Meiotic Chromosome Structure, the Synaptonemal Complex, and Infertility.

In meiosis, homologous chromosome synapsis is mediated by a supramolecular protein structure, the synaptonemal complex (SC), that assembles between homologous chromosome axes. The mammalian SC comprises at least eight largely coiled-coil proteins that interact and self-assemble to generate a long, zipper-like structure that holds homologous chromosomes in close proximity and promotes the formation of genetic crossovers and accurate meiotic chromosome segregation. In recent years, numerous mutations in human SC genes have been associated with different types of male and female infertility. Here, we integrate structural information on the human SC with mouse and human genetics to describe the molecular mechanisms by which SC mutations can result in human infertility. We outline certain themes in which different SC proteins are susceptible to different types of disease mutation and how genetic variants with seemingly minor effects on SC proteins may act as dominant-negative mutations in which the heterozygous state is pathogenic.

Structural basis for Mis18 complex assembly and its implications for centromere maintenance.

The centromere, defined by the enrichment of CENP-A (a Histone H3 variant) containing nucleosomes, is a specialised chromosomal locus that acts as a microtubule attachment site. To preserve centromere identity, CENP-A levels must be maintained through active CENP-A loading during the cell cycle. A central player mediating this process is the Mis18 complex (Mis18α, Mis18ß and Mis18BP1), which recruits the CENP-A-specific chaperone HJURP to centromeres for CENP-A deposition. Here, using a multi-pronged approach, we characterise the structure of the Mis18 complex and show that multiple hetero- and homo-oligomeric interfaces facilitate the hetero-octameric Mis18 complex assembly composed of 4 Mis18α, 2 Mis18ß and 2 Mis18BP1. Evaluation of structure-guided/separation-of-function mutants reveals structural determinants essential for cell cycle controlled Mis18 complex assembly and centromere maintenance. Our results provide new mechanistic insights on centromere maintenance, highlighting that while Mis18α can associate with centromeres and deposit CENP-A independently of Mis18ß, the latter is indispensable for the optimal level of CENP-A loading required for preserving the centromere identity.

Protein target highlights in CASP15: Analysis of models by structure providers.

We present an in-depth analysis of selected CASP15 targets, focusing on their biological and functional significance. The authors of the structures identify and discuss key protein features and evaluate how effectively these aspects were captured in the submitted predictions. While the overall ability to predict three-dimensional protein structures continues to impress, reproducing uncommon features not previously observed in experimental structures is still a challenge. Furthermore, instances with conformational flexibility and large multimeric complexes highlight the need for novel scoring strategies to better emphasize biologically relevant structural regions. Looking ahead, closer integration of computational and experimental techniques will play a key role in determining the next challenges to be unraveled in the field of structural molecular biology.

Crossover recombination and synapsis are linked by adjacent regions within the N terminus of the Zip1 synaptonemal complex protein.

Accurate chromosome segregation during meiosis relies on the prior establishment of at least one crossover recombination event between homologous chromosomes. Most meiotic recombination intermediates that give rise to interhomolog crossovers are embedded within a hallmark chromosomal structure called the synaptonemal complex (SC), but the mechanisms that coordinate the processes of SC assembly (synapsis) and crossover recombination remain poorly understood. Among known structural components of the budding yeast SC, the Zip1 protein is unique for its independent role in promoting crossover recombination; Zip1 is specifically required for the large subset of crossovers that also rely on the meiosis-specific MutSγ complex. Here we report that adjacent regions within Zip1's N terminus encompass its crossover and synapsis functions. We previously showed that deletion of Zip1 residues 21-163 abolishes tripartite SC assembly and prevents robust SUMOylation of the SC central element component, Ecm11, but allows excess MutSγ crossover recombination. We find the reciprocal phenotype when Zip1 residues 2-9 or 10-14 are deleted; in these mutants SC assembles and Ecm11 is hyperSUMOylated, but MutSγ crossovers are strongly diminished. Interestingly, Zip1 residues 2-9 or 2-14 are required for the normal localization of Zip3, a putative E3 SUMO ligase and pro-MutSγ crossover factor, to Zip1 polycomplex structures and to recombination initiation sites. By contrast, deletion of Zip1 residues 15-20 does not detectably prevent Zip3's localization at Zip1 polycomplex and supports some MutSγ crossing over but prevents normal SC assembly and Ecm11 SUMOylation. Our results highlight distinct N terminal regions that are differentially critical for Zip1's roles in crossing over and SC assembly; we speculate that the adjacency of these regions enables Zip1 to serve as a liaison, facilitating crosstalk between the two processes by bringing crossover recombination and synapsis factors within close proximity of one another.

