vector was employed (Fig. 2b). These 93 constructs were then transfected into Sf9 cells to generate baculoviruses. 94 Individually or as core MCCs in bigBac. An affinity column was used for the 95 purification of the core MCC, and we observed three bands with expected sizes 96 of approximately 92.2, 46.3 and 24.5 kDa for MBP AtCDC20, 6his AtBubR1 and 97 AtMad2 6his, respectively (Supplementary Figure 1). Analogous results were 98 obtained in the second purification step via size-exclusion chromatography (SEC) 99 on a Superose 6 column, and the results were analysed by SDS‒PAGE (Fig. 2d– 100 e). These results revealed that in _Arabidopsis thaliana,_ the core MCC [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 101 components (AtMad2, AtCDC20 and AtBubR1) interact reciprocally, as observed 102 in yeast (Chao et al., 2012) and humans (Alfieri et al., 2016). In eukaryotes, the 103 cell division cycle 20 protein (CDC20) is an essential component of the SAC. 104 ( Musacchio and Salmon, 2007, Tian et al., 2012). In _A. thaliana_, AtCDC20 has 105 four hundred and fifty-seven amino acids, with a MIM motif in the N-terminal 106 region and a short C-terminal region. Its central region is a classical β-propeller 107 domain (Fig. 2e,f and Supplementary Figure 2), with seven WD40 repeats 108 (blades) each formed by four antiparallel β-sheets stabilized by hydrophobic 109 interactions (Tian et al., 2012; Xu & Mim, 2011; Yu, 2007). Our analysis of 110 sequences from different plant species (Supplementary Figure 3) revealed a 111 classic AtCDC20 MIM motif (MIM1) in the N-terminal region (R88-P96); two 112 further MIM motifs were identified in the β-propeller domain, namely, a 113 noncanonical MIM motif (MIM2) on blade 3 and, using the ELM tool ( Kumar et 114 al., 2020), we identified a third motif (MIM3) on blade 7 (R404-P412), as shown 115 in Figure 2 e,f). In plants, compared with MIM2, MIM1 is more variable. However, 116 MIM3 is conserved, suggesting its biological importance. Nevertheless, to 117 function in a SAC context, the MIM3 motif must be accessible to Mad2. This 118 requires transient and partial unfolding of the β-propeller domain. In this context, 119 we identified a conserved sequence (G333-R339) referred to here as the glycine 120 box (Fig. 2 e,f). Additionally, the structural characteristics of the β-sheets of blade 121 1 (E129-D133) and blade 7 (E424-W430) are shown in supplementary Figure 4. 122 Both will be detailed later. In _Arabidopsis thaliana,_ BubR1 (AtBubR1) has 396 123 residues and is composed of eight antiparallel α-helices, with the TPR domain in 124 the N-terminal region (Fig. 2g, h) ( D'Arcy et al., 2010; Bolanos-Garcia et al., 125 2011). This TPR domain presents six α-helices with a concave shape, forming [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 126 subdomains TPR1 (P84-K105), TPR2 (L126-H152) and TPR3 (A159-R188). 127 AtBubR1 has three classical KEN boxes: KEN1 (K31–N33) in the N-terminal 128 region before the TPR domain and KEN2 (K220–N222) and KEN3 (K305–N307) 129 after the TPR domain (Fig. 2g, h and Supplementary Figure 5 and 6). We also 130 identified a new conserved KEN box (K376-T378) in which asparagine was 131 replaced by threonine, named here the KET box. Only KEN box 2 is not 132 conserved in plants. (Figures 2 g, h and Supplementary Figure 5). Mad2 is a 133 HORMA protein ( Rosenberg & Corbett, 2015; Aravind, L., & Koonin, 1998), and 134 _Arabidopsis thaliana_ (AtMad2) has 207 residues and a HORMA domain (S17– 135 H192) (Figure 2 i,j and Supplementary Figure 7 and 8). We identified two regions 136 in AtMad2 that mediate classical and no canonical interactions with AtCDC20, 137 AtBubR1 and AtMad2, suggesting that it functions in SAC regulation. (Figure 2 i, 138 j and Supplementary Figure 7 and 8). Both will be detailed later. Thus, our results 139 reveal that even considering the difference in size of BubR1, the components of 140 the core MCC of _A. thaliana_ (AtMad2, AtBuBR1 and AtCDC20) exhibit the same 141 general composition of domains and motifs for SAC functions, which has already 142 been identified in other eukaryotes ( Kops et al., 2020; Musacchio & Salmon, 143 2007; Hardwick et al., 2000). The complete MCCs characterized in other 144 eukaryotes include the proteins Bub1 and Bub3 ( Kops et al., 2020; Musacchio, 145 2015; Musacchio & Salmon, 2207; van Hooff et al., 2017). Therefore, to 146 investigate whether this composition is also applicable to _A. thaliana_, we 147 performed size-exclusion chromatography (SEC) on a Superose 6 column and 148 performed pull-down experiments. In both experiments, we used the core MCC 149 plus AtBub1 (BMF1) and AtBub3:1. In the case of the pull-down, amylose and 150 MBP-AtCDC20 (as bait) were used. The two experiments were analysed by [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 151 SDS‒PAGE. Our combined pulldown and SEC results revealed that neither 152 AtBub1 nor AtBub3;1 bind the core MCC (Fig 3 a, b and Supplementary Figure 153 9). These results corroborate those of previous studies in _A. thaliana_ ( Komaki 154 and Schnittger, 2017) . Chemical crosslinking (Pan et al., 2019), when combined 155 with bigBac approaches, becomes an efficient strategy for _in vitro_ studies of 156 protein complex formation. Therefore, to identify protein‒protein interactions 157 (PPIs) between AtCDC20, AtMad2, and AtBubR1 in the core MCC of _A. thaliana_, 158 we performed chemical crosslinking (Fig. 3c–h) with disuccinimidyl dibutyric urea 159 (DSBU) (Pan et al., 2018). Different combinations of full-length proteins were 160 used: a) core MCC with MBP AtCDC20 plus AtBub3:1 and AtBub1; b) just core 161 MCC with MBP AtCDC20; c) MBP AtCDC20 plus AtMad2; and d) core MCC with 162 8TEV AtCDC20 (Fig. 3 c-h). A total of six thousand and five hundred and twenty 163 four interactions were identified, of which sixty-three were nonredundant 164 crosslinks. Among these three proteins, twenty-five are intramolecular, and thirty 165 eight are intermolecular. Nonredundant crosslinks were used to validate the 166 models (Chao et al., 2012; Alfieri et al., 2016; Mapelli et al., 2007) applied here 167 (Supplementary Tables 1–6). The nonredundant cross-links C3–C7, C12–C17, 168 C32–C36, C40, C41, B1-B7, and M1-M7 confirm the composition of the MCC 169 core in _A. thaliana_ with AtMad2, AtCDC20 and AtBubR1 and reciprocal 170 interactions between these proteins (Fig. 3a–n; Supplementary Fig. 10-13 and 171 Supplementary Tables 1–6), corroborating the results of studies in other 172 eukaryotes. Chao et al., 2012; Alfieri et al., 2016; Mapelli et al., 2007). We 173 identified the hierarchical order of the interactions between Mad2 and the other 174 genes (Fig. 3 I, k and n). In the presence of AtBubR1, residues K114 and K190 175 of AtMad2 interact with residues K376 and T378 on the KETbox of AtBubR1 (Fig. [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 176 3i,n and supplementary table 5). However, in the absence of AtBubR1, residues 177 K114, S117, and K183 of AtMad2 interact with residue K221 of AtCDC20 (Fig. 178 3k and Supplementary Table 4). This interaction site corresponds to MIM2 e and 179 is shown in the nonredundant cross-links C41–C43 and B9–10 (Fig. 3j, 180 Supplementary Fig. 7 and Supplementary Table 4). The AtMad2 sequence 181 identified here as a KIM (for KET box Interact Motif) with a preferred affinity for 182 binding to BubR1 (I105–R118) includes a conserved sequence (E110–R118) 183 corresponding to T109–R117 in human cells. To create “stable” O-Mad2 184 (Mad2 [LL] ), this sequence was deleted (Mapelli et al., 2007). Thus, our findings 185 suggest that AtBubR1 can block this region and thereby regulate the 186 stoichiometry of O-Mad2 and C-Mad2 in a SAC context. Future studies will be 187 necessary to confirm or refute this hypothesis. Additionally, the alignments show 188 that these cross-linked regions involved in this hierarchy of interactions are 189 conserved in plants, corroborating our hypothesis (Fig. 3 I, k, n and 190 Supplementary Figures 3, 5 and 7). 191 192 **Homocomplexes of AtMad2 in** _**Arabidopsis thaliana**_ 193 Mad2 plays a central role in SAC signalling because of its ability to switch 194 its structure from open to closed ( Luo et al., 2000; Sironi et al., 2001) to form 195 homocomplexes as dimers (Mapelli et al., 2007) or tetramers (Fang et al., 1998). 