I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Ralph
Adams
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Callum
Joyce
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Mikhail
Kuravskiy
,
Katriona
Harrison
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Zainab
Ahdash
,
Matthew
Balmforth
,
Kelda
Chia
,
Cinzia
Marceddu
,
Matthew
Coates
,
James
Snowden
,
Emmanuel
Goursaud
,
Karelle
Ménochet
,
Jean
Van Den Elsen
,
Richard J.
Payne
,
Alastair D. G.
Lawson
,
Anthony
Scott-Tucker
,
Alex
Macpherson
Diamond Proposal Number(s):
[29404, 5956]
Open Access
Abstract: Background: Serum albumin binding is an established mechanism to extend the serum half-life of antibody fragments and peptides. The cysteine rich knob domains, isolated from bovine antibody ultralong CDRH3, are the smallest single chain antibody fragments described to date and versatile tools for protein engineering.
Methods: Here, we used phage display of bovine immune material to derive knob domains against human and rodent serum albumins. These were used to engineer bispecific Fab fragments, by using the framework III loop as a site for knob domain insertion.
Results: By this route, neutralisation of the canonical antigen (TNFα) was retained but extended pharmacokinetics in-vivo were achieved through albumin binding. Structural characterisation revealed correct folding of the knob domain and identified broadly common but non-cross-reactive epitopes. Additionally, we show that these albumin binding knob domains can be chemically synthesised to achieve dual IL-17A neutralisation and albumin binding in a single chemical entity.
Conclusions: This study enables antibody and chemical engineering from bovine immune material, via an accessible discovery platform.
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May 2023
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23269]
Open Access
Abstract: Staphylococcus aureus is an opportunistic pathogen that is able to thwart an effective host immune response by producing a range of immune evasion molecules, including S. aureus binder of IgG (Sbi) which interacts directly with the central complement component C3, its fragments and associated regulators. Recently we reported the first structure of a disulfide-linked human C3d17C dimer and highlighted its potential role in modulating B-cell activation. Here we present an X-ray crystal structure of a disulfide-linked human C3d17C dimer, which undergoes a structurally stabilising N-terminal 3D domain swap when in complex with Sbi. These structural studies, in combination with circular dichroism and fluorescence spectroscopic analyses, reveal the mechanism underpinning this unique helix swap event and could explain the origins of a previously discovered N-terminally truncated C3dg dimer isolated from rat serum. Overall, our study unveils a novel staphylococcal complement evasion mechanism which enables the pathogen to harness the ability of dimeric C3d to modulate B-cell activation.
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May 2022
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I03-Macromolecular Crystallography
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Alex
Macpherson
,
James R.
Birtley
,
Robert J.
Broadbridge
,
Kevin
Brady
,
Monika-Sarah E. D.
Schulze
,
Yalan
Tang
,
Callum
Joyce
,
Kenneth
Saunders
,
Gregory
Bogle
,
John
Horton
,
Sebastian
Kelm
,
Richard D.
Taylor
,
Richard J.
Franklin
,
Matthew D.
Selby
,
Maisem
Laabei
,
Toska
Wonfor
,
Adam
Hold
,
Phil
Stanley
,
Douangsone
Vadysirisack
,
Jiye
Shi
,
Jean
Van Den Elsen
,
Alastair D. G.
Lawson
Open Access
Abstract: Cysteine-rich knob domains found in the ultralong complementarity determining regions of a subset of bovine antibodies are capable of functioning autonomously as 3–6 kDa peptides. While they can be expressed recombinantly in cellular systems, in this paper we show that knob domains are also readily amenable to a chemical synthesis, with a co-crystal structure of a chemically synthesized knob domain in complex with an antigen showing structural equivalence to the biological product. For drug discovery, following the immunization of cattle, knob domain peptides can be synthesized directly from antibody sequence data, combining the power and diversity of the bovine immune repertoire with the ability to rapidly incorporate nonbiological modifications. We demonstrate that, through rational design with non-natural amino acids, a paratope diversity can be massively expanded, in this case improving the efficacy of an allosteric peptide. As a potential route to further improve stability, we also performed head-to-tail cyclizations, exploiting the proximity of the N and C termini to synthesize functional, fully cyclic antibody fragments. Lastly, we highlight the stability of knob domains in plasma and, through pharmacokinetic studies, use palmitoylation as a route to extend the plasma half-life of knob domains in vivo. This study presents an antibody-derived medicinal chemistry platform, with protocols for solid-phase synthesis of knob domains, together with the characterization of their molecular structures, in vitro pharmacology, and pharmacokinetics.
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Sep 2021
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I04-Macromolecular Crystallography
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Ayla A.
Wahid
,
Rhys W.
Dunphy
,
Alex
Macpherson
,
Beth G.
Gibson
,
Liudmila
Kulik
,
Kevin
Whale
,
Catherine
Back
,
Thomas M.
Hallam
,
Bayan
Alkhawaja
,
Rebecca L.
Martin
,
Ingrid
Meschede
,
Maisem
Laabei
,
Alastair D. G.
Lawson
,
V. Michael
Holers
,
Andrew G.
Watts
,
Susan J.
Crennell
,
Claire L.
Harris
,
Kevin J.
Marchbank
,
Jean M. H.
Van Den Elsen
Diamond Proposal Number(s):
[17212]
Open Access
Abstract: Cleavage of C3 to C3a and C3b plays a central role in the generation of complement-mediated defences. Although the thioester-mediated surface deposition of C3b has been well-studied, fluid phase dimers of C3 fragments remain largely unexplored. Here we show C3 cleavage results in the spontaneous formation of C3b dimers and present the first X-ray crystal structure of a disulphide-linked human C3d dimer. Binding studies reveal these dimers are capable of crosslinking complement receptor 2 and preliminary cell-based analyses suggest they could modulate B cell activation to influence tolerogenic pathways. Altogether, insights into the physiologically-relevant functions of C3d(g) dimers gained from our findings will pave the way to enhancing our understanding surrounding the importance of complement in the fluid phase and could inform the design of novel therapies for immune system disorders in the future.
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Aug 2021
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I03-Macromolecular Crystallography
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Alex
Macpherson
,
Maisem
Laabei
,
Zainab
Ahdash
,
Melissa A
Graewert
,
James R
Birtley
,
Monika-Sarah
Schulze
,
Susan
Crennell
,
Sarah A
Robinson
,
Ben
Holmes
,
Vladas
Oleinikovas
,
Per H.
Nilsson
,
James
Snowden
,
Victoria
Ellis
,
Tom Eirik
Mollnes
,
Charlotte M.
Deane
,
Dmitri
Svergun
,
Alastair D. G.
Lawson
,
Jean M. H.
Van Den Elsen
Diamond Proposal Number(s):
[20029]
Open Access
Abstract: Bovines have evolved a subset of antibodies with ultra-long CDRH3 regions that harbour cysteine-rich knob domains. To produce high affinity peptides, we previously isolated autonomous 3-6 kDa knob domains from bovine antibodies. Here, we show that binding of four knob domain peptides elicits a range of effects on the clinically validated drug target complement C5. Allosteric mechanisms predominated, with one peptide selectively inhibiting C5 cleavage by the alternative pathway C5 convertase, revealing a targetable mechanistic difference between the classical and alternative pathway C5 convertases. Taking a hybrid biophysical approach, we present C5-