Gastrointestinal nematodes in two galliform birds from South Africa: patterns associated with host sex and age.

Parasite ecology has recently focused on elucidating patterns and processes that shape helminth communities in avian hosts. However, helminths parasitizing gamebirds are still poorly understood. Here we describe the gastrointestinal nematode fauna of Swainson's spurfowl, Pternistis swainsonii (Phasianidae) and helmeted guineafowl, Numida meleagris (Numididae), collected at three and four localities, respectively, in South Africa and analyze the prevalence, mean abundance and diversity of their helminth communities. Eleven nematode species were collected from spurfowl, which had a mean number of nematode species per host of 3.01 ± 0.18, whereas guineafowl harboured 15 nematode species, with a mean number of nematode species per host of 3.93 ± 0.12. Focusing on the most prevalent species, we also asked if host sex and/or age were associated with infracommunity structure with regard to nematode counts and species richness, as well as the species and taxonomic composition of infracommunities. While pooling data of nematode species masked the influence of host characteristics on helminth communities, analysis of individual nematode species revealed a number of patterns. In particular, adult female bias was seen in Tetrameres swainsonii in spurfowl and in Allodapa dentigera and Gongylonema congolense in guineafowl; Acuaria gruveli reached higher numbers in adult spurfowl than in juveniles, and helminth infracommunities in juvenile male guineafowl were more species rich than those in adult males. Combined, our results suggest that helminth communities of spurfowl and guineafowl are associated with a complex interplay of numerous factors, including host characteristics, parasite traits and environmental conditions.

A molecular model for self-assembly of the synaptonemal complex protein SYCE3.

The synaptonemal complex (SC) is a supramolecular protein assembly that mediates homologous chromosome synapsis during meiosis. This zipper-like structure assembles in a continuous manner between homologous chromosome axes, enforcing a 100-nm separation along their entire length and providing the necessary three-dimensional framework for cross-over formation. The mammalian SC comprises eight components-synaptonemal complex protein 1-3 (SYCP1-3), synaptonemal complex central element protein 1-3 (SYCE1-3), testis-expressed 12 (TEX12), and six6 opposite strand transcript 1 (SIX6OS1)-arranged in transverse and longitudinal structures. These largely α-helical, coiled-coil proteins undergo heterotypic interactions, coupled with recursive self-assembly of SYCP1, SYCE2-TEX12, and SYCP2-SYCP3, to achieve the vast supramolecular SC structure. Here, we report a novel self-assembly mechanism of the SC central element component SYCE3, identified through multi-angle light scattering and small-angle X-ray scattering (SAXS) experiments. These analyses revealed that SYCE3 adopts a dimeric four-helical bundle structure that acts as the building block for concentration-dependent self-assembly into a series of discrete higher-order oligomers. We observed that this is achieved through staggered lateral interactions between self-assembly surfaces of SYCE3 dimers and through end-on interactions that likely occur through intermolecular domain swapping between dimer folds. These mechanisms are combined to achieve potentially limitless SYCE3 assembly, particularly favoring formation of dodecamers of three laterally associated end-on tetramers. Our findings extend the family of self-assembling proteins within the SC and reveal additional means for structural stabilization of the SC central element.