196 Our lack of redundant crosslinks B9, B10 and C9-43 and C28–C32 revealed that 197 in _A. thaliana_, Mad2 can interact with both CDC202 and BubR1 in its central and 198 C-terminal regions (Supplementary figure 10-14 and Supplementary tables 4 and 199 5). In the dimer of AtMad2, the sequence delimited by R134-Y142 has a 200 substantial hydrophobic content (Supplementary Figure 7). The residues R134, [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 201 S138, T141 and Y142 of C-Mad2 can form strong hydrogen bonds (≤ 3 Å) with 202 Y34, N30, P53 and Q130 of I-Mad2 (Fig. 4a–d), analogous to hMad2 R133, 203 Q134, T136 and T140 ( Hara et al., 2015; Mariani et al., 2012). In human cells, 204 T136, A137, T140 and F141 (S137, S138, T141 and Y142 in _A. thaliana_ ), located 205 at the asymmetric contact interface of hMad2, have implications for 206 conformational change (Mapelli et al., 2007; Hara et al., 2015; Mariani et al., 207 2012). The sequence of residues that interact with Mad2 and has implications for 208 the functionality of the SAC, is called an MIM motif ( Hara et al., 2015; Mariani et 209 al., 2012). Therefore, by definition, the interaction region in the AtMad2 dimer is 210 composed of the amino acids R134–Y142 (R133–F141 in hMad2) and is a 211 noncanonical MIM motif (Fig. 4c,d). Additionally, the observation of crosslinks 212 with DSBU and measured distances between K114, S117, and K183 (AtMad2) 213 and K221 (AtCDC20) above forty angstroms suggests that these interactions are 214 possible only with an AtMad2 dimer (or tetramer) in the core MCC. The 215 implications of the core MCC composition involving the dimer will be discussed 216 later. Although the structure and formation of Mad2 dimers have been studied 217 (Mapelli et al., 2007; Hara et al., 2015; Mariani et al., 2012), aspects of tetramer 218 formation remain unknown. In human cells, tetramers of Mad2 inhibit cell cycle 219 progression _in vitro_ through the inhibition of APC/C activity ( Fang, et al., 1998). 220 Crosslinks M8 among amino acids S61 and K123 and M9 and M10 (K92–K114 221 and K92–S117, respectively) revealed that the distance between the residues 222 “should” be greater than 12.5 Å (Fig. 4g, Supplementary Figure 14). We used O 223 Mad2 in model 2v64, where K114 and S117 are missing **.** (Mapelli et al., 2007). 224 N109 and E110 are shown as a reference for a hypothetical position of these 225 amino acids (Supplementary Figure 14). In this case, our data revealed that a [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 226 tetramer of AtMad2 formed with two dimers docked in an overlapping 227 arrangement. The MIM motif is a contact point between AtMad2 dimers (Fig. 4a– 228 c) and is composed mostly of hydrophobic residues (Fig. 4a–c) and 229 Supplementary Figure 10). Thus, the AtMad2 tetramer forms a primary 230 hydrophobic core delimited longitudinally by four arginine (R134) residues (Fig. 231 4a–h). 232 233 **Structure of the core MCC** 234 As observed in yeast and humans (Chao et al., 2012; Alfieri et al., 2016) 235 and shown previously here. In _A. thaliana_, the core MCC is also composed of the 236 proteins Mad2, BubR1, and CDC20. However, in plants, the interactions between 237 the components of the core MCC have been much less studied than those in 238 other eukaryotic models and, consequently, are poorly understood. Two Cdc20 239 binding sites on BubR1 were demonstrated in studies with human cells, with 240 implications for SAG regulation. ( Davenport et al., 2006). The lack of redundant 241 C9-C25 crosslinks (Supplementary Table 4) indicates the formation of a complex 242 of AtBubR1 and two AtCDC20 molecules (Fig. 5a–i and Supplementary Table 4), 243 referred to here as CDC20 of mitotic complex 2 (CDC20 [MC1] and CDC20MC2). 244 Interestingly, the concentration of AtMad2 does not appear to be sufficient to 245 interact with all AtBubR1 and AtCDC20 molecules. In this context, the 246 nonredundant crosslinks (C9-C11, C18, C19, C26-C37, C39, B10, B11, and M8 247 M10) revealed that some interaction sites between CDC20 and BubR1 are also 248 shared by AtMad2 (Supplementary Tables 4 and 5). Thus, the absence of 249 AtMad2 enables the interaction of multiple AtCDC20 and AtBubR1 units. This 250 explanation is corroborated by the observation of SEC/S6 in slot 1, corresponding [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 251 to AtCDC20 and AtBubR1 complexes with a molecular mass greater than the 252 core MCC in slots 3-13 (Fig. 1c, d and Fig. 2a, b). We speculate that the presence 253 of AtMad2 predominantly in the form of dimers or tetramers explains this finding. 254 Further investigations will be needed to determine the stoichiometry of the 255 interactions between AtCDC20 and AtBubR1 in the presence and absence of 256 AtMad2 homocomplexes. In human cells, APC/C and Mad2 compete for KILR 257 sites in CDC20 ( Izawa & Pines, 2012; O verlack et al., 2015) . The presenc e of 258 nonredundant crosslinks (C13–C16 and C26–C36) reveale d that AtMad2 and 259 AtBubR1 b in d AtCDC20 similarly in the conserved m otifs R88, I89, L90 and A91 260 (Supplementary Tables 4 and 5). T hese findings suggest that AtBubR1 regulates 261 the interaction between AtMad2 and AtCDC20 at this site. A tCDC20 binds the C 262 terminus of AtBubR1 or AtMad2 at the top of the β-propeller and shows 263 conserved interactions ( Izawa & Pines, 2012; O verlack et al., 2015). In contrast 264 to KEN box 2, KEN boxes 1 and 3 are conserved in plants (Fig. 2g; Fig. 3m, n 265 Fig. 6f and Supplementary Fig. 5). Our results revealed that AtCDC20 bound 266 AtBubR1 in the N-terminal and C-terminal regions, as well as the TPR3 domain 267 (Supplementary Figures 10, 11 and 13 and Supplementary Tables 1 and 2). At 268 the C-terminus, AtCDC20 interacts with AtBubR1 on KEN box 3 and the KET box 269 (Supplementary Figure 10 and Supplementary Tables 1 and 2). AtCDC20 also 270 bound AtMad2 in the Horma domain in the central and C-terminal regions 271 (Supplementary Figure 10, Supplementary Table 3). The crosslinking of C13 272 (Supplementary Table 4) s uggests the _in vitro_ conditions under which AtBubR1 273 itself can block the RILA-mediated impairment of bindin g between AtMad2 and 274 AtCDC20 (C9–C11 and C26–C29). This involves hotspot interaction sites of KEN 275 boxes (C23–C25 and B1–B8) with possible implications for APC/C activity (King [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 276 et al., 2007; Keskin et al., 2005). and autoinhibitory binding with a second CDC20 277 in the functional SAC ( Tipton et al., 2001). Examples are provided by the 278 nonredundant crosslinking o f B1 between the first and second KEN boxes or B7 279 between the second and third KEN boxes with the new KET box (Fig. 3e–g, l-n; 280 Supplementary Figure 12; Supplementary Table 5). As previously mentioned, our 281 ELM analyses revealed an MIM motif (MIM3) in AtCDC20 on blade 7 (Fig. 2 e, f). 282 However, MIM3 is buried within the β-propeller, preventing access by AtMad2 283 (Supplementary Figure 4). It has been demonstrated that the absence of the 284 “velcro” formed by hydrophobic interactions between the β-sheets of blades 1 285 and 7 can allow for a transient structural state, granting substrates access to the 286 central cavity of the β-propeller domain. (Xu and Mim., 2011). This feature is 287 found in AtCDC20, where these hydrophobic interactions between blades 1 and 288 7 are unlikely (Fig. 6a, b). Additionally, we identified a conserved glycine box 289 (G333–R339) in AtCDC20 that may facilitate partial unfolding of the β-propeller 290 domain because of its intrinsic flexibility (Fig. 6c–e). Studies have already shown 291 unfolding and refolding of the β-propeller for Tc toxin activation (Gatsogiannis et 292 al., 2018). In a SAC context (Chao et al., 2012), this glycine box can form 293 hydrogen bonds with both AtBubR1 in KEN box 1 (E32) and the AtMad2 dimer 294 interface (R134, S138 and T141). However, in the interaction of the core MCC 295 with the APC/C (Alfieri et al., 2016) **,** these hydrogen bonds are unlikely (Fig. 6c– 296 f). As previously shown, the Mad2 dimer interface is an MIM motif (Fig. 4a–d). 297 Considering the measured distances and the models applied here, interactions 298 between K221 (AtCDC20) and K114, S117 and K183 (AtMad2) of the 299 nonredundant crosslinks C38, C42 and C43 are unlikely (Fig 7a, b and. They 300 would only be possible through the coupling of a dimer (or Mad2 tetramer). This [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 301 would place at least two MIM motifs (antiparallel) of AtMad2 near the glycine box 302 (AtCDC20) and the KEN box of AtBubR1 (Fig. 6 a, b and Supplementary Table 303 4). Thus, all the difficulties involved in representing a three-dimensional structure 304 in a two-dimensional illustration should be considered. We hypothesize a model 305 for the composition of the core MCC in _Arabidopsis thaliana_, featuring AtMad2 as 306 a homocomplex (dimer or tetramer) and two AtCDC20 molecules and using 307 AtBubR1 as a platform for both proteins (Figure 6 c). Its composition not only 308 reflects features unique to this plant species but also provides new perspectives 309 regarding core MCC organization in other organisms. 