B21-High Throughput SAXS
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Diamond Proposal Number(s):
[14435, 15580, 15836, 15897]
Abstract: 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.
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Jan 2023
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[13587, 18598, 14435, 15580, 15897, 15836, 21777]
Open Access
Abstract: 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.
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Sep 2022
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[15836]
Open Access
Abstract: 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.
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Dec 2021
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[13587, 14435, 15580, 15836, 15897, 18598, 21777]
Abstract: 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.
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Aug 2021
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B16-Test Beamline
B21-High Throughput SAXS
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Open Access
Abstract: The DNA repair factor CtIP has a critical function in double-strand break (DSB) repair by homologous recombination, promoting the assembly of the repair apparatus at DNA ends and participating in DNA-end resection. However, the molecular mechanisms of CtIP function in DSB repair remain unclear. Here, we present an atomic model for the three-dimensional architecture of human CtIP, derived from a multi-disciplinary approach that includes X-ray crystallography, small-angle X-ray scattering (SAXS) and diffracted X-ray tracking (DXT). Our data show that CtIP adopts an extended dimer-of-dimers structure, in agreement with a role in bridging distant sites on chromosomal DNA during the recombinational repair. The zinc-binding motif in the CtIP N-terminus alters dynamically the coiled-coil structure, with functional implications for the long-range interactions of CtIP with DNA. Our results provide a structural basis for the three-dimensional arrangement of chains in the CtIP tetramer, a key aspect of CtIP function in DNA DSB repair.
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Jun 2021
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B21-High Throughput SAXS
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13587, 18598, 15836]
Open Access
Abstract: The Linker of Nucleoskeleton and Cytoskeleton (LINC) complex mechanically couples cytoskeletal and nuclear components across the nuclear envelope to fulfil a myriad of cellular functions, including nuclear shape and positioning, hearing, and meiotic chromosome movements. The canonical model is that 3:3 interactions between SUN and KASH proteins underlie the nucleocytoskeletal linkages provided by the LINC complex. Here, we provide crystallographic and biophysical evidence that SUN-KASH is a constitutive 6:6 complex in which two constituent 3:3 complexes interact head-to-head. A common SUN-KASH topology is achieved through structurally diverse 6:6 interaction mechanisms by distinct KASH proteins, including zinc-coordination by Nesprin-4. The SUN-KASH 6:6 interface provides a molecular mechanism for the establishment of integrative and distributive connections between 3:3 structures within a branched LINC complex network. In this model, SUN-KASH 6:6 complexes act as nodes for force distribution and integration between adjacent SUN and KASH molecules, enabling the coordinated transduction of large forces across the nuclear envelope.
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Jan 2021
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Open Access
Abstract: 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.
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Oct 2020
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[15836, 21777, 23510]
Open Access
Abstract: 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.
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Sep 2020
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B21-High Throughput SAXS
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Jingjing
Zhang
,
Manickam
Gurusaran
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Yasuhiro
Fujiwara
,
Kexin
Zhang
,
Meriem
Echbarthi
,
Egor
Vorontsov
,
Rui
Guo
,
Devon F.
Pendlebury
,
Intekhab
Alam
,
Gabriel
Livera
,
Martini
Emmanuelle
,
P. Jeremy
Wang
,
Jayakrishnan
Nandakumar
,
Owen
Davies
,
Hiroki
Shibuya
Open Access
Abstract: 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.
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Apr 2020
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Rosalba
Lepore
,
Andriy
Kryshtafovych
,
Markus
Alahuhta
,
Harshul A.
Veraszto
,
Yannick J.
Bomble
,
Joshua C.
Bufton
,
Alex N.
Bullock
,
Cody
Caba
,
Hongnan
Cao
,
Owen R.
Davies
,
Ambroise
Desfosses
,
Matthew
Dunne
,
Krzysztof
Fidelis
,
Celia W.
Goulding
,
Manickam
Gurusaran
,
Irina
Gutsche
,
Christopher J.
Harding
,
Marcus D.
Hartmann
,
Christopher S.
Hayes
,
Andrzej
Joachimiak
,
Petr G.
Leiman
,
Peter
Loppnau
,
Andrew L.
Lovering
,
Vladimir V.
Lunin
,
Karolina
Michalska
,
Ignacio
Mir‐sanchis
,
Alok
Mitra
,
John
Moult
,
George N.
Phillips Jr
,
Daniel
Pinkas
,
Phoebe A.
Rice
,
Yufeng
Tong
,
Maya
Topf
,
Jonathan D.
Walton
,
Torsten
Schwede
Diamond Proposal Number(s):
[14692]
Open Access
Abstract: 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.
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Sep 2019
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