I02-Macromolecular Crystallography
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Open Access
Abstract: The RIO kinases (RIOKs) are a universal family of atypical kinases that are essential for assembly of the pre-40S ribosome complex. Here, we present the crystal structure of human RIO kinase 2 (RIOK2) bound to a specific inhibitor. This first crystal structure of an inhibitor-bound RIO kinase reveals the binding mode of the inhibitor and explains the structure–activity relationship of the inhibitor series. The inhibitor binds in the ATP-binding site and forms extensive hydrophobic interactions with residues at the entrance to the ATP-binding site. Analysis of the conservation of active site residues reveals the reasons for the specificity of the inhibitor for RIOK2 over RIOK1 and RIOK3, and it provides a template for inhibitor design against the human RIOK family.
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Apr 2019
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I03-Macromolecular Crystallography
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Open Access
Abstract: A dynamic multi-protein assembly known as the replisome is responsible for DNA synthesis in eukaryotic cells. In yeast, the hub protein Ctf4 bridges DNA helicase and DNA polymerase and recruits factors with roles in metabolic processes coupled to DNA replication. An important question in DNA replication is the extent to which the molecular architecture of the replisome is conserved between yeast and higher eukaryotes. Here, we describe the biochemical basis for the interaction of the human CTF4-orthologue AND-1 with DNA polymerase α (Pol α)/primase, the replicative polymerase that initiates DNA synthesis. AND-1 has maintained the trimeric structure of yeast Ctf4, driven by its conserved SepB domain. However, the primary interaction of AND-1 with Pol α/primase is mediated by its C-terminal HMG box, unique to mammalian AND-1, which binds the B subunit, at the same site targeted by the SV40 T-antigen for viral replication. In addition, we report a novel DNA-binding activity in AND-1, which might promote the correct positioning of Pol α/primase on the lagging-strand template at the replication fork. Our findings provide a biochemical basis for the specific interaction between two critical components of the human replisome, and indicate that important principles of replisome architecture have changed significantly in evolution.
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Nov 2017
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Julijus
Bogomolovas
,
Jennifer R.
Fleming
,
Brian R.
Anderson
,
Rhys
Williams
,
Stephan
Lange
,
Bernd
Simon
,
Muzamil M.
Khan
,
Rüdiger
Rudolf
,
Barbara
Franke
,
Belinda
Bullard
,
Daniel J.
Rigden
,
Henk
Granzier
,
Siegfried
Labeit
,
Olga
Mayans
Diamond Proposal Number(s):
[7146]
Open Access
Abstract: Missense single-nucleotide polymorphisms (mSNPs) in titin are emerging as a main causative factor of heart failure. However, distinguishing between benign and disease-causing mSNPs is a substantial challenge. Here, we research the question of whether a single mSNP in a generic domain of titin can affect heart function as a whole and, if so, how. For this, we studied the mSNP T2850I, seemingly linked to arrhythmogenic right ventricular cardiomyopathy (ARVC). We used structural biology, computational simulations and transgenic muscle in vivo methods to track the effect of the mutation from the molecular to the organismal level. The data show that the T2850I exchange is compatible with the domain three-dimensional fold, but that it strongly destabilizes it. Further, it induces a change in the conformational dynamics of the titin chain that alters its reactivity, causing the formation of aberrant interactions in the sarcomere. Echocardiography of knock-in mice indicated a mild diastolic dysfunction arising from increased myocardial stiffness. In conclusion, our data provide evidence that single mSNPs in titin's I-band can alter overall muscle behaviour. Our suggested mechanisms of disease are the development of non-native sarcomeric interactions and titin instability leading to a reduced I-band compliance. However, understanding the T2850I-induced ARVC pathology mechanistically remains a complex problem and will require a deeper understanding of the sarcomeric context of the titin region affected.
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Sep 2016
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: As part of a programme of synthesizing and investigating the biological properties of new fluoroquinolone antibacterials and their targeting of topoisomerase IV from Streptococcus pneumoniae, we have solved the X-ray structure of the complexes of two new 7,8-bridged fluoroquinolones (with restricted C7 group rotation favouring tight binding) in complex with the topoisomerase IV from S. pneumoniae and an 18-base-pair DNA binding site—the E-site—found by our DNA mapping studies to bind drug strongly in the presence of topoisomerase IV (Leo et al. 2005 J. Biol. Chem. 280, 14 252–14 263, doi:10.1074/jbc.M500156200). Although the degree of antibiotic resistance towards fluoroquinolones is much lower than that of β-lactams and a range of ribosome-bound antibiotics, there is a pressing need to increase the diversity of members of this successful clinically used class of drugs. The quinolone moiety of the new 7,8-bridged agents ACHN-245 and ACHN-454 binds similarly to that of clinafloxocin, levofloxacin, moxifloxacin and trovofloxacin but the cyclic scaffold offers the possibility of chemical modification to produce interactions with other topoisomerase residues at the active site.
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Sep 2016
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Glyn
Hemsworth
,
Andrew J.
Thompson
,
Judith
Stepper
,
Lukasz
Sobala
,
Travis
Coyle
,
Johan
Larsbrink
,
Oliver
Spadiut
,
Ethan D.
Goddard-borger
,
Keith A.
Stubbs
,
Harry
Brumer
,
Gideon
Grogan
Diamond Proposal Number(s):
[7864]
Open Access
Abstract: The human gastrointestinal tract harbours myriad bacterial species, collectively termed the microbiota, that strongly influence human health. Symbiotic members of our microbiota play a pivotal role in the digestion of complex carbohydrates that are otherwise recalcitrant to assimilation. Indeed, the intrinsic human polysaccharide-degrading enzyme repertoire is limited to various starch-based substrates; more complex polysaccharides demand microbial degradation. Select Bacteroidetes are responsible for the degradation of the ubiquitous vegetable xyloglucans (XyGs), through the concerted action of cohorts of enzymes and glycan-binding proteins encoded by specific xyloglucan utilization loci (XyGULs). Extending recent (meta)genomic, transcriptomic and biochemical analyses, significant questions remain regarding the structural biology of the molecular machinery required for XyG saccharification. Here, we reveal the three-dimensional structures of an α-xylosidase, a β-glucosidase, and two α-l-arabinofuranosidases from the Bacteroides ovatus XyGUL. Aided by bespoke ligand synthesis, our analyses highlight key adaptations in these enzymes that confer individual specificity for xyloglucan side chains and dictate concerted, stepwise disassembly of xyloglucan oligosaccharides. In harness with our recent structural characterization of the vanguard endo-xyloglucanse and cell-surface glycan-binding proteins, the present analysis provides a near-complete structural view of xyloglucan recognition and catalysis by XyGUL proteins.
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Jul 2016
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[10369]
Open Access
Abstract: The vast majority of clinically approved protein kinase inhibitors target the ATP-binding pocket directly. Consequently, many inhibitors have broad selectivity profiles and most have significant off-target effects. Allosteric inhibitors are generally more selective, but are difficult to identify because allosteric binding sites are often unknown or poorly characterized. Aurora-A is activated through binding of TPX2 to an allosteric site on the kinase catalytic domain, and this knowledge could be exploited to generate an inhibitor. Here, we generated an allosteric inhibitor of Aurora-A kinase based on a synthetic, vNAR single domain scaffold, vNAR-D01. Biochemical studies and a crystal structure of the Aurora-A/vNAR-D01 complex show that the vNAR domain overlaps with the TPX2 binding site. In contrast with the binding of TPX2, which stabilizes an active conformation of the kinase, binding of the vNAR domain stabilizes an inactive conformation, in which the αC-helix is distorted, the canonical Lys-Glu salt bridge is broken and the regulatory (R-) spine is disrupted by an additional hydrophobic side chain from the activation loop. These studies illustrate how single domain antibodies can be used to characterize the regulatory mechanisms of kinases and provide a rational basis for structure-guided design of allosteric Aurora-A kinase inhibitors.
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Jul 2016
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[6388]
Open Access
Abstract: Mycobacterium tuberculosis (Mtb), the aetiological agent of tuberculosis, has evolved to scavenge nutrients from the confined environment of host macrophages with mycobacterial ATP-binding cassette (ABC) transporters playing a key role in nutrient acquisition. Mtb-UspC (Rv2318) is the solute-binding protein of the essential transporter UspABC, one of four Mtb ABC transporters implicated by homology in sugar acquisition. Herein, we report the structural and functional characterization of Mtb-UspC. The 1.5 Å resolution structure of UspC reveals a two subdomain architecture that forms a highly acidic carbohydrate-substrate binding cleft. This has allowed a distinct preference of Mtb-UspC for amino sugars as determined by thermal shift analysis and solution saturation transfer difference-NMR. Taken together our data support the functional assignment of UspABC as an amino-sugar transporter. Given the limited availability of carbohydrates within the phagosomal environmental niche during Mtb intracellular infection, our studies suggest that UspABC enables Mtb to optimize the use of scarce nutrients during intracellular infection, linking essentiality of this protein to a potential role in recycling components of cell-wall peptidoglycan.
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Jun 2016
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B21-High Throughput SAXS
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: The vertebrate-specific proteins astrotactin-1 and 2 (ASTN-1 and ASTN-2) are integral membrane perforin-like proteins known to play critical roles in neurodevelopment, while ASTN-2 has been linked to the planar cell polarity pathway in hair cells. Genetic variations associated with them are linked to a variety of neurodevelopmental disorders and other neurological pathologies, including an advanced onset of Alzheimer's disease. Here we present the structure of the majority endosomal region of ASTN-2, showing it to consist of a unique combination of polypeptide folds: a perforin-like domain, a minimal epidermal growth factor-like module, a unique form of fibronectin type III domain and an annexin-like domain. The perforin-like domain differs from that of other members of the membrane attack complex-perforin (MACPF) protein family in ways that suggest ASTN-2 does not form pores. Structural and biophysical data show that ASTN-2 (but not ASTN-1) binds inositol triphosphates, suggesting a mechanism for membrane recognition or secondary messenger regulation of its activity. The annexin-like domain is closest in fold to repeat three of human annexin V and similarly binds calcium, and yet shares no sequence homology with it. Overall, our structure provides the first atomic-resolution description of a MACPF protein involved in development, while highlighting distinctive features of ASTN-2 responsible for its activity.
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May 2016
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I18-Microfocus Spectroscopy
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Diamond Proposal Number(s):
[7837]
Open Access
Abstract: Woodlice efficiently sequester copper (Cu) in ‘cuprosomes' within hepatopancreatic ‘S' cells. Binuclear ‘B’ cells in the hepatopancreas form iron (Fe) deposits; these cells apparently undergo an apocrine secretory diurnal cycle linked to nocturnal feeding. Synchrotron-based µ-focus X-ray spectroscopy undertaken on thin sections was used to characterize the ligands binding Cu and Fe in S and B cells of Oniscus asellus (Isopoda). Main findings were: (i) morphometry confirmed a diurnal B-cell apocrine cycle; (ii) X-ray fluorescence (XRF) mapping indicated that Cu was co-distributed with sulfur (mainly in S cells), and Fe was co-distributed with phosphate (mainly in B cells); (iii) XRF mapping revealed an intimate morphological relationship between the basal regions of adjacent S and B cells; (iv) molecular modelling and Fourier transform analyses indicated that Cu in the reduced Cu+ state is mainly coordinated to thiol-rich ligands (Cu–S bond length 2.3 Å) in both cell types, while Fe in the oxidized Fe3+ state is predominantly oxygen coordinated (estimated Fe–O bond length of approx. 2 Å), with an outer shell of Fe scatterers at approximately 3.05 Å; and (v) no significant differences occur in Cu or Fe speciation at key nodes in the apocrine cycle. Findings imply that S and B cells form integrated unit-pairs; a functional role for secretions from these cellular units in the digestion of recalcitrant dietary components is hypothesized.
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Mar 2016
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I02-Macromolecular Crystallography
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Open Access
Abstract: The folliculin/Fnip complex has been demonstrated to play a crucial role in the mechanisms underlying Birt–Hogg–Dubé (BHD) syndrome, a rare inherited cancer syndrome. Lst4 has been previously proposed to be the Fnip1/2 orthologue in yeast and therefore a member of the DENN family. In order to confirm this, we solved the crystal structure of the N-terminal region of Lst4 from Kluyveromyces lactis and show it contains a longin domain, the first domain of the full DENN module. Furthermore, we demonstrate that Lst4 through its DENN domain interacts with Lst7, the yeast folliculin orthologue. Like its human counterpart, the Lst7/Lst4 complex relocates to the vacuolar membrane in response to nutrient starvation, most notably in carbon starvation. Finally, we express and purify the recombinant Lst7/Lst4 complex and show that it exists as a 1 : 1 heterodimer in solution. This work confirms the membership of Lst4 and the Fnip proteins in the DENN family, and provides a basis for using the Lst7/Lst4 complex to understand the molecular function of folliculin and its role in the pathogenesis of BHD syndrome.
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Dec 2015
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