Molecular structure of human synaptonemal complex protein SYCE1.

The reduction in chromosome number during meiosis is essential for the production of haploid germ cells and thereby fertility. To achieve this, homologous chromosomes are first synapsed together by a protein assembly, the synaptonemal complex (SC), which permits genetic exchange by crossing over and the subsequent accurate segregation of homologues. The mammalian SC is formed of a zipper-like array of SYCP1 molecules that bind together homologous chromosomes through self-assembly in the midline that is structurally supported by the central element. The SC central element contains five proteins-SYCE1, SYCE3, SIX6OS1, and SYCE2-TEX12-that permit SYCP1 assembly to extend along the chromosome length to achieve full synapsis. Here, we report the structure of human SYCE1 through solution biophysical methods including multi-angle light scattering and small-angle X-ray scattering. The structural core of SYCE1 is formed by amino acids 25-179, within the N-terminal half of the protein, which mediates SYCE1 dimerization. This α-helical core adopts a curved coiled-coil structure of 20-nm length in which the two chains are arranged in an anti-parallel configuration. This structure is retained within full-length SYCE1, in which long C-termini adopt extended conformations to achieve an elongated molecule of over 50 nm in length. The SYCE1 structure is compatible with it functioning as a physical strut that tethers other components to achieve structural stability of the SC central element.

Maintenance of clinical remission in early axial spondyloarthritis following certolizumab pegol dose reduction.

BACKGROUND: The best strategy for maintaining clinical remission in patients with axial spondyloarthritis (axSpA) has not been defined. C-OPTIMISE compared dose continuation, reduction and withdrawal of the tumour necrosis factor inhibitor certolizumab pegol (CZP) following achievement of sustained remission in patients with early axSpA. METHODS: C-OPTIMISE was a two-part, multicentre phase 3b study in adults with early active axSpA (radiographic or non-radiographic). During the 48-week open-label induction period, patients received CZP 200 mg every 2 weeks (Q2W). At Week 48, patients in sustained remission (Ankylosing Spondylitis Disease Activity Score (ASDAS) <1.3 at Weeks 32/36 and 48) were randomised to double-blind CZP 200 mg Q2W (full maintenance dose), CZP 200 mg every 4 weeks (Q4W; reduced maintenance dose) or placebo (withdrawal) for a further 48 weeks. The primary endpoint was remaining flare-free (flare: ASDAS ≥2.1 at two consecutive visits or ASDAS >3.5 at any time point) during the double-blind period. RESULTS: At Week 48, 43.9% (323/736) patients achieved sustained remission, of whom 313 were randomised to CZP full maintenance dose, CZP reduced maintenance dose or placebo. During Weeks 48 to 96, 83.7% (87/104), 79.0% (83/105) and 20.2% (21/104) of patients receiving the full maintenance dose, reduced maintenance dose or placebo, respectively, were flare-free (p<0.001 vs placebo in both CZP groups). Responses in radiographic and non-radiographic axSpA patients were comparable. CONCLUSIONS: Patients with early axSpA who achieve sustained remission at 48 weeks can reduce their CZP maintenance dose; however, treatment should not be completely discontinued due to the high risk of flare following CZP withdrawal. TRIAL REGISTRATION NUMBER: NCT02505542, ClinicalTrials.gov.

Target highlights in CASP13: Experimental target structures through the eyes of their authors.

The functional and biological significance of selected CASP13 targets are described by the authors of the structures. The structural biologists discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP13 experiment.

An Engineered Complement Factor H Construct for Treatment of C3 Glomerulopathy.