310 311 **Discussion** 312 Plants modulate the development of their body plan by mediating signals 313 from the immediate environment. These signals can be positive, such as nutrient 314 availability (Liu and Lee., 2022), or originate from biotic and abiotic stresses (Zhu, 315 J. K., 2016; Huang et al., 2019). This process requires the convergence of 316 different signalling pathways linked to various phases of the cell cycle 317 (Shimotohno et al., 2021) to synchronize the duplication and segregation of 318 genetic material with conditions favourable f or development. Despite significant 319 advances in recent decades (Sablowski, R., & Gutierrez, C., 2022), our 320 understanding of plant cell cycle control lags behind what is known about other 321 eukaryotes. Adopting an evolutionary perspective broadens the horizons of our 322 research. Identifying convergent and divergent aspects makes it possible to trace 323 details that are otherwise difficult to observe in specific study models. This 324 enables us to overcome barriers in understanding complex molecular 325 mechanisms and points towards alternative methodological approaches. In this [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 326 context, identifying intermolecular interactions and understanding the formation 327 of protein complexes involved in the control of chromosome segregation in plants 328 is important for elucidating the functions of these interactions, whether they are 329 conserved or not (Liu et al., 2022). Our results revealed that in _A. thaliana_, the 330 CDC20 and Mad2 proteins, with respect to size, domain composition and cell 331 cycle-related interaction motifs (Fig. 1e–j; Supplementary Figures 3-9), are 332 conserved (McAinsh and Kops, 2023). The cross-links of the N-terminal region of 333 AtCDC20 with those of both AtMad2 and AtBubR1 are consistent with previous 334 findings in related studies with canonical interactions, such as those involving the 335 MIM motif, β-propeller domain, KEN-boxes and the TPR domain ( Sironi et al., 336 2001; Tang et al.; 2001; King et al., 2007; Lara-Gonzalez et al. 2011 3 5–38). For 337 example, studies have shown that CDC20 can interact with Mad2 on R88 and 338 E170 ( Chao et al., 2012) and crosslink C28-C32 on a “safety belt”, indicating that 339 AtCDC20 can interact with AtMad2 in the same form (Fig. 4d–n and 340 Supplementary Figures 10-14). Compared with HsBubR1 (McAinsh and Kops, 341 2023), AtBubR1 presents significant differences in length and domain 342 composition (Fig. 2 g, h; Supplementary Fig. 10). In human cells, BubR1 is 343 phosphorylated at different sites (on the C-terminal domain) related to 344 microtubule attachment and chromosome congregation (Huang et al., 2008; 345 Elowe et al., 2009). AtBubR1 (BMF2) is minor and does not have these sites, 346 suggesting that it does not play a role in the docking of microtubules to the 347 kinetochore, corroborating the findings of Komaki et al. (2017). Our findings 348 suggest that in _A. thaliana_, AtBubR1 functions only in MCC assembly and SAC 349 signalling (Elowe et al., 2009). Although the N-terminal region of AtBubR1 350 presents the same structural characteristics observed in human cells and yeast [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 351 ( D'Arcy et al., 2010 and Bolanos-Garcia et la., 2011), how the TPR domain and 352 KEN boxes (Fig. 2g,h) are present remains unclear. In eukaryotes, complete and 353 functional MCCs include Bub1 and Bub3 ( McAinsh & Kops, 2023). Our 354 experiments revealed that in _A. thaliana,_ Bub1 (BMF1) and Bub3:1 do not bind to 355 the protein complex formed by AtCDC20, AtMad2 and AtBubR1 (Fig. 1c). The 356 GLEBS motif is related to the interaction with Bub3 in the WD40 of the beta 357 propeller domain not found in AtBubR1 (Fig. 2 g, h). Therefore, even though it is 358 relatively small, AtBubR1 (BMF2) is sufficient for core MCC formation and does 359 not act to recruit AtBub1 and AtBub3.1 (Figs. 3 and 5). Compared with yeast and 360 animal SAC signalling, MCC assembly is significantly different (McAinsh and 361 Kops, 2023). However, further studies are needed to identify whether crucial 362 underlying factors can (even if transiently) promote the association of the proteins 363 Bub1 and Bub3:1 with the MCC core in _A. thaliana_ . Studies on MCC formation in 364 human cells have shown that the Mad1-Mad2 and Mad1-Bub1 complexes are 365 required as physical components of the catalytic mechanism of formation 366 (Faesen et al., 2017). Our results show that Mad1 and Bub1 (BMF3) are not 367 necessary for _in vitro_ core MCC formation in _A. thaliana_ . Thus, the role of these 368 proteins in MCC functionality in plants requires investigation. An essential and 369 pivotal characteristic of Mad2 is its ability to undergo a topological switch, in which 370 two different conformations, namely, open (O-Mad2) and closed (C-Mad2), after 371 interaction with Mad1 and CDC20 on the MIM motif ( Sironi et al., 2001; Kim et al. 372 2002; Yu e t a l., 2025 Luo et al., 2002). Another important characteristic of Mad2 373 is the formation of homocomplexes: dimers (Mapelli et al., 2006; Fang et al., 374 1998) and tetramers (Fang et al., 1998). Disruptions in Mad2 dimer and tetramer 375 formation affect SAC progression in eukaryotic cells. (Mapelli et al., 2006; Fang., [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 376 1998; Mapelli et al., 2006) bition of APC/C activity. Therefore, we aimed to 377 analyse protein‒protein interactions (PPIs) in AtMad2 homocomplexes. We 378 found that AtMad2 can be organized into dimers and tetramers (Fig. 4; 379 Supplementary Fig. 10 and Supplementary Table 6). The phosphorylation of 380 S190 blocks Mad2 dimerization (Kim et al., 2010), and BubR1 has been 381 previously shown to block Mad2 dimerization in the MCC (Mariani et al., 2012). 382 Furthermore, the interaction of Mad3 (BubR1 in yeast) with the mad2 dimer is 383 functional for SAC progression (Mariani et al., 2012). We found that the 384 interaction between AtMad2 (central region) and CDC20 (blade 3) is blocked by 385 BubR1, more specifically by the conserved KET box motif (Fig. 3c–I,k,n; and 386 Supplementary Tables 4–6). These results corroborate studies on SAC 387 regulation via the interaction of BubR1 with the Mad2 homocomplex (Mapelli et 388 al., 2007; Mariani et al., 2012; Faesen et al., 2017). Our findings suggest a 389 regulatory mechanism in which AtBubR1 can block the interaction of the AtMad2 390 dimer (or tetramer) with AtCDC20, interfering with SAC signalling. The Mad2 391 dimer is formed by two antiparallel Mad2 molecules, where the hydrophobic core 392 is important for the interaction among the open and closed structural 393 conformations of these molecules (Mapelli et al., 2007). In this context, the 394 formation of a tetramer can generate a structural arrangement with a central 395 hydrophobic core delimited by four R134 residues (MIM of AtMad2). The 396 importance of hydrophobic amino acids for interactions with the CDC20 MIM and 397 the conversion of Mad2 from the open state to the closed state has been 398 demonstrated recently (Yu et al., 2025). Some of these residues, L13, F23 and 399 F24 (L14, F24 and F25 in AtMad2), are part of the hydrophobic core of Mad2 400 (Mapelli et al., 2007) **.** Thus, we can speculate that the hydrophobic core in the [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 401 AtMad2 tetramer may combine with hydrophobic interactions and hydrogen 402 binding to trigger structural rearrangement, enabling autocatalytic conversion of 403 O-Mad2 into C-Mad2 in the absence of Mad1 or Cdc20. However, a structural 404 model of the AtMad2 tetramer is needed to analyse the implications of the 405 assembly of four AtMad2 MIM motifs within this homocomplex, thereby 406 confirming or refuting the hypothesis of an autocatalytic mechanism. In this 407 context, Mad2 tetramers arrest cell cycle progression _in vitro_ by inhibiting APC/C 408 (Fang et al., 1998), and residues R133 and Q134 are required for the interaction 409 between BubR1 and the Mad2 dimer. The presence of B9 and B10 crosslinks 410 (Fig. 4e–h; Supplementary Table 5) suggests that the C-terminus of AtBubR1 can 411 stabilize AtMad2 oligomers (i.e., dimers or tetramers) at the core MCC, 412 supporting the hypothesis that the Mad2 dimer is a component of the MCC 413 (Mariani et al., 2012). However, whether BubR1 can stabilize Mad2 tetramers in 414 the MCC in _A. thaliana_ and prevent cell cycle progression by impairing MCC 415 APC/C (Fang et al., 1998) still needs to be investigated in plants. A dditionally **,** t he 416 MIM motif sequence of AtMad2 is predicted to interact with 14-3-3 and casein 417 kinases (Fig. 4i). In plants, these two kinases are related to cell cycle regulation, 418 tissue development, stress responses, metabolism regulation, and growth control 419 ( Youn et al., 2015, Zhao et al., 2021). Mutations in R134 analogues are sufficient 420 to disrupt the SAC (Sironi et al., 2001; Mapelli et al., 2007; Fang et al., 1998; Hara 421 et al., 2015). We speculate that phosphorylation at this specific point has the 422 potential to disturb SAC progression. This could connect cell cycle control to 423 variations in the immediate environment. In yeast and human cells, the core MCC 424 adopts the same general structure, forming a heterotrimer with CDC20, Mad2 425 and BubR1 (Kops et al., 2020; Musacchio 2015; Musacchio and Salmon, 2007; [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 426 McAinsh and Kops 2023). Here, we used full-length proteins and showed that 427 more than one AtCDC20 can interact with AtBubR1, together with AtMad2 428 homocomplexes, to form the core MCC (Figs. 2, 3, and 5; Supplementary Figs. 429 11-14 and Supplementary Tables 4-6). In this context, we identified not only 430 canonical PPIs but also novel plant-conserved and nonconserved motif binding. 