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.

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.

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.

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.

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.

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.

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.

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.

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.

Centrosome dysfunction associated with somatic expression of the synaptonemal complex protein TEX12.

The synaptonemal complex (SC) is a supramolecular protein scaffold that mediates chromosome synapsis and facilitates crossing over during meiosis. In mammals, SC proteins are generally assumed to have no other function. Here, we show that SC protein TEX12 also localises to centrosomes during meiosis independently of chromosome synapsis. In somatic cells, ectopically expressed TEX12 similarly localises to centrosomes, where it is associated with centrosome amplification, a pathology correlated with cancer development. Indeed, TEX12 is identified as a cancer-testis antigen and proliferation of some cancer cells is TEX12-dependent. Moreover, somatic expression of TEX12 is aberrantly activated via retinoic acid signalling, which is commonly disregulated in cancer. Structure-function analysis reveals that phosphorylation of TEX12 on tyrosine 48 is important for centrosome amplification but not for recruitment of TEX12 to centrosomes. We conclude that TEX12 normally localises to meiotic centrosomes, but its misexpression in somatic cells can contribute to pathological amplification and dysfunction of centrosomes in cancers.

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.

Structural basis of meiotic chromosome synaptic elongation through hierarchical fibrous assembly of SYCE2-TEX12.

The synaptonemal complex (SC) is a supramolecular protein assembly that mediates synapsis between homologous chromosomes during meiosis. SC elongation along the chromosome length (up to 24 µm) depends on its midline α-fibrous component SYCE2-TEX12. Here, we report X-ray crystal structures of human SYCE2-TEX12 as an individual building block and on assembly within a fibrous lattice. We combine these structures with mutagenesis, biophysics and electron microscopy to reveal the hierarchical mechanism of SYCE2-TEX12 fiber assembly. SYCE2-TEX12's building blocks are 2:2 coiled coils that dimerize into 4:4 hetero-oligomers and interact end-to-end and laterally to form 10-nm fibers that intertwine within 40-nm bundled micrometer-long fibers that define the SC's midline structure. This assembly mechanism bears striking resemblance with intermediate filament proteins vimentin, lamin and keratin. Thus, SYCE2-TEX12 exhibits behavior typical of cytoskeletal proteins to provide an α-fibrous SC backbone that structurally underpins synaptic elongation along meiotic chromosomes.

A prometaphase mechanism of securin destruction is essential for meiotic progression in mouse oocytes.

Successful cell division relies on the timely removal of key cell cycle proteins such as securin. Securin inhibits separase, which cleaves the cohesin rings holding chromosomes together. Securin must be depleted before anaphase to ensure chromosome segregation occurs with anaphase. Here we find that in meiosis I, mouse oocytes contain an excess of securin over separase. We reveal a mechanism that promotes excess securin destruction in prometaphase I. Importantly, this mechanism relies on two phenylalanine residues within the separase-interacting segment (SIS) of securin that are only exposed when securin is not bound to separase. We suggest that these residues facilitate the removal of non-separase-bound securin ahead of metaphase, as inhibiting this period of destruction by mutating both residues causes the majority of oocytes to arrest in meiosis I. We further propose that cellular securin levels exceed the amount an oocyte is capable of removing in metaphase alone, such that the prometaphase destruction mechanism identified here is essential for correct meiotic progression in mouse oocytes.

Helical ensembles outperform ideal helices in molecular replacement.

The conventional approach in molecular replacement is the use of a related structure as a search model. However, this is not always possible as the availability of such structures can be scarce for poorly characterized families of proteins. In these cases, alternative approaches can be explored, such as the use of small ideal fragments that share high, albeit local, structural similarity with the unknown protein. Earlier versions of AMPLE enabled the trialling of a library of ideal helices, which worked well for largely helical proteins at suitable resolutions. Here, the performance of libraries of helical ensembles created by clustering helical segments is explored. The impacts of different B-factor treatments and different degrees of structural heterogeneity are explored. A 30% increase in the number of solutions obtained by AMPLE was observed when using this new set of ensembles compared with the performance with ideal helices. The boost in performance was notable across three different fold classes: transmembrane, globular and coiled-coil structures. Furthermore, the increased effectiveness of these ensembles was coupled to a reduction in the time required by AMPLE to reach a solution. AMPLE users can now take full advantage of this new library of search models by activating the `helical ensembles' mode.

Meiotic chromosome synapsis depends on multivalent SYCE1-SIX6OS1 interactions that are disrupted in cases of human infertility.

