I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[15916]
Open Access
Abstract: Retroviruses can integrate their DNA into the host-cell genome. Inherited retroviral DNA and other transposable elements account for more than half of the human genome. Transposable elements must be tightly regulated to restrict their proliferation and prevent toxic gene expression. KAP1/TRIM28 is an essential regulator of transposable element transcription. We determined the crystal structure of the KAP1 TRIM. The structure identifies a protein–protein interaction site required for recruitment of KAP1 to transposable elements. An epigenetic gene silencing assay confirms the importance of this site for KAP1-dependent silencing. We also show that KAP1 self-assembles in solution, but this self-assembly is not required for silencing. Our work provides insights into KAP1-dependent silencing and tools for expanding our mechanistic understanding of this process.
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Jul 2019
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Danguole
Kureisaite-ciziene
,
Aravindan
Varadajan
,
Stephen H.
Mclaughlin
,
Marjolein
Glas
,
Alejandro
Montón Silva
,
Rosa
Luirink
,
Carolin
Mueller
,
Tanneke
Den Blaauwen
,
Tom N.
Grossmann
,
Joen
Luirink
,
Jan
Lowe
Open Access
Abstract: Most bacteria and archaea use the tubulin homologue FtsZ as its central organizer of cell division. In Gram-negative Escherichia coli bacteria, FtsZ recruits cytosolic, transmembrane, periplasmic, and outer membrane proteins, assembling the divisome that facilitates bacterial cell division. One such divisome component, FtsQ, a bitopic membrane protein with a globular domain in the periplasm, has been shown to interact with many other divisome proteins. Despite its otherwise unknown function, it has been shown to be a major divisome interaction hub. Here, we investigated the interactions of FtsQ with FtsB and FtsL, two small bitopic membrane proteins that act immediately downstream of FtsQ. We show in biochemical assays that the periplasmic domains of E. coli FtsB and FtsL interact with FtsQ, but not with each other. Our crystal structure of FtsB bound to the β domain of FtsQ shows that only residues 64 to 87 of FtsB interact with FtsQ. A synthetic peptide comprising those 24 FtsB residues recapitulates the FtsQ-FtsB interactions. Protein deletions and structure-guided mutant analyses validate the structure. Furthermore, the same structure-guided mutants show cell division defects in vivo that are consistent with our structure of the FtsQ-FtsB complex that shows their interactions as they occur during cell division. Our work provides intricate details of the interactions within the divisome and also provides a tantalizing view of a highly conserved protein interaction in the periplasm of bacteria that is an excellent target for cell division inhibitor searches.
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Sep 2018
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8547, 11235]
Abstract: Mmi1 is an essential RNA-binding protein in the fission yeast Schizosaccharomyces pombe that eliminates meiotic transcripts during normal vegetative growth. Mmi1 contains a YTH domain that binds specific RNA sequences, targeting mRNAs for degradation. The YTH domain of Mmi1 uses a noncanonical RNA-binding surface that includes contacts outside the conserved fold. Here, we report that an N-terminal extension that is proximal to the YTH domain enhances RNA binding. Using X-ray crystallography, NMR and biophysical methods, we show that this low-complexity region becomes more ordered upon RNA binding. This enhances the affinity of the interaction of the Mmi1 YTH domain with specific RNAs by reducing the dissociation rate of the Mmi1–RNA complex. We propose that the low-complexity region influences RNA binding indirectly by reducing dynamic motions of the RNA binding groove and stabilizing a conformation of the YTH domain that binds to RNA with high affinity. Taken together, our work reveals how a low-complexity region proximal to a conserved folded domain can adopt an ordered structure to aid nucleic acid binding.
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Apr 2018
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Open Access
Abstract: In Saccharomyces cerevisiae generation of export-competent mRNPs terminates the nuclear phase of the gene expression pathway and facilitates transport to the cytoplasm for translation. Nab2 functions in this process to control both mRNP compaction that facilitates movement through nuclear pore complexes and the length of transcript poly(A) tails. Nab2 has a modular structure that includes seven CCCH Zn fingers that bind to A-rich RNAs and fingers 5–7 are critical for these functions. Here, we demonstrate, using both biophysical and structural methods, that binding A11G RNA induces dimerization of Zn fingers 5–7 mediated by the novel spatial arrangement of the fingers promoting each RNA chain binding two protein chains. The dimerization of Nab2 induced by RNA binding provides a basis for understanding its function in both poly(A) tail length regulation and in the compaction of mature transcripts to facilitate nuclear export.
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Dec 2016
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[11235]
Open Access
Abstract: Cilia are thin cell projections with essential roles in cell motility, fluid movement, sensing, and signaling. They are templated from centrioles that dock against the plasma membrane and subsequently extend their peripheral microtubule array. The molecular mechanisms underpinning cilia assembly are incompletely understood. Cep104 is a key factor involved in cilia formation and length regulation that rides on the ends of elongating and shrinking cilia. It is mutated in Joubert syndrome, a genetically heterogeneous ciliopathy. Here we provide structural and biochemical data that Cep104 contains a tubulin-binding TOG (tumor overexpressed gene) domain and a novel C2HC zinc finger array. Furthermore, we identify the kinase Nek1, another ciliopathy-associated protein, as a potential binding partner of this array. Finally, we show that Nek1 competes for binding to Cep104 with the distal centriole-capping protein CP110. Our data suggest a model for Cep104 activity during ciliogenesis and provide a novel link between Cep104 and Nek1.