431 For instance, “motfis” KEN boxes are crucial for MCC training and SAC functions 432 (King et al., 2007 and Tipton et al., 2011). With respect to AtBubR1 (BMF2), we 433 identified three KEN boxes and one noncanonical KET box (Fig. 3e–g, l–n; Fig. 434 6f, Supplementary Fig. 5, Supplementary Table 5). We also revealed that there 435 are shared interaction sites among AtMad2, AtCDC20 and AtBubR1, suggesting 436 a complex mechanism of reciprocal regulation (Supplementary Figures 10-11). 437 The identification of two functional binding sites for CDC20 on BubR1 with 438 implications for the functionality of the SAC is not recent (Davenport et al., 2006). 439 In addition, sequential coordination of the interaction between Mad2, BubR1 and 440 CDC20 was suggested, where Mad2 interacts with CDC20, and the latter is then 441 able to bind to BubR1 (Davenport et al., 2006). The binding affinities of AtMad2, 442 AtCDc20 and AtBubR1 differ. The KIM motif of AtMad2 interacts preferentially 443 with AtBubR1 in the KET box motif (Fig. 3e–g). The interaction of the KIM motif 444 with the β-propeller domain occurs only in the absence of AtBubR1, which binds 445 to the same region of AtCDC20. These findings suggest that AtBubR1 may 446 regulate the interaction between AtMad2 and AtCDC20. These findings indicate 447 that higher concentrations of AtMad2 and AtCDC20 are required for reciprocal 448 interactions in a scenario in which the AtBubR1 concentration remains constant. 449 In this context, the interaction of AtBubR1 with two AtCDC20 molecules in the 450 absence of APC/C suggests that the stoichiometric balance of AtCDC20 is crucial [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 451 for the functionality of the core MCC in _A. thaliana_ . Corroborating this hypothesis, 452 AtBubR1 can interact with itself (Supplementary Figure 13), suggesting that the 453 C-terminus of AtBubR1 can “close” in a coiled-coil structure without AtCDC20 454 being available. The structural and functional characteristics of the CDC20 455 protein, its interactions with SAC proteins (BubR1 and Mad2) and its implications 456 for SAC signalling have been extensively studied (Luo et al., 2000; Sironi et al., 457 2001; Tian et al., 2012; Hardwick et al., 2000; Fang et al., 1998; Davenport et al., 458 2006; Izawa et al., 2012; Tang et al., 2001; King et al., 2007; Yu et al., 2025, Luo 459 et al., 2002). However, the identification of conserved MIM3 prompted us to 460 investigate structural elements that enable the interaction of AtCDC20’s MIM3 461 with AtMad2 (Fig. 2e, f; Supplementary Figure 3). In this context, several 462 examples of transient localized flexibility within the β-propeller domain as a 463 requirement for specific functions exist. For instance, studies involving G proteins 464 have shown that interactions with phosducin induce state changes in the β 465 propeller domain, specifically in blades 6 and 7 (Loew et al., 1998). More recently, 466 it was shown that this type of change in the β-propeller domain can be reversed 467 (Gatsogiannis et al., 2018). The absence of “Velcro” closure between the β 468 sheets of blades 1 and 7 allows for a certain degree of flexibility and enables 469 access to the interior of the β-propeller domain on prolyl oligopeptidase (Fulop et 470 al., 1998). In the β-sheets that interact between blades 1 and 7 of AtCDC20, 471 strong hydrophobic interactions are unlikely, indicating a loosening of the “velcro” 472 in this region. (Fig. 6a, b). Interestingly, we identified a conserved glycine-rich 473 sequence (glycine box) that, in the context of the SAC, can form hydrogen bonds 474 with KEN box 1 (AtBubR1) and the Mad2 dimer interface (Fig. 6c–e). We 475 speculate that the interactions of the glycine box (AtCDC20) with both AtBubR1 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 476 and Mad2 homocomplexes may facilitate a transient state of disarray in blades 6 477 and 7. This could cause a slight displacement of blade 6, creating space for 478 separation between the β-sheets of blades 1 and 7 (Fig. 6 a–e; Supplementary 479 Figs. 3 and 4). In this way, an additional MIM motif could be exposed, resulting in 480 CDC20 acting as another template for the conversion of O-Mad2 to C-Mad2. 481 Acting as a “catalytic step” outside kinetochores (Musacchio 2015). This 482 mechanism has the potential to accelerate the availability of C-Mad2 and may 483 shift the kinetic balance towards the completion of SAC signalling. Our findings 484 corroborate those of previous studies that revealed that the formation of Mad2 485 dimers accelerates the binding of Mad2 and CDC20 (Simonetta et al., 2009). We 486 recognize the need for a specific structural model of the core MCC in _A. thaliana_ 487 to overcome limitations regarding further details on the interactions between 488 AtCDC20, AtBubR1 and AtMad2. Taken together, our results show that in _A._ 489 _thaliana_, AtBubR1 can function as a platform that accommodates more than one 490 AtCDC20. In this context, AtCDC20 itself, in turn, also acts as a platform to 491 accommodate homocomplexes (dimers or tetramers) of AtMad2 (Fig 7c). This 492 reveals a significant difference in the composition of the core MCC compared with 493 what is known in other eukaryotic models. In conclusion, despite structural 494 similarities to those of other eukaryotes, we also identified interactions that are 495 novel and specific to _A. thaliana_ . While laying the structural foundation for core 496 MCC formation in plants, our results also offer new insights into core MCC 497 assembly in other eukaryotes. 498 499 **Methods** 500 **Protein expression and purification** [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 501 The full-length sequences of the following genes optimized for insect cells 502 were obtained (GeneScript, New Jersey, EUA): AtCDC20 (At4g33270), AtBubR1 503 (AT2g33560), AtMad2 (At3g25980), AtBub1 (AT2g20635) and AtBub3;1 504 (AT3g19590). These genes were subsequently cloned and inserted into the pLIB 505 vector ( Weissmann et al., 2016). AtCDC20 was tagged with maltose-binding 506 protein (MBP), and all the other proteins were tagged with 6His. To generate the 507 plant core MCC, the following components were combined in pBIG1a 508 ( Weissmann et al., 2016) : pLIB MBP AtCDC20, pLIB 6his AtBubR1 and pLIB 509 AtMad2 6his. Additionally, individual pLIB 6his AtBub1 and pLIB 6his Bub3;1 510 constructs were produced. Baculoviruses were generated in SF9 insect cells and 511 expressed in a 250 ml BTI-Tna038 insect cell suspension ( Trowitzsch et al., 512 2010) in SF-900 III 1 medium (Gibco). After 96 h, these cells were pooled by 513 centrifugation (4,000 rpm, 5 min, 4 °C). Afterwards, the supernatant was 514 discarded, and the cells were resuspended in buffer containing 50 mM HEPES 515 (pH 7.5), 300 mM NaCl, 5% glycerol, 10 mM imidazole and 2 mM TCEP (B300i), 516 lysed by sonication and cleared by centrifugation (78,800  g, 30 min and 4 °C). 