Background C3 glomerulopathy (C3G) is associated with dysregulation of the alternative pathway of complement activation, and treatment options for C3G remain limited. Complement factor H (FH) is a potent regulator of the alternative pathway and might offer a solution, but the mass and complexity of FH makes generation of full-length FH far from trivial. We previously generated a mini-FH construct, with FH short consensus repeats 1-5 linked to repeats 18-20 (FH1-5^18-20), that was effective in experimental C3G. However, the serum t1/2 of FH1-5^18-20 was significantly shorter than that of serum-purified FH.Methods We introduced the oligomerization domain of human FH-related protein 1 (denoted by R1-2) at the carboxy or amino terminus of human FH1-5^18-20 to generate two homodimeric mini-FH constructs (FHR1-2^1-5^18-20 and FH1-5^18-20^R1-2, respectively) in Chinese hamster ovary cells and tested these constructs using binding, fluid-phase, and erythrocyte lysis assays, followed by experiments in FH-deficient Cfh-/- mice.Results FHR1-2^1-5^18-20 and FH1-5^18-20^R1-2 homodimerized in solution and displayed avid binding profiles on clustered C3b surfaces, particularly FHR1-2^1-5^18-20 Each construct was >10-fold more effective than FH at inhibiting cell surface complement activity in vitro and restricted glomerular basement membrane C3 deposition in vivo significantly better than FH or FH1-5^18-20 FH1-5^18-20^R1-2 had a C3 breakdown fragment binding profile similar to that of FH, a >5-fold increase in serum t1/2 compared with that of FH1-5^18-20, and significantly better retention in the kidney than FH or FH1-5^18-20Conclusions FH1-5^18-20^R1-2 may have utility as a treatment option for C3G or other complement-mediated diseases.

Limited radiographic progression and sustained reductions in MRI inflammation in patients with axial spondyloarthritis: 4-year imaging outcomes from the RAPID-axSpA phase III randomised trial.

OBJECTIVES: To report 4-year imaging outcomes in the RAPID-axSpA (NCT01087762) study of patients with ankylosing spondylitis (AS) and non-radiographic axial spondyloarthritis (nr-axSpA), treated with certolizumab pegol (CZP). METHODS: This phase III, randomised trial was placebo-controlled and double-blind to week 24, dose-blind to week 48 and open-label to week 204. Patients fulfilling the Assessment of Spondyloarthritis International Society (ASAS) axSpA criteria with active disease were stratified (AS/nr-axSpA) according to the modified New York (mNY) criteria at randomisation. Spinal radiographs were assessed using the modified Stoke Ankylosing Spondylitis Spine Score (mSASSS). MRI inflammation used the Spondyloarthritis Research Consortium of Canada (SPARCC) score for sacroiliac joints (SIJ) and the Berlin spinal score (remission defined as SPARCC <2 and Berlin ≤2, respectively). RESULTS: MRI improvements from baseline (BL) to week 12 were maintained to week 204 (SPARCC BL: AS=8.5, nr-axSpA=7.5; SPARCC week 204: AS=1.3, nr-axSpA=2.4; Berlin BL: AS=7.4, nr-axSpA=4.4; Berlin week 204: AS=2.6, nr-axSpA=1.9). 66.7% of patients with AS and 69.6% of patients with nr-axSpA with BL SPARCC scores ≥2, and 65.4% of patients with AS and 57.3% of patients with nr-axSpA with BL Berlin score >2, achieved remission at week 204. Mean mSASSS change in AS from BL to week 204 was 0.98 (95% CI 0.34, 1.63); 0.67 (95% CI 0.21,1.13) from BL to week 96; and 0.31 (95% CI 0.02,0.60) from week 96 to week 204. Corresponding nr-axSpA changes were 0.06 (95% CI -0.17,0.28), -0.01 (95% CI -0.19,0.17) and 0.07 (95% CI -0.07,0.20). 4.5% of patients with nr-axSpA fulfilled the mNY criteria at week 204, while 4.3% of patients with AS no longer did so. CONCLUSIONS: In patients with CZP-treated axSpA, rapid decreases in spinal and SIJ MRI inflammation were maintained to week 204. Overall, 4-year spinal progression was low, with less progression during years 2-4 than 0-2. Radiographic SIJ grading changes demonstrated limited progression. TRIAL REGISTRATION NUMBER: NCT01087762; Post-results.