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Dec 2016
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[11235]
Open Access
Abstract: The similarity of eukaryotic actin to crenactin, a filament-forming protein from the crenarchaeon Pyrobaculum calidifontis supports the theory of a common origin of Crenarchaea and Eukaryotes. Monomeric structures of crenactin and actin are similar, although their filament architectures were suggested to be different. Here we report that crenactin forms bona fide double helical filaments that show exceptional similarity to eukaryotic F-actin. With cryo-electron microscopy and helical reconstruction we solved the structure of the crenactin filament to 3.8 Å resolution. When forming double filaments, the 'hydrophobic plug' loop in crenactin rearranges. Arcadin-2, also encoded by the arcade gene cluster, binds tightly with its C-terminus to the hydrophobic groove of crenactin. Binding is reminiscent of eukaryotic actin modulators such as cofilin and thymosin β4 and arcadin-2 is a depolymeriser of crenactin filaments. Our work further supports the theory of shared ancestry of Eukaryotes and Crenarchaea.
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Nov 2016
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I02-Macromolecular Crystallography
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Paul R.
Elliott
,
Derek
Leske
,
Matous
Hrdinka
,
Katrin
Bagola
,
Berthe k.
Fiil
,
Stephen h.
Mclaughlin
,
Jane
Wagstaff
,
Norbert
Volkmar
,
John c.
Christianson
,
Benedikt m.
Kessler
,
Stefan m. V.
Freund
,
David
Komander
,
Mads
Gyrd-hansen
Diamond Proposal Number(s):
[11235]
Open Access
Abstract: The linear ubiquitin chain assembly complex (LUBAC) regulates immune signaling, and its function is regulated by the deubiquitinases OTULIN and CYLD, which associate with the catalytic subunit HOIP. However, the mechanism through which CYLD interacts with HOIP is unclear. We here show that CYLD interacts with HOIP via spermatogenesis-associated protein 2 (SPATA2). SPATA2 interacts with CYLD through its non-canonical PUB domain, which binds the catalytic CYLD USP domain in a CYLD B-box-dependent manner. Significantly, SPATA2 binding activates CYLD-mediated hydrolysis of ubiquitin chains. SPATA2 also harbors a conserved PUB-interacting motif that selectively docks into the HOIP PUB domain. In cells, SPATA2 is recruited to the TNF receptor 1 signaling complex and is required for CYLD recruitment. Loss of SPATA2 increases ubiquitination of LUBAC substrates and results in enhanced NOD2 signaling. Our data reveal SPATA2 as a high-affinity binding partner of CYLD and HOIP, and a regulatory component of LUBAC-mediated NF-κB signaling.
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Sep 2016
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[11235]
Open Access
Abstract: The GTPase Arf1 is the major regulator of vesicle traffic at both the cis- and trans-Golgi. Arf1 is activated at the cis-Golgi by the guanine nucleotide exchange factor (GEF) GBF1 and at the trans-Golgi by the related GEF BIG1 or its paralog, BIG2. The trans-Golgi-specific targeting of BIG1 and BIG2 depends on the Arf-like GTPase Arl1. We find that Arl1 binds to the dimerization and cyclophilin binding (DCB) domain in BIG1 and report a crystal structure of human Arl1 bound to this domain. Residues in the DCB domain that bind Arl1 are required for BIG1 to locate to the Golgi in vivo. DCB domain-binding residues in Arl1 have a distinct conformation from those in known Arl1-effector complexes, and this plasticity allows Arl1 to interact with different effectors of unrelated structure. The findings provide structural insight into how Arf1 GEFs, and hence active Arf1, achieve their correct subcellular distribution.
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Jul 2016
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I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: Bacterial cell division involves a contractile ring that organises downstream proteins at the division site and which contains the tubulin homologue FtsZ. ZapC has been discovered as a non-essential regulator of FtsZ. It localises to the septal ring and deletion of zapC leads to a mild phenotype, while overexpression inhibits cell division. Interference with cell division is facilitated by an interaction with FtsZ. Here, we present the 2.9 Å crystal structure of ZapC from Escherichia coli. ZapC forms a dimer and comprises two domains that belong to the Royal superfamily of which many members bind methylated arginines or lysines. ZapC contains an N-terminal chromo-like domain and a Tudor-like C-terminal domain. We show by ITC that ZapC binds the C-terminal tail of FtsZ.
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Dec 2015
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Abstract: Evolution and design of
protein complexes are frequently viewed
through the lens of amino acid mutations
at protein interfaces, but we showed previously
that residues distant from interfaces
are also commonly involved in the evolution
of alternative quaternary structures.
We hypothesized that in these protein
families, the difference in oligomeric state
is due to a change in intersubunit geometry.
The indirect mutations would act by
changing protein conformation and dynamics,
similar to the way in which allosteric
small molecules introduce functional
conformational change. We refer to these
substitutions as “allosteric mutations.”
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Dec 2014
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