517 After the sediment was discarded, the supernatant was subjected to affinity 518 chromatography (1 h, 4 °C and constant agitation) with a complete His-tag 519 (Sigma‒Aldrich) according to the manufacturer’s instructions utilizing B300i 520 buffer. The electrophoretic profile of this sample was analysed via 12% tricine‒ 521 SDS‒PAGE and stained with Coomassie brilliant blue. Afterwards, the eluted 522 sample was concentrated (Amicon tube 10 kDa), aliquoted, frozen in liquid 523 nitrogen and stored at −80 °C. 524 525 **Size-exclusion chromatography** [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 526 SEC was performed in a Superose 6, 10/300 GL column (GE Healthcare) 527 equilibrated in 25 mM HEPES buffer (pH 7.5), 50 mM NaCl, 5% glycerol and 2 528 mM TCEP (B50) according to the recommended flow rates and isocratic 529 conditions in a cold room (4 °C). After analyses of the chromatogram and 530 electrophoretic profile obtained via 12% tricine-SDS‒PAGE with Coomassie 531 brilliant blue staining, the fractions of interest were pooled, concentrated (Amicon 532 tube 10 kDa), frozen in liquid nitrogen and stored at −80 °C. 533 534 **Pull-down assay using amylose resin** 535 A pull-down assay was performed as described by Pan and collaborators 536 (Pan et al., 2019), with a few modifications. The proteins of the plant core MCC, 537 either alone or together with AtBub1 or Atbub3;1, were diluted to a concentration 538 of 6 µM in 40 µl of 20 mM HEPES buffer (pH 7.5) containing 50 mM NaCl, 5% 539 glycerol and 2 mM TCEP (B50) and gently mixed with 25 µl of amylose resin 540 (NEB) that was previously equilibrated with buffer B50. Afterwards, 20 µl of this 541 mixture was taken as the input sample, and the remaining sample (40 µl) was 542 incubated for 30 min at 4 °C with constant agitation. The mixture was 543 subsequently washed by centrifugation with 1 ml of buffer B50 three times to 544 separate the amylose-bound proteins from the unbound fraction. After the 545 proteins were eluted from the amylose resin by incubation with buffer B50 plus 546 10 mM maltose (Sigma‒Aldrich), they were incubated for 1 h at 4 °C with constant 547 agitation. Next, the amylose was separated by centrifugation and discarded. The 548 supernatant (eluted) was recovered and analysed via tricine‒SDS‒PAGE (12%) 549 with Coomassie brilliant blue staining. 550 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 551 **Chemical crosslinking with DSBU** 552 A chemical crosslinking assay was performed as described by Pan and 553 collaborators (2018) with a few modifications. Proteins of the plant core MCC 554 complex at a concentration of 3 µM (0.34 mg/ml) were dissolved in buffer 555 containing 20 mM HEPES (pH 7.5), 50 mM NaCl, 5% glycerol and 2 mM TCEP 556 (B50). Additionally, the plant MCC complex (2 µM (2.4 mg/ml)) plus 1 µM (1.2 557 mg/ml) AtBub1 or 1 µM (1.4 mg/ml) AtBub3:1 was dissolved in the same buffer 558 (B50). Before the crosslinking reaction began, a 10 µl aliquot was collected for 559 electrophoretic analysis. DSBU (Alinda Chemical Limited) was added to the 560 protein solution at a final concentration of 3 mM. The samples were incubated at 561 25 °C for 1 h. The reaction was then stopped by the addition of Tris-HCl (pH 8.0) 562 at a final concentration of 100 mM, followed by incubation at 25 °C for 30 min. A 563 10 µl aliquot was subsequently collected for analysis via tricine-SDS‒PAGE 564 (12%) with Coomassie blue staining. 565 566 **Mass spectrometry analysis** 567 Mass spectrometry analysis was performed as described by Hubner and 568 collaborators (Hubner et al., 2010) with a few modifications. The samples were 569 subsequently centrifuged at 15,000 rpm for 5 min, after which the supernatant 570 was removed. Each sample was subsequently subjected to LysC/trypsin 571 digestion, denaturation in 25 μL of denaturation–reduction buffer (8 M urea + 1 572 mM DTT) and vortexing at 25 °C for 30 min. The sample was subsequently 573 subjected to alkylation with 5.5 mM chloroacetamide, followed by 20 min of 574 vortexing at 25 °C. Next, 25 μL of 20 mM ammonium bicarbonate (ABC) buffer 575 was added to achieve a final urea concentration of < 4 M, after which 2 μg of [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 576 LysC was added, and the mixture was vortexed for 3 h at 25 °C (0.5 μg/μl LysC, 577 4 μl). Afterwards, 1 µg of trypsin (10 μl stock solution of 0.1 μg/μl + 40 μl dilution 578 buffer) was added, and the sample was incubated at 25 °C overnight in dilution 579 buffer (100 mM Tris, 1 mM CaCl2, pH 8.5). The next day, the digestion was 580 stopped, with a final TFA concentration of 0.2%. The pH was approximately 2.5, 581 and the final volume of the mixture was approximately 100 μL. The samples were 582 then subjected to LC MS/MS analysis. MaxQuant searches to check the purity of 583 the complexes were performed in parallel in a bacterial database, an insect 584 database, and among the taxonomies of _Trichoplusia ni_ and _Arabidopsis thaliana_ . 585 The sequences were submitted to a database of known contaminants with a false 586 discovery rate (FDR) of 1%. The MeroX output was filtered to the experimentally 587 observed mass deviation as indicated in the data file name, and the minimum 588 score was set according to an FDR of 5%. Graphical representations showing 589 intra- and intermolecular nonredundant crosslinks in circular plots were 590 [generated using xVis (https://xvis.genzentrum.lmu.de/CrossVisNoLogin.php).](https://xvis.genzentrum.lmu.de/CrossVisNoLogin.php) 591 592 **Computational tools** 593 Sequence alingments were performed with MUSCLE with defaults in 594 jalview v 2.11.5.1 (Waterhouse et al., 2009). Sequence logo charts were 595 generated with a local installation of weblogo v3 (Crooks et al., 2004). Protein 596 structure models were generated using Modeller protocols (Webb & Sali, 2017) . 597 Cartoon representations of protein structures and measurements between Cα– 598 Cα with nonredundant crosslinks were prepared using PyMOL (available at 599 [https://pymol.org/2) following the instructions in the user guide. To predict](https://pymol.org/2) protein 600 domains and short linear motifs (SLiMs), we used ELM (Kumar, et al., 2020). [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 601 Cartoons for the topologic representation of protein domains and different motifs 602 were generated using Domain Graph version 1.0 (DOG) (Ren et al., 2009). 603 604 **References** 605 1. Howard, A., & Pelc, S. R. Synthesis of desoxyribonucleic acid in normal and 606 irradiated cells and its relation to chromosome breakage. _International Journal of_ 607 _Radiation Biology and Related Studies in Physics, Chemistry and Medicine_ 49(2), 608 207–218 (1986). 609 610 2. Stals, H., & Inzé, D. When plant cells decide to divide. _Trends in Plant_ 611 _Science_ 6(8), 359–364 (2001). 612 613 3. Kops, G. J., Snel, B., & Tromer, E. C. (2020). Evolutionary dynamics of the 614 spindle assembly checkpoint in eukaryotes. _Current Biology_, _30_ (10), R589-R602. 615 616 4. Dewitte, W., & Murray, J. A. The plant cell cycle. _Annual Review of Plant_ 617 _Biology_ 54(1), 235–264 (2003). 618 619 5. De Veylder, L., Beeckman, T., & Inzé, D. The ins and outs of the plant cell 620 cycle. _Nature Reviews Molecular Cell Biology_ 8(8), 655–665 (2007). 621 622 6. Carneiro, A. K., Montessoro, P. D. F., Fusaro, A. F., Araújo, B. G., & Hemerly, 623 A. S. (2021). Plant CDKs—driving the cell cycle through climate 624 change. _Plants_, _10_ (9), 1804. 625 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 626 7. Musacchio, A. The molecular biology of spindle assembly checkpoint signalling 627 dynamics. _Current Biology_ 25(20), R1002–R1018 (2015). 628 629 8. Van Leene, J., Boruc, J., De Jaeger, G., Russinova, E., & De Veylder, L. A 630 kaleidoscopic view of the Arabidopsis core cell cycle interactome. _Trends in Plant_ 631 _Science_ 16(3), 141–150 (2011). 632 633 9. Kapanidou, M., Curtis, N. L., & Bolanos-Garcia, V. M. (2017). Cdc20: at the 634 crossroads between chromosome segregation and mitotic exit. _Trends in_ 635 _biochemical sciences_, _42_ (3), 193-205. 636 637 10. D'Arcy, S., Davies, O. R., Blundell, T. L., & Bolanos-Garcia, V. M. Defining 638 the molecular basis of BubR1 kinetochore interactions and APC/C-CDC20 639 inhibition. _Journal of Biological Chemistry_ 285(19), 14764–14776 (2010). 640 641 11. Sczaniecka, M., Feoktistova, A., May, K. M., Chen, J. S., Blyth, J., Gould, K. 642 L., & Hardwick, K. G. The spindle checkpoint functions of Mad3 and Mad2 depend 643 on a Mad3 KEN box-mediated interaction with Cdc20-anaphase-promoting 644 complex (APC/C). _Journal of Biological Chemistry_ 283(34), 23039–23047 645 (2008). 646 647 12. Rosenberg, S. C., & Corbett, K. D. The multifaceted roles of the HORMA 648 domain in cellular signalling. _Journal of Cell Biology_ 211(4), 745–755 (2015). 649 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 650 13. Luo, X., Fang, G., Coldiron, M., Lin, Y., Yu, H., Kirschner, M. W., & Wagner, 651 G. Structure of the Mad2 spindle assembly checkpoint protein and its interaction 652 with Cdc20. _Nature Structural Biology_ _7_ (3), 224–229 (2000). 653 654 14. Sironi, L., Melixetian, M., Faretta, M., Prosperini, E., Helin, K., & Musacchio, 655 A. (2001). Mad2 binding to Mad1 and Cdc20, rather than oligomerization, is 656 required for the spindle checkpoint. _The EMBO journal_, _20_ (22), 6371-6382. 657 658 15. O’Reilly, F. J., & Rappsilber, J. Cross-linking mass spectrometry: Methods 659 and applications in structural, molecular and systems biology. _Nature Structural_ 660 _& Molecular Biology_ 25(11), 1000–1008 (2018). 