The crystal structure of SUN1-KASH6 reveals an asymmetric LINC complex architecture compatible with nuclear membrane insertion.

The LINC complex transmits cytoskeletal forces into the nucleus to control the structure and movement of nuclear contents. It is formed of nuclear SUN and cytoplasmic KASH proteins, which interact within the nuclear lumen, immediately below the outer nuclear membrane. However, the symmetrical location of KASH molecules within SUN-KASH complexes in previous crystal structures has been difficult to reconcile with the steric requirements for insertion of their immediately upstream transmembrane helices into the outer nuclear membrane. Here, we report the crystal structure of the SUN-KASH complex between SUN1 and JAW1/LRMP (KASH6) in an asymmetric 9:6 configuration. This intertwined assembly involves two distinct KASH conformations such that all six KASH molecules emerge on the same molecular surface. Hence, they are ideally positioned for insertion of upstream sequences into the outer nuclear membrane. Thus, we report a SUN-KASH complex architecture that appears to be directly compatible with its biological role.

A conserved CENP-E region mediates BubR1-independent recruitment to the outer corona at mitotic onset.

The outer corona plays an essential role at the onset of mitosis by expanding to maximize microtubule attachment to kinetochores.1,2 The low-density structure of the corona forms through the expansion of unattached kinetochores. It comprises the RZZ complex, the dynein adaptor Spindly, the plus-end directed microtubule motor centromere protein E (CENP-E), and the Mad1/Mad2 spindle-assembly checkpoint proteins.3,4,5,6,7,8,9,10 CENP-E specifically associates with unattached kinetochores to facilitate chromosome congression,11,12,13,14,15,16 interacting with BubR1 at the kinetochore through its C-terminal region (2091-2358).17,18,19,20,21 We recently showed that CENP-E recruitment to BubR1 at the kinetochores is both rapid and essential for correct chromosome alignment. However, CENP-E is also recruited to the outer corona by a second, slower pathway that is currently undefined.19 Here, we show that BubR1-independent localization of CENP-E is mediated by a conserved loop that is essential for outer-corona targeting. We provide a structural model of the entire CENP-E kinetochore-targeting domain combining X-ray crystallography and Alphafold2. We reveal that maximal recruitment of CENP-E to unattached kinetochores critically depends on BubR1 and the outer corona, including dynein. Ectopic expression of the CENP-E C-terminal domain recruits the RZZ complex, Mad1, and Spindly, and prevents kinetochore biorientation in cells. We propose that BubR1-recruited CENP-E, in addition to its essential role in chromosome alignment to the metaphase plate, contributes to the recruitment of outer corona proteins through interactions with the CENP-E corona-targeting domain to facilitate the rapid capture of microtubules for efficient chromosome alignment and mitotic progression.

Molecular insights into LINC complex architecture through the crystal structure of a luminal trimeric coiled-coil domain of SUN1.

The LINC complex, consisting of interacting SUN and KASH proteins, mechanically couples nuclear contents to the cytoskeleton. In meiosis, the LINC complex transmits microtubule-generated forces to chromosome ends, driving the rapid chromosome movements that are necessary for synapsis and crossing over. In somatic cells, it defines nuclear shape and positioning, and has a number of specialised roles, including hearing. Here, we report the X-ray crystal structure of a coiled-coiled domain of SUN1's luminal region, providing an architectural foundation for how SUN1 traverses the nuclear lumen, from the inner nuclear membrane to its interaction with KASH proteins at the outer nuclear membrane. In combination with light and X-ray scattering, molecular dynamics and structure-directed modelling, we present a model of SUN1's entire luminal region. This model highlights inherent flexibility between structured domains, and raises the possibility that domain-swap interactions may establish a LINC complex network for the coordinated transmission of cytoskeletal forces.

