B21-High Throughput SAXS
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Abstract: Little information is available concerning the structural features of nucleotide pyrophosphatase/phosphodiesterases (NPPs) of plant origin and the crystal structures of these proteins have not yet been reported. The aim of this study was to obtain insight into these aspects by carrying out a comparative analysis of the sequences of two different fragments of an NPP from the latex of the Mediterranean shrub Euphorbia characias (ELNPP) and by studying the low-resolution structure of the purified protein in solution by means of small-angle X-ray scattering. This is the first structure of a plant NPP in solution that has been reported to date. It is shown that the ELNPP sequence is highly conserved in many other plant species. Of note, the catalytic domains of these plant NPPs have the same highly conserved PDE-domain organization as mammalian NPPs. Moreover, ELNPP is a dimer in solution and this oligomerization state is likely to be common to other plant enzymes.
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Sep 2020
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B21-High Throughput SAXS
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Nathan P.
Cowieson
,
Charlotte J. C.
Edwards-gayle
,
Katsuaki
Inoue
,
Nikul S.
Khunti
,
James
Doutch
,
Eugene
Williams
,
Steven
Daniels
,
Geoff
Preece
,
Nicholas A.
Krumpa
,
John P.
Sutter
,
Mark D.
Tully
,
Nick J.
Terrill
,
Robert P.
Rambo
Open Access
Abstract: B21 is a small-angle X-ray scattering (SAXS) beamline with a bending magnet source in the 3 GeV storage ring at the Diamond Light Source Ltd synchrotron in the UK. The beamline utilizes a double multi-layer monochromator and a toroidal focusing optic to deliver 2 × 1012 photons per second to a 34 × 40 µm (FWHM) focal spot at the in-vacuum Eiger 4M (Dectris) detector. A high-performance liquid chromatography system and a liquid-handling robot make it possible to load solution samples into a temperature-controlled in-vacuum sample cell with a high level of automation. Alternatively, a range of viscous or solid materials may be loaded manually using a range of custom sample cells. A default scattering vector range from 0.0026 to 0.34 Å−1 and low instrument background make B21 convenient for measuring a wide range of biological macromolecules. The beamline has run a full user programme since 2013.
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Sep 2020
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B21-High Throughput SAXS
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[15868, 22906]
Abstract: In tryptophan biosynthesis, the reaction catalyzed by the enzyme indole-3-glycerol phosphate synthase (IGP synthase, IGPS) starts with a condensation step where the substrate’s carboxylated phenyl group makes a nucleophilic attack to form the pyrrole ring of the indole, followed by a decarboxylation which restores the aromaticity of the phenyl. IGPS from Pseudomonas aeruginosa has the highest turnover number of all characterized IGPS enzymes, providing an excellent model system to test the necessity of the decarboxylation step. This step was since the 1960s considered to be mechanistically essential based on studies of the IGPS:PRAI fusion protein from Escherichia coli. Here, we present the crystal structure of P. aeruginosa IGPS in complex with reduced CdRP, a non-reactive substrate analogue, and using a sensitive discontinuous assay, we demonstrate weak promiscuous activity on the decarboxylated substrate 1-(phenylamino)-1-deoxyribulose-5-phosphate (PAdRP) – with approximately 1000' lower rate of IGP formation than from the native substrate. We also show that E. coli IGPS, at even lower rate, can produce IGP from decarboxylated substrate. Our structure of P. aeruginosa IGPS has eight molecules in the asymmetric unit, out of which seven contain ligand, and one displays a previously unobserved conformation closer to the reactive state. One of the few non-conserved active-site residues, Phe201 in P. aeruginosa IGPS, is by mutagenesis demonstrated to be important for the higher turnover of this enzyme on both substrates. Our results demonstrate that, despite IGPS’s classification as a carboxylyase (i.e. decarboxylase), decarboxylation not a completely essential step in its catalysis.
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Sep 2020
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Alice J.
Bochel
,
Christopher
Williams
,
Airlie J.
Mccoy
,
Hans-jürgen
Hoppe
,
Ashley J.