661 662 16. Rozbesky, D., Rosulek, M., Kukacka, Z., Chmelík, J., Man, P., & Novák, P. ́ 663 Impact of chemical cross-linking on protein structure and function. _Analytical_ 664 _Chemistry_ 90(2), 1104–1113 (2018). 665 666 17. Botticelli, L., Bakhtina, A. A., Kaiser, N. K., Keller, A., McNutt, S., Bruce, J. 667 E., & Chu, F. Chemical cross-linking and mass spectrometry enabled systems 668 level structural biology. _Current Opinion in Structural Biology_ 87, 102872 (2024). 669 670 18. Graziadei, A., & Rappsilber, J. Leveraging crosslinking mass spectrometry 671 in structural and cell biology. _Structure_ 30(1), 37–54 (2022). 672 673 19. Pan, D., Brockmeyer, A., Mueller, F., Musacchio, A., & Bange, T. Simplified 674 protocol for cross-linking mass spectrometry using the MS-cleavable cross-linker [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 675 DSBU with efficient cross-link identification. _Analytical Chemistry_ 90(18), 10990– 676 10999 (2018). 677 678 20. Weissmann, F., Petzold, G., VanderLinden, R., Huis In't Veld, P. J., Brown, 679 N. G., Lampert, F., ... & Peters, J. M. (2016). biGBac enables rapid gene 680 assembly for the expression of large multisubunit protein 681 complexes. _Proceedings of the National Academy of Sciences_, _113_ (19), E2564 682 E2569. 683 684 21. Trowitzsch, S., Bieniossek, C., Nie, Y., Garzoni, F., & Berger, I. New 685 baculovirus expression tools for recombinant protein complex production. _Journal_ 686 _of Structural Biology_ 172(1), 45–54 (2010). 687 688 22. Chao, W. C., Kulkarni, K., Zhang, Z., Kong, E. H., & Barford, D. Structure of 689 the mitotic checkpoint complex. _Nature_ 484(7393), 208–213 (2012). 690 691 23. Alfieri, C., Chang, L., Zhang, Z., Yang, J., Maslen, S., Skehel, M., & Barford, 692 D. Molecular basis of APC/C regulation by the spindle assembly 693 checkpoint. _Nature_ 536(7617), 431–436 (2016). 694 695 24. Musacchio, A., & Salmon, E. D. The spindle-assembly checkpoint in space 696 and time. _Nature Reviews Molecular Cell Biology_ _8_ (5), 379–393 (2007). 697 698 25. Tian, W., Li, B., Warrington, R., Tomchick, D. R., Yu, H., & Luo, X. Structural 699 analysis of human Cdc20 supports multisite degron recognition by [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 700 APC/C. _Proceedings of the National Academy of Sciences_ 109(45), 18419– 701 18424 (2012). 702 703 26. Xu, C., & Min, J. (2011). Structure and function of WD40 domain proteins. 704 _Protein & cell_, _2_ (3), 202-214. 705 706 27. Yu, H. Cdc20: A WD40 activator for a cell cycle degradation 707 machine. _Molecular Cell_ 27(1), 3–16 (2007). 708 709 28. Kumar, M., Gouw, M., Michael, S., Sámano-Sánchez, H., Pancsa, R., 710 Glavina, J., ... & Gibson, T. J. (2020). ELM—the eukaryotic linear motif resource 711 in 2020. _Nucleic acids research_, _48_ (D1), D296-D306. 712 713 29. Bolanos-Garcia, V. M., Lischetti, T., Matak-Vinković, D., Cota, E., Simpson, 714 P. J., Chirgadze, D. Y., ... & Blundell, T. L. (2011). Structure of a Blinkin-BUBR1 715 complex reveals an interaction crucial for kinetochore-mitotic checkpoint 716 regulation via an unanticipated binding Site. _Structure_, _19_ (11), 1691-1700. 717 718 30. Aravind, L., & Koonin, E. V. The HORMA domain: A common structural 719 denominator in mitotic checkpoints, chromosome synapsis and DNA 720 repair. _Trends in Biochemical Sciences_ 23(8), 284–286 (1998). 721 722 31. Hardwick, K. G., Johnston, R. C., Smith, D. L., & Murray, A. W. (2000). MAD3 723 encodes a novel component of the spindle checkpoint which interacts with Bub3p, 724 Cdc20p, and Mad2p. _The Journal of cell biology_, _148_ (5), 871-882. [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 725 32. van Hooff, J. J., Tromer, E., van Wijk, L. M., Snel, B., & Kops, G. J. (2017). 726 Evolutionary dynamics of the kinetochore network in eukaryotes as revealed by 727 comparative genomics. _The EMBO Reports_, _18_ (9), 1559-1571. 728 729 33. Komaki, Shinichiro, and Arp Schnittger. "The spindle assembly checkpoint in 730 Arabidopsis is rapidly shut off during severe stress." _Developmental cell_ 43.2 731 (2017): 172-185. 732 733 34. Pan, D., Walstein, K., Take, A., Bier, D., Kaiser, N., & Musacchio, A. 734 Mechanism of centromere recruitment of the CENP-A chaperone HJURP and its 735 implications for centromere licensing. _Nature Communications_ 10(1), 4046 736 (2019). 737 738 35. Mapelli, M., Massimiliano, L., Santaguida, S., & Musacchio, A. The Mad2 739 conformational dimer: Structure and implications for the spindle assembly 740 checkpoint. _Cell_ 131(4), 730–743 (2007). 741 742 36. Fang, G., Yu, H., & Kirschner, M. W. The checkpoint protein MAD2 and the 743 mitotic regulator CDC20 form a ternary complex with the anaphase-promoting 744 complex to control anaphase initiation. _Genes & Development_ 12(12), 1871– 745 1883 (1998). 746 747 37. Hara, M., Özkan, E., Sun, H., Yu, H., & Luo, X. Structure of an intermediate 748 conformer of the spindle checkpoint protein Mad2. _Proceedings of the National_ 749 _Academy of Sciences_ 112(36), 11252–11257 (2015). [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 750 38. Mariani, L., Chiroli, E., Nezi, L., Muller, H., Piatti, S., Musacchio, A., & 751 Ciliberto, A. Role of the Mad2 dimerization interface in the spindle assembly 752 checkpoint independent of kinetochores. _Current Biology_ 22(20), 1900–1908 753 (2012). 754 755 39. Davenport, J., Harris, L. D., & Goorha, R. (2006). Spindle checkpoint function 756 requires Mad2-dependent Cdc20 binding to the Mad3 homology domain of 757 BubR1. _Experimental cell research_, _312_ (10), 1831-1842. 758 759 40. Izawa, D., & Pines, J. Mad2 and the APC/C compete for the same site on 760 Cdc20 to ensure proper chromosome segregation. _Journal_ _of_ _Cell_ 761 _Biology_ 199(1), 27–37 (2012). 762 763 41. O verlack, K., Primorac, I., Vleugel, M., Krenn, V., Maffini, S., Hoffmann, I., 764 Kops, G. J. P. L., & Musacchio, A. A molecular basis for the differential roles of 765 Bub1 and BubR1 in the spindle assembly checkpoint. _eLife_ 4, e05269 (2015). 766 767 42. King, E. M., van der Sar, S. J., & Hardwick, K. G. Mad3 KEN boxes mediate 768 both and Mad3 turnover, and are critical for the spindle checkpoint. _PLoS_ 769 _One_ _2_ (4), e342 (2007). 770 771 43. Keskin, O., Ma, B., & Nussinov, R. Hot regions in protein–protein interactions: 772 The organization and contribution of structurally conserved hot spot 773 residues. _Journal of Molecular Biology_ 345(5), 1281–1294 (2005). 774 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 775 44. Tipton, A. R., Wang, K., Link, L., Bellizzi, J. J., Huang, H., Yen, T., & Liu, S. 776 T. BUBR1 and closed MAD2 (C-MAD2) interact directly to assemble a functional 777 mitotic checkpoint complex. _Journal of Biological Chemistry_ 286(24), 21173– 778 21179 (2011). 779 780 45. Xu, C., & Min, J. (2011). _Structure and function of WD40 domain proteins._ 781 _Protein Cell 2: 202–214_ . 782 783 46. Gatsogiannis, C., Merino, F., Roderer, D., Balchin, D., Schubert, E., Kuhlee, 784 A., ... & Raunser, S. (2018). Tc toxin activation requires unfolding and refolding 785 of a β-propeller. _Nature_, _563_ (7730), 209-213. 786 787 47. Liu, B., & Lee, Y. R. J. (2022). Spindle assembly and mitosis in plants. _Annual_ 788 _Review of Plant Biology_, _73_ (1), 227-254. 789 790 48. Zhu, J. K. (2016). Abiotic stress signalling and responses in 791 plants. _Cell_, _167_ (2), 313-324 792 793 49. Huang, H., Ullah, F., Zhou, D. X., Yi, M., & Zhao, Y. (2019). Mechanisms of 794 ROS regulation of plant development and stress responses. _Frontiers in plant_ 795 _science_, _10_, 800. 796 797 50. Shimotohno, A., Aki, S. S., Takahashi, N., & Umeda, M. (2021). Regulation 798 of the plant cell cycle in response to hormones and the environment. _Annual_ 799 _review of plant biology_, _72_ (1), 273-296. [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 800 51. Sablowski, R., & Gutierrez, C. (2022). Cycling in a crowd: Coordination of 801 plant cell division, growth, and cell fate. _The Plant Cell_, _34_ (1), 193-208. 802 803 52. McAinsh, A. D., & Kops, G. J. Principles and dynamics of spindle assembly 804 checkpoint signalling. _Nature Reviews Molecular Cell Biology_ 24(8), 543–559 805 (2023). 806 807 53. Tang, Z., Bharadwaj, R., Li, B., & Yu, H. (2001). Mad2-Independent inhibition 808 of APCCdc20 by the mitotic checkpoint protein BubR1. _Developmental cell_, _1_ (2), 809 227-237. 810 811 54. King, E. M., van der Sar, S. J., & Hardwick, K. G. (2007). Mad3 KEN boxes 812 mediate both Cdc20 and Mad3 turnover, and are critical for the spindle 813 checkpoint. _PloS one_, _2_ (4), e342. 814 815 55. Lara-Gonzalez, P., Scott, M. I., Diez, M., Sen, O., & Taylor, S. S. BubR1 816 blocks substrate recruitment to the APC/C in a KEN-box-dependent 817 manner. _Journal of Cell Science_ 124(24), 4332–4345 (2011). 