Structural maturation of SYCP1-mediated meiotic chromosome synapsis by SYCE3.

In meiosis, a supramolecular protein structure, the synaptonemal complex (SC), assembles between homologous chromosomes to facilitate their recombination. Mammalian SC formation is thought to involve hierarchical zipper-like assembly of an SYCP1 protein lattice that recruits stabilizing central element (CE) proteins as it extends. Here we combine biochemical approaches with separation-of-function mutagenesis in mice to show that, rather than stabilizing the SYCP1 lattice, the CE protein SYCE3 actively remodels this structure during synapsis. We find that SYCP1 tetramers undergo conformational change into 2:1 heterotrimers on SYCE3 binding, removing their assembly interfaces and disrupting the SYCP1 lattice. SYCE3 then establishes a new lattice by its self-assembly mimicking the role of the disrupted interface in tethering together SYCP1 dimers. SYCE3 also interacts with CE complexes SYCE1-SIX6OS1 and SYCE2-TEX12, providing a mechanism for their recruitment. Thus, SYCE3 remodels the SYCP1 lattice into a CE-binding integrated SYCP1-SYCE3 lattice to achieve long-range synapsis by a mature SC.

The meiotic LINC complex component KASH5 is an activating adaptor for cytoplasmic dynein.

Cytoplasmic dynein-driven movement of chromosomes during prophase I of mammalian meiosis is essential for synapsis and genetic exchange. Dynein connects to chromosome telomeres via KASH5 and SUN1 or SUN2, which together span the nuclear envelope. Here, we show that KASH5 promotes dynein motility in vitro, and cytosolic KASH5 inhibits dynein's interphase functions. KASH5 interacts with a dynein light intermediate chain (DYNC1LI1 or DYNC1LI2) via a conserved helix in the LIC C-terminal, and this region is also needed for dynein's recruitment to other cellular membranes. KASH5's N-terminal EF-hands are essential as the interaction with dynein is disrupted by mutation of key calcium-binding residues, although it is not regulated by cellular calcium levels. Dynein can be recruited to KASH5 at the nuclear envelope independently of dynactin, while LIS1 is essential for dynactin incorporation into the KASH5-dynein complex. Altogether, we show that the transmembrane protein KASH5 is an activating adaptor for dynein and shed light on the hierarchy of assembly of KASH5-dynein-dynactin complexes.

Crystal structure of human XLF/Cernunnos reveals unexpected differences from XRCC4 with implications for NHEJ.

The recently characterised 299-residue human XLF/Cernunnos protein plays a crucial role in DNA repair by non-homologous end joining (NHEJ) and interacts with the XRCC4-DNA Ligase IV complex. Here, we report the crystal structure of the XLF (1-233) homodimer at 2.3 A resolution, confirming the predicted structural similarity to XRCC4. The XLF coiled-coil, however, is shorter than that of XRCC4 and undergoes an unexpected reverse in direction giving rise to a short distorted four helical bundle and a C-terminal helical structure wedged between the coiled-coil and head domain. The existence of a dimer as the major species is confirmed by size-exclusion chromatography, analytical ultracentrifugation, small-angle X-ray scattering and other biophysical methods. We show that the XLF structure is not easily compatible with a proposed XRCC4:XLF heterodimer. However, we demonstrate interactions between dimers of XLF and XRCC4 by surface plasmon resonance and analyse these in terms of surface properties, amino-acid conservation and mutations in immunodeficient patients. Our data are most consistent with head-to-head interactions in a 2:2:1 XRCC4:XLF:Ligase IV complex.

Efficacy and safety of certolizumab pegol in a broad population of patients with active rheumatoid arthritis: results from the REALISTIC phase IIIb study.