Meiotic reductional division depends on the synaptonemal complex (SC), a supramolecular protein assembly that mediates homologous chromosomes synapsis and promotes crossover formation. The mammalian SC has eight structural components, including SYCE1, the only central element protein with known causative mutations in human infertility. We combine mouse genetics, cellular, and biochemical studies to reveal that SYCE1 undergoes multivalent interactions with SC component SIX6OS1. The N terminus of SIX6OS1 binds and disrupts SYCE1's core dimeric structure to form a 1:1 complex, while their downstream sequences provide a distinct second interface. These interfaces are separately disrupted by SYCE1 mutations associated with nonobstructive azoospermia and premature ovarian failure (POF), respectively. Mice harboring SYCE1's POF mutation and a targeted deletion within SIX6OS1's N terminus are infertile with failure of chromosome synapsis. We conclude that both SYCE1-SIX6OS1 binding interfaces are essential for SC assembly, thus explaining how SYCE1's reported clinical mutations give rise to human infertility.

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.

The BRCA2-MEILB2-BRME1 complex governs meiotic recombination and impairs the mitotic BRCA2-RAD51 function in cancer cells.

Breast cancer susceptibility gene II (BRCA2) is central in homologous recombination (HR). In meiosis, BRCA2 binds to MEILB2 to localize to DNA double-strand breaks (DSBs). Here, we identify BRCA2 and MEILB2-associating protein 1 (BRME1), which functions as a stabilizer of MEILB2 by binding to an α-helical N-terminus of MEILB2 and preventing MEILB2 self-association. BRCA2 binds to the C-terminus of MEILB2, resulting in the formation of the BRCA2-MEILB2-BRME1 ternary complex. In Brme1 knockout (Brme1-/-) mice, the BRCA2-MEILB2 complex is destabilized, leading to defects in DSB repair, homolog synapsis, and crossover formation. Persistent DSBs in Brme1-/- reactivate the somatic-like DNA-damage response, which repairs DSBs but cannot complement the crossover formation defects. Further, MEILB2-BRME1 is activated in many human cancers, and somatically expressed MEILB2-BRME1 impairs mitotic HR. Thus, the meiotic BRCA2 complex is central in meiotic HR, and its misregulation is implicated in cancer development.

Extending the scope of coiled-coil crystal structure solution by AMPLE through improved ab initio modelling.

The phase problem remains a major barrier to overcome in protein structure solution by X-ray crystallography. In recent years, new molecular-replacement approaches using ab initio models and ideal secondary-structure components have greatly contributed to the solution of novel structures in the absence of clear homologues in the PDB or experimental phasing information. This has been particularly successful for highly α-helical structures, and especially coiled-coils, in which the relatively rigid α-helices provide very useful molecular-replacement fragments. This has been seen within the program AMPLE, which uses clustered and truncated ensembles of numerous ab initio models in structure solution, and is already accomplished for α-helical and coiled-coil structures. Here, an expansion in the scope of coiled-coil structure solution by AMPLE is reported, which has been achieved through general improvements in the pipeline, the removal of tNCS correction in molecular replacement and two improved methods for ab initio modelling. Of the latter improvements, enforcing the modelling of elongated helices overcame the bias towards globular folds and provided a rapid method (equivalent to the time requirements of the existing modelling procedures in AMPLE) for enhanced solution. Further, the modelling of two-, three- and four-helical oligomeric coiled-coils, and the use of full/partial oligomers in molecular replacement, provided additional success in difficult and lower resolution cases. Together, these approaches have enabled the solution of a number of parallel/antiparallel dimeric, trimeric and tetrameric coiled-coils at resolutions as low as 3.3 Å, and have thus overcome previous limitations in AMPLE and provided a new functionality in coiled-coil structure solution at lower resolutions. These new approaches have been incorporated into a new release of AMPLE in which automated elongated monomer and oligomer modelling may be activated by selecting `coiled-coil' mode.

Obtaining Tertiary Protein Structures by the ab Initio Interpretation of Small Angle X-ray Scattering Data.

Small angle X-ray scattering (SAXS) is an important tool for investigating the structure of proteins in solution. We present a novel ab initio method representing polypeptide chains as discrete curves used to derive a meaningful three-dimensional model from only the primary sequence and SAXS data. High resolution structures were used to generate probability density functions for each common secondary structural element found in proteins, which are used to place realistic restraints on the model curve's geometry. This is coupled with a novel explicit hydration shell model in order to derive physically meaningful three-dimensional models by optimizing against experimental SAXS data. The efficacy of this model is verified on an established benchmark protein set, and then it is used to predict the lysozyme structure using only its primary sequence and SAXS data. The method is used to generate a biologically plausible model of the coiled-coil component of the human synaptonemal complex central element protein.

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.