818 819 56. Huang, H., Hittle, J., Zappacosta, F., Annan, R. S., Hershko, A., & Yen, T. J. 820 (2008). Phosphorylation sites in BubR1 that regulate kinetochore attachment, 821 tension, and mitotic exit. _The Journal of cell biology_, _183_ (4), 667-680. 822 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 823 57. Elowe, S., Dulla, K., Uldschmid, A., Li, X., Dou, Z., & Nigg, E. A. (2010). 824 Uncoupling of the spindle-checkpoint and chromosome-congression functions of 825 BubR1. _Journal of cell science_, _123_ (1), 84-94. 826 827 58. Bolanos-Garcia, V. M., & Blundell, T. L. (2011). BUB1 and BUBR1: 828 multifaceted kinases of the cell cycle. _Trends in biochemical sciences_, _36_ (3), 829 141-150. 830 831 59. Faesen, A. C., Thanasoula, M., Maffini, S., Breit, C., Müller, F., Van Gerwen, 832 S., ... & Musacchio, A. (2017). Basis of catalytic assembly of the mitotic 833 checkpoint complex. _Nature_, _542_ (7642), 498-502. 834 835 60. Kim, S., Sun, H., Ball, H. L., Wassmann, K., Luo, X., & Yu, H. Phosphorylation 836 of the spindle checkpoint protein Mad2 regulates its conformational 837 transition. _Proceedings of the National Academy of Sciences_ 107(46), 19772– 838 19777 (2010). 839 840 61. Yu, C. W., Fischer, E. S., Greener, J. G., Yang, J., Zhang, Z., Freund, S. M., 841 & Barford, D. (2025). Molecular mechanism of Mad2 conformational conversion 842 promoted by the Mad2 ‐ interaction motif of Cdc20. _Protein Science_, _34_ (4), 843 e70099. 844 845 62. Luo, X., Tang, Z., Rizo, J., & Yu, H. The Mad2 spindle checkpoint protein 846 undergoes similar major conformational changes upon binding to either Mad1 or 847 Cdc20. _Molecular Cell_ _9_ (1), 59–71 (2002). [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 848 63. Youn, J. H., & Kim, T. W. Functional insights of plant GSK3-like kinases: 849 Multitaskers in diverse cellular signal transduction pathways. _Molecular_ 850 _Plant_ 8(4), 552–565 (2015). 851 852 64. Zhao, X., Li, F., & Li, K. The 14 ‐ 3 ‐ 3 proteins: Regulators of plant metabolism 853 and stress responses. _Plant Biology_ 23(4), 531–539 (2021). 854 855 65. Loew, A., Ho, Y. K., Blundell, T., & Bax, B. (1998). Phosducin induces a 856 structural change in transducin βγ. _Structure_, _6_ (8), 1007-1019. 857 858 66. Fülöp, V., Böcskei, Z., & Polgár, L. (1998). Prolyl oligopeptidase: an unusual 859 β-propeller domain regulates proteolysis. _Cell_, _94_ (2), 161-170. 860 67. Simonetta, M., Manzoni, R., Mosca, R., Mapelli, M., Massimiliano, L., Vink, 861 M., ... & Ciliberto, A. (2009). The influence of catalysis on mad2 activation 862 dynamics. _PLoS biology_, _7_ (1), e1000010. 863 864 68. Hubner, N. C., Bird, A. W., Cox, J., Splettstoesser, B., Bandilla, P., Poser, I., 865 ... & Mann, M. (2010). Quantitative proteomics combined with BAC 866 TransgeneOmics reveals _in vivo_ protein interactions. _Journal of Cell_ 867 _Biology_, _189_ (4), 739-754. 868 869 69. Webb, B., & Sali, A. Protein structure modelling with MODELLER. 870 In _Functional Genomics: Methods and Protocols_ (Springer, New York, NY, 2017), 871 pp. 39–54. 872 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 873 70. Ren, J., Wen, L., Gao, X., Jin, C., Xue, Y., & Yao, X. DOG 1.0: Illustrator of 874 protein domain structures. _Cell Research_ 19(2), 271–273 (2009). 875 876 **Acknowledgements** 877 Thank you to Andrea Musacchio for inviting me and giving me the 878 opportunity to achieve this work. We are very grateful for working at the Max 879 Planck Institute in Dortmund. Special thanks to Dongqing Pan for his excellent 880 tips and relevant discussions. We also thank Sabine Wohlgemuth, Isabelle 881 Stender, Dorothee Vogt, Carolin Koerne and Franziska Müller for their technical 882 support. Thanks to Gilliana Dutra Neves Cruz for her comments and suggestions 883 in the preparation of this manuscript. We thank the Max Planck Institute of 884 Molecular Physiology and all of Department 1. This work was supported by Max 885 Planck Society - Max-Planck-Gesellschaft (127493). 886 887 **Ethics** 888 **Author contributions** 889 E.A.G.O. and E.V.N.G. collected the data and performed the models; I.S.S. 890 collected the data and performed the computational analysis; G. P. S and P.G. 891 C. They carried out data collection and performed alignments, consensus 892 analyses, and sequence topologies; M.A.L.C. conceived the project, performed 893 protein expression and purification, designed the protein experiments, and 894 prepared and wrote the manuscript. 895 896 **Competing interests** 897 The authors declare that they have no competing interests. [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 898 899 **Data availability** 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 923 **Figures and Legends** 924 **Figure 1** 925 926 927 **Figure 1- Graphical representation of the strategy for identifying protein‒** 928 **protein interactions (PPIs) involved in the formation of the MCC core in** _**A.**_ 929 _**thaliana**_ **.** First, the MCC core components (AtBubR1, AtCDC20, and AtMad2) 930 are expressed and purified * _in vitro_ - as full-length proteins. Next, a cross-linking 931 reaction is performed using disuccinimidyl dibutyric urea (DSBU). An enzymatic 932 digestion protocol is subsequently applied, followed by peptide identification via 933 mass spectrometry. The process concludes with the mapping of cross-links and 934 a comparative analysis against structural models. 935 936 937 938 939 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 940 **Figure 2** 941 942 943 944 945 946 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 947 **Figure 2 – Expression, purification and structural analysis of core MCC** 948 **proteins of** _**Arabidopsis thaliana**_ **– a,** Schematic representation of the pLIB 949 vector constructs of SAC proteins, including MBP AtCDC20 (green), orange 950 (6his-AtBub1), 6his AtBubR1 (blue), AtMad2 6his (yellow), AtBub1 6his (pink) 951 and AtBub3:1 6his (lilac). **b**, Schematic representation of the bigBac assembly of 952 the _Arabidopsis thaliana_ core MCC proteins, including MBP AtCDC20 (green), 953 6his AtBubR1 (blue) and AtMad2 6his (yellow). **c**, Size-exclusion 954 chromatography with core MCC proteins (MBP AtCDC20, 6His AtBubR1 and 955 6His AtMad2) using Sepharose S6 and isocratic conditions. Red arrows indicate 956 only AtCDC20 and AtBubR1 in a complex. **d**, SDS‒PAGE analysis of size 957 exclusion chromatography with sample A (core MCC proteins). Red arrows 958 indicate only AtCDC20 and AtBubR1 in a complex. The coloured bars indicate 959 the relative positions of the SAC proteins: green (MBP-AtCDC20), blue (6-His 960 AtBubR1), and yellow (6His-AtMad2). The red arrow indicates a slot with only the 961 MBP AtCDC20 and 6his AtBubR1. **e**, Cartoon representation of the conserved 962 topology of plant CDC20 showing the KEN box (salmon), MIM motifs (black), 963 seven WD40 repeat blades: 1 (red), 2 (green), 3 (blue), 4 (orange), 5 (magenta), 964 6 (cyan) and 7 (yellow) and the glycine box (brown). **f**, Cartoon representation of 965 the β-propeller of AtCDc20 showing seven WD40 repeat blades, MIM motifs, and 966 glycine boxes. The colour scheme is the same as that in Figure 2e. N- and C 967 termini were omitted. **g**, Cartoon representation of the AtBubR1 N-terminal region 968 topology with KEN box 1 and KEN box 2 (orange) separated by the conserved 969 TPR domain composed of TPR1 (lilac), TPR2 (green), and TPR3 (salmon). **h**, 970 Cartoon representation of the TPR domain of AtBubR1. The colour scheme is the 971 same as that in Figure 2g. **i**, Cartoon representation of the conserved topology of [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 972 AtMad2 with the HORMA domain (yellow) and the MIM motif (red). **j**, Cartoon 973 representation of AtMad2: HORMA domain. The colour scheme is the same as 974 that in Figure 2i. Plant species: At ( _Arabidopsis thaliana_ ), Pv ( _Phaseolus vulgaris_ ), 975 Sl ( _Solanum lycopersicum_ ), Os ( _Oryza sativa_ ) and Sb ( _Sorghum bicolor_ ). 976 977 **Figure 3** 978 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 979 **Figure 3 – Protein‒protein interactions for core MCC formation in** 980 _**Arabidopsis**_ _**thaliana**_ **in** _**Arabidopsis**_ _**thaliana**_ **—a,** Size-exclusion 981 chromatography with core MCC proteins plus AtBubu3:1 and AtBub1. **b**, SDS‒ 982 PAGE analysis of size-exclusion chromatography with the core MCC proteins 983 plus AtBubu3:1 and AtBub1. The coloured bars indicate the relative positions of 984 the SAC proteins: green (MBP-AtCDC20), orange (6his-AtBub1), blue (6-his 985 AtBubR1), purple (6his-Bub3:1), and yellow (6his-AtMad2). **c**, SDS‒PAGE 986 analysis of DSBU crosslinking results with SAC proteins. 