OBJECTIVE: To investigate the efficacy and safety of certolizumab pegol (CZP) in a broad population of patients with active RA. METHODS: In this 12-week, double-blind period of the phase IIIb trial, RA patients with inadequate response to at least one DMARD were randomized 4:1 to CZP (400 mg at weeks 0, 2 and 4, followed by 200 mg every 2 weeks) or placebo (every 2 weeks) plus current therapy stratified by previous TNF inhibitor use, concomitant methotrexate use and disease duration (<2 vs ≥2 years). The primary outcome was ACR20 response rate at week 12. RESULTS: Of 1063 patients (CZP = 851; placebo = 212), 37.6% had previous TNF inhibitor use. Baseline mean HAQ Disability Index (HAQ-DI) and DAS 28-joint assessment-ESR [DAS28(ESR)] values were 1.5 and 6.4 in the CZP group, and 1.6 and 6.4 in the placebo group, respectively. The primary endpoint was significant (week 12 ACR20, CZP vs placebo: 51.1 vs 25.9%; P < 0.001); differences were noted at week 2 (31.8 vs 8.5%; P < 0.001). HAQ-DI and DAS28(ESR) change from baseline and ACR50 were significant from week 2. Week 12 ACR20 responses were similar across CZP patient subgroups regardless of concomitant DMARD use at baseline. Adverse and serious adverse events were comparable between CZP and placebo, with no new safety signals. CONCLUSION: CZP was associated with rapid and consistent clinical responses and improved physical function in a diverse group of RA patients, irrespective of concomitant or previous therapy. TRIAL REGISTRATION: ClinicalTrials.gov, http://clinicaltrials.gov/, NCT00717236.

Coiled-coil structure of meiosis protein TEX12 and conformational regulation by its C-terminal tip.

Meiosis protein TEX12 is an essential component of the synaptonemal complex (SC), which mediates homologous chromosome synapsis. It is also recruited to centrosomes in meiosis, and aberrantly in certain cancers, leading to centrosome dysfunction. Within the SC, TEX12 forms an intertwined complex with SYCE2 that undergoes fibrous assembly, driven by TEX12's C-terminal tip. However, we hitherto lack structural information regarding SYCE2-independent functions of TEX12. Here, we report X-ray crystal structures of TEX12 mutants in three distinct conformations, and utilise solution light and X-ray scattering to determine its wild-type dimeric four-helical coiled-coil structure. TEX12 undergoes conformational change upon C-terminal tip mutations, indicating that the sequence responsible for driving SYCE2-TEX12 assembly within the SC also controls the oligomeric state and conformation of isolated TEX12. Our findings provide the structural basis for SYCE2-independent roles of TEX12, including the possible regulation of SC assembly, and its known functions in meiotic centrosomes and cancer.

Defining the molecular basis of BubR1 kinetochore interactions and APC/C-CDC20 inhibition.

BubR1 is essential for the mitotic checkpoint that prevents aneuploidy in cellular progeny by triggering anaphase delay in response to kinetochores incorrectly/not attached to the mitotic spindle. Here, we define the molecular architecture of the functionally significant N-terminal region of human BubR1 and present the 1.8 A crystal structure of its tetratricopeptide repeat (TPR) domain. The structure reveals divergence from the classical TPR fold and is highly similar to the TPR domain of budding yeast Bub1. Shared distinctive features include a disordered loop insertion, a 3(10)-helix, a tight turn involving glycine positive Phi angles, and noncanonical packing of and between the TPR motifs. We also define the molecular determinants of the interaction between BubR1 and kinetochore protein Blinkin. We identify a shallow groove on the concave surface of the BubR1 TPR domain that forms multiple discrete and potentially cooperative interactions with Blinkin. Finally, we present evidence for a direct interaction between BubR1 and Bub1 mediated by regions C-terminal to their TPR domains. This interaction provides a mechanism for Bub1-dependent kinetochore recruitment of BubR1. We thus present novel molecular insights into the structure of BubR1 and its interactions at the kinetochore-microtubule interface. Our studies pave the way for future structure-directed engineering aimed at dissecting the roles of kinetochore-bound and other pools of BubR1 in vivo.