Winter
,
Ryan D.
Nicholls
,
Karl
Harlos
,
E. Yvonne
Jones
,
Imre
Berger
,
A. Bassim
Hassan
,
Matthew P.
Crump
Diamond Proposal Number(s):
[8423]
Abstract: The cation-independent mannose 6-phosphate (M6P)/Insulin-like growth factor-2 receptor (CI-MPR/IGF2R) is an ∼300 kDa transmembrane protein responsible for trafficking M6P-tagged lysosomal hydrolases and internalizing IGF2. The extracellular region of the CI-MPR has 15 homologous domains, including M6P-binding domains (D) 3, 5, 9, and 15 and IGF2-binding domain 11. We have focused on solving the first structures of human D7–10 within two multi-domain constructs, D9–10 and D7–11, and provide the first high-resolution description of the high-affinity M6P-binding D9. Moreover, D9 stabilizes a well-defined hub formed by D7–11 whereby two penta-domains intertwine to form a dimeric helical-type coil via an N-glycan bridge on D9. Remarkably the D7–11 structure matches an IGF2-bound state of the receptor, suggesting this may be an intrinsically stable conformation at neutral pH. Interdomain clusters of histidine and proline residues may impart receptor rigidity and play a role in structural transitions at low pH.
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Sep 2020
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[19951]
Open Access
Abstract: Importance of disordered protein regions is increasingly recognized in biology, but their characterization remains challenging due to the lack of suitable experimental and theoretical methods. NMR experiments can detect multiple timescale dynamics and structural details of disordered protein regions, but their detailed interpretation is often difficult. Here we combine protein backbone 15N spin relaxation data with molecular dynamics (MD) simulations to detect not only heterogeneous dynamics of large partially disordered proteins but also their conformational ensembles. We observed that the rotational dynamics of folded regions in partially disordered proteins is dominated by similar rigid body rotation as in globular proteins, thereby being largely independent of flexible disordered linkers. Disordered regions, on the other hand, exhibit complex rotational motions with multiple timescales below ∼30 ns which are difficult to detect from experimental data alone, but can be captured by MD simulations. Combining MD simulations and backbone 15N spin relaxation data, measured applying segmental isotopic labeling with salt-inducible split intein, we resolved the conformational ensemble and dynamics of partially disordered periplasmic domain of TonB protein from Helicobacter pylori containing 250 residues. To demonstrate the universality of our approach, it was applied also to the partially disordered region of chicken Engrailed 2. Our results pave the way in understanding how TonB transfers energy from inner membrane to the outer membrane receptors in Gram-negative bacteria, as well as the function of other proteins with disordered domains.
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Sep 2020
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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):
[16258]
Open Access
Abstract: Cellular cross-talk between ubiquitination and other posttranslational modifications contributes to the regulation of numerous processes. One example is ADP-ribosylation of the carboxyl terminus of ubiquitin by the E3 DTX3L/ADP-ribosyltransferase PARP9 heterodimer, but the mechanism remains elusive. Here, we show that independently of PARP9, the conserved carboxyl-terminal RING and DTC (Deltex carboxyl-terminal) domains of DTX3L and other human Deltex proteins (DTX1 to DTX4) catalyze ADP-ribosylation of ubiquitin’s Gly76. Structural studies reveal a hitherto unknown function of the DTC domain in binding NAD+. Deltex RING domain recruits E2 thioesterified with ubiquitin and juxtaposes it with NAD+ bound to the DTC domain to facilitate ADP-ribosylation of ubiquitin. This ubiquitin modification prevents its activation but is reversed by the linkage nonspecific deubiquitinases. Our study provides mechanistic insights into ADP-ribosylation of ubiquitin by Deltex E3s and will enable future studies directed at understanding the increasingly complex network of ubiquitin cross-talk.