1 without and 2 with 987 DSBU. The coloured bars indicate the relative positions of the SAC proteins: red 988 (cross-linked proteins), green (MBP-AtCDC20), orange (6his-AtBub1), blue (6 989 his-AtBubR1), purple (6his-Bub3:1), and yellow (6his-AtMad2). **d**, Table 1, 990 Summary of the nonredundant crosslinks obtained in experiments A, B, C and D, 991 distributed according to different regions of the proteins. **e**, Circular plot showing 992 the nonredundant crosslinks obtained in experiments with MBP-AtCDC20, 6His 993 AtBubR1, AtMad2, AtBub1 and 6His AtBub3:1 (A); **f** Circular plot showing the 994 nonredundant crosslinks obtained in experiments with MBP-AtCDC20, 6His 995 AtBubR1 and AtMad2 (B). **g**, Circular plot showing the nonredundant crosslinks 996 obtained in experiments with 8TEV-AtCDC20 and AtMad2-6his (C). **h**, Circular 997 plot showing the nonredundant crosslinks obtained in experiments with 8TEV 998 AtCDC20,6-HHis- and AtMad2-6His (D). Red and blue lines indicate intra- and 999 intermolecular nonredundant crosslinks, respectively. The dashed black circle in 1000 Figure 3 e-h indicates the regions of AtCDC20, AtMad2 and AtBubR1 that 1001 participate in hierarchical interactions. Consensus sequence logos with 1002 interaction motifs included **i** KIM (AtMad2), **j** MIM1 (AtCDC20), **k** MIM2 1003 (AtCDC20), **l** KEN box 2 (AtBubR1), **m** KEN box 3 (AtBubR1), and **n** KET box [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1004 (AtBubR1). The black circles indicate the amino acids included in the peptide 1005 identified in the crosslinking experiment. Red circles indicate a conserved 1006 sequence that, in HsMad2, has been replaced by a GSG to generate stabilized 1007 O-Mad2 (Mapelli et al., 2007). Red boxes indicate regions containing peptides 1008 identified within the hierarchical interaction between AtMad2, AtBubR1 and 1009 AtCDC20. 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020 1021 1022 1023 1024 1025 1026 1027 1028 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1029 **Figure 4** 1030 1031 1032 1033 1034 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1035 **Figure 4 – Mad2 dimer and tetramer structure in** _**Arabidopsis thaliana**_ **based** 1036 **on crooslink experiments—a,** Cartoon representation showing an overview of 1037 the Mad2 dimer. O-Mad2 (orange and MIM motif magenta), C-Mad2 (yellow and 1038 MIM motif cyan) and “safety belt” (black). **b and c**, Cartoon representation 1039 showing detailed views of the interaction motif (MIM motif) in the AtMad2 dimer 1040 interface. The colour scheme is the same as that in Figure 4a. Colours elements: 1041 nitrogen (blue), oxygen (red) and potential hydrogen bond formation (≤ 3 Å) 1042 between the amino acids of the primary hydrophobic core are dashed black lines; 1043 **d**, Consensus sequence logos with AtMad2’s MIM motifs included; pink circles 1044 indicate a prediction for 14-3-3 phosphorylation; **e** and **f**, Surface view from the 1045 bottom and top, respectively, with 180° rotation and exposed amino acids S61 1046 and K123. Colours elements at S61 and K123: nitrogen (blue) and oxygen (red); 1047 **g**, Cartoon representation of the Mad2 dimer showing the distances in angstroms 1048 between amino acids S61 and K123; **h**, Cartoon representation of the proposed 1049 model of AtMad2 tetramer formation with the interaction point between the two 1050 Mad2 dimers on S61 and K123, indicated by black dashed lines. The model used 1051 2v64 (Mapelli et al., 2007). 1052 1053 1054 1055 1056 1057 1058 1059 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1060 **Figure 5** 1061 1062 1063 1064 1065 1066 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1067 **Figure 5 – Positioning of two AtCDC20 molecules that interact with** 1068 **AtBubR1 and AtMad2 for core MCC formation in** _**Arabidopsis thaliana**_ **. a,** 1069 Cartoon representation of two AtCDC20 molecules, AtBubR1, and AtMad2, 1070 based on cross-linking experiments. AtCDC20 [MC1] (green), AtCDC20 [MC2] (gray), 1071 AtMad2 (yellow) and BubR1 (blue). MIM2 (red), MIM3 (magenta), glycine box 1072 (salmon), AtMad2’s MIM (cyan), and KEN boxes 1 and 2 of AtBubR1 (orange) of 1073 AtCDC20. **b,** Intermolecular crosslinking between AtCDC20 [MC1] (S300 and T303) 1074 and AtMad2 (K123); distance measured in angstroms, 18,7 Å and 16,9 Å, 1075 respectively; **c,** intermolecular crosslinking between AtCDC20 [MC1] (T87) and 1076 AtMad2 (K114), 17,2 Å; **d**, intermolecular crosslinking between AtCDC20 [MC1] 1077 (K394) and AtBubR1 (K227) and between AtCDC20 [MC2] (K221) and AtBubR1 1078 (K176), 22,4 Å and 14,2 Å, respectively. **e**, Cartoon representation of two 1079 AtCDC20 molecules, AtBubR1, and AtMad2, based on crosslinking experiments 1080 with 180° rotation and using the same colour scheme as in Figure 5a. **f**, 1081 Intramolecular crosslinking between amino acids S123, K123 and K190 1082 (AtMad2), 9,1 Å and 13,1 Å, respectively. **g**, Intramolecular crosslinking between 1083 amino acids K221, S226 and S256 (AtCDC20 [ MC1] ), 10,6 Å and 10,3 Å, 1084 respectively. **h**, Intramolecular crosslinking between amino acids K123 and K191 1085 (AtBubR1), 14,1 Å. **i**, Intramolecular crosslinking between amino acids K221, 1086 S226 and S256 (AtCDC20 [ MC2] ), 10,5 Å and 10,0 Å, respectively. Colours: nitrogen 1087 (blue), oxygen (red). The 5LCW model was used (Alfieri et al., 2017). 1088 1089 1090 1091 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1092 **Figure 6** 1093 1094 1095 1096 1097 1098 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1099 **Figure 6 – Characteristics of the AtCDC20 protein that enable structural** 1100 **flexibility of the β-propeller domain. a**, Cartoon representation of the β 1101 propeller of AtCDc20 showing seven WD40 repeat blades (gray); AtCDC20’s 1102 MIM2 and MIM3; red and magenta, respectively; the amino acid L132 (β-sheet of 1103 blade1) is shown in red; and the amino acids L426, F428 and W429 (β-sheet of 1104 blade 7) are shown in green. Amino acid shown in stick **b**, Detailed views of the 1105 amino acid L132 (β-sheet of blade1) shown in red; the amino acids L426, F428 1106 and W429 (β-sheet of blade 7) are shown in green. Colours elements: nitrogen 1107 (blue), oxygen (red), the distances in angstroms between hydrophobic amino 1108 acids (red): L132 and the amino acids L426, F428 and W429 are dashed black 1109 lines. **c,** Detailed views of the amino acids with potential hydrogen bond formation 1110 between components of the core MCC in the context of the SAC: E33 KEN box1 1111 (AtBubR1 in orange); G335, G336, G337 and D338 glycine boxes (AtCDC20 in 1112 salmon); and R134, S138 and T141 AtMad2’s MIM (cyan). The distances in 1113 angstroms are shown in red. The model used 4aez (Chao et al., 2007). **d**, 1114 Detailed views of the amino acids with unlikely hydrogen bond formation between 1115 components of the core MCC in the context of APC/C: E33 KEN box1 on 1116 AtBubR1 (orange); G335, G336, G337 and D338 glycine boxes (salmon); and 1117 R134, S138 and T141 AtMad2’s MIM (cyan). The distances in angstroms are 1118 shown in red. The 5LCW model was used (Alfieri et al., 2017). **e**, Consensus 1119 sequence logos with the glycine box included; amino acids with potential 1120 hydrogen bonds are indicated by coloured circles, with E33 on KEN box 1 at 1121 AtBubR1 (orange) or with R134, 138 and T141 on the AtMad2 of the MIM (cyan). 1122 **f**, Consensus sequence logos with KEN box 1 (AtBubR1). The black circles [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1123 indicate the amino acids included in the peptide identified in the crosslinking 1124 experiment. 1125 1126 **Figure 7** 1127 1128 1129 1130 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.06.764920; this version posted November 9, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.06.764920) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1131 **Figure 7 – Positioning of the AtMad2 dimer in the core MCC of** _**Arabidopsis**_ 1132 _**thaliana**_ **. a**, Cartoon representation showing measurements of the distances 1133 between amino acid S221 of AtCDC20 (red) and amino acids K114, S117 and 1134 S183 of AtMad2 (yellow). The measurements in angstroms (dashed black lines) 1135 are shown in red. Amino acids are also shown: KEN box 1 of AtBubR1 (orange), 1136 AtMad2’s MIM (cyan) and the glycine box of AtCDC20 (salmon). **b**, Detailed 1137 views showing the amino acids K31, E32 and N33 (KEN box 1 of AtBubR1) in 1138 orange; R134, S138, S139, V140 and S141 of AtMad2’s MIM (cyan); and G333, 1139 G334, G335, G336 and G336 (Glycine box of AtCDC20) in salmon. **c**, Schematic 1140 model for the composition and organization of the core MCC in _Arabidopsis_ 1141 _thaliana_ on the basis of the crosslinking results. 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155