Structure and assembly of the NOT10:11 module of the CCR4-NOT complex

Abstract

NOT1, NOT10, and NOT11 form a conserved module in the CCR4-NOT complex, critical for post-transcriptional regulation in eukaryotes, but how this module contributes to the functions of the CCR4-NOT remains poorly understood. Here, we present cryo-EM structures of human and chicken NOT1:NOT10:NOT11 ternary complexes to sub-3 Å resolution, revealing an evolutionarily conserved, flexible structure. Through biochemical dissection studies, which include the Drosophila orthologs, we show that the module assembly is hierarchical, with NOT11 binding to NOT10, which then organizes it for binding to NOT1. A short proline-rich motif in NOT11 stabilizes the entire module assembly.

Fig. 1. Structure of the vertebrate NOT10:NOT11 module.

a Domain organization scheme of human NOT10 and NOT11 and the N-terminal portion of NOT1. NOT1 comprises two HEAT repeat domains, HEAT_N and HEAT_C. NOT10 consists of one large TPR repeat domain. NOT11 features an ɑ-helical N-terminal domain NOT11_N and a C-terminal MIF4G domain connected by an extended linker, which harbors the NOT10-binding motif NOT10-BM. b Cryo-EM reconstruction of the human NOT1:10:11 complex. c Cryo-EM reconstruction of the chicken NOT1:10:11 complex. d Structure of the human complex. NOT1 comprises two helical repeat domains, and NOT10 is one highly curved TPR repeat domain. Only the extended NOT11_Linker, bound to NOT10, is ordered, while the NOT11_MIF4G domain is flexible and disordered. Boxed regions are shown in more detail in Fig. 2 and Supplementary Fig. 1. e Structure of the chicken complex. In contrast to the human complex, the NOT1-HEAT_C domain is disordered, but the NOT11_N domain is ordered. f Schematic representation of the NOT10:11 module. The module assembles around NOT11_Linker and NOT10. NOT11_MIF4G and NOT1_C domains are flexible. A pair of critical prolines in NOT11_Linker is marked in red.

Fig. 2. The conserved proline-rich motif in NOT11 stabilizes binding to NOT10.

a Closeup view of the C-terminal part of the human NOT11_Linker bound to NOT10. b Closeup view of the human NOT11 proline-rich motif bound to NOT10. c CryoEM density for the C-terminal part of the human NOT11_Linker. d CryoEM density for the human NOT11 proline-rich motif. e CryoEM density for the C-terminal part of the chicken NOT11_Linker. f CryoEM density for the chicken NOT11 proline-rich motif.

Fig. 3. The NOT10:NOT11 module assembles sequentially.

a Schematic representation of the constructs used in this study. b GST pulldown assays to identify the NOT11-binding region in NOT10. c GST pulldown assay to identify the NOT10-binding region in NOT11. d GST pulldown assay showing that the NOT11 NOT10-binding motif (N10BM) is necessary and sufficient for binding NOT10. e A NOT11 P289A–P290A double mutant disrupts NOT10 binding. f The NOT10-BM is also necessary and sufficient for the NOT10:NOT11 interaction in Drosophila. g Unlike the NOT10:NOT11 heterodimer, NOT11 alone cannot associate with NOT1. h NOT10 alone cannot bind NOT1, but requires association with NOT11. Source data displaying uncropped gel images corresponding to b–h are available in Supplementary Figs. 7 and 8.