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Sep 2020
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[22925]
Open Access
Abstract: The designed arginine-rich surfactant-like peptide R3L12 (arginine3–leucine12) is shown to form a remarkable diversity of self-assembled nanostructures in aqueous solution, depending on pH, including nanotubes, mesh-like tubular networks in three-dimensions and square planar arrays in two-dimensions. These structures are built from α-helical antiparallel coiled–coil peptide dimers arranged perpendicular to the nanotube axis, in a “cross-α” nanotube structure. The aggregation behavior is rationalized based on the effects of dimensionality, and the balance of hydrophobic and electrostatic interactions. The nanotube and nanomesh structures display arginine at high density on their surfaces, which may be valuable for future applications.
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Sep 2020
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[17773]
Abstract: Elastic fibres are essential components of all mammalian elastic tissues such as blood vessels, lung and skin, and are critically important for the mechanical properties they endow. The main components of elastic fibres are elastin and fibrillin, where correct formation of elastic fibres requires a fibrillin microfibril scaffold for the deposition of elastin. It has been demonstrated previously that the interaction between fibrillin and tropoelastin, the elastin precursor, increases the rate of assembly of tropoelastin. Furthermore, tropoelastin and fibrillin can be cross-linked by transglutaminase-2 but the function of cross-linking on their elastic properties is yet to be elucidated. Here we show that transglutaminase cross-linking supports formation of a 1:1 stoichiometric fibrillin-tropoelastin complex. SAXS data show that the complex retains features of the individual proteins, but is elongated supporting end-to-end assembly. Elastic network models were constructed to compare the dynamics of tropoelastin and fibrillin individually as well as in the cross-linked complex. Normal mode analysis was performed to determine the structures' most energetically favourable, biologically accessible motions which show that within the complex, tropoelastin is less mobile and this molecular stabilisation extends along the length of the tropoelastin molecule to regions remote from the cross-linking site. Together, these data suggest a long-range stabilising effect of cross-linking that occurs due to the covalent linkage of fibrillin to tropoelastin. This work provides insight into the interactions of tropoelastin and fibrillin and how cross-link formation stabilises the elastin precursor so it is primed for elastic fibre assembly.
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Sep 2020
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[24693]
Abstract: Tuning the enzymatic degradation and disassembly rates of polymeric amphiphiles and their assemblies is crucial for designing enzyme-responsive nanocarriers for controlled drug delivery applications. The common methods to control the enzymatic degradation of amphiphilic polymers are to tune the molecular weights and ratios of the hydrophilic and hydrophobic blocks. In addition, to these approaches, the architecture of the hydrophilic block can also serve as a tool to tune enzymatic degradation and disassembly. To gain deeper understanding of the effect of the molecular architecture of the hydrophilic block we prepared two types of well-defined PEG-dendron amphiphiles bearing linear or V-shaped PEG chains as the hydrophilic blocks. The high molecular precision of these amphiphiles, which emerges from the utilization of dendrons as the hydrophobic blocks allowed us to study the self-assembly and enzymatic degradation and disassembly of the two types of amphiphiles with high resolution. Interestingly, the micelles of the V-shaped amphiphiles were significantly smaller and disassembled faster than those of the amphiphiles based on linear PEG. However, the complete enzymatic cleavage of the hydrophobic end-groups was significantly slower for the V-shaped amphiphiles. Our results show that the V-shape architecture can stabilize the unimer state and hence plays a double role in the enzymatic degradation and the induced disassembly and how it can be utilized to control the release of encapsulated or bound molecular cargo.
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Aug 2020
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B21-High Throughput SAXS
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Abstract: Efficient gene transfer is necessary for advanced biotechnologies ranging from gene therapy to synthetic biology. Peptide nanoparticles provide suitable packaging systems promoting targeted gene expression or silencing. Though these systems have yet to match the transfection efficacy of viruses, they are typically devoid of drawbacks characteristic of virus-based vectors, including insertional mutagenesis, low packaging capacities, and strong immune responses. Given the promise nanoparticle formulations hold for gene delivery, methods of their preparation and accurate analysis of their physicochemical and biological properties become indispensable for progress toward systems that seek to outperform viral vectors. Herein, we report a comprehensive protocol for the preparation and characterization of archetypal peptide nanoparticles resulting from nonspecific and noncovalent complexation with RNA and DNA.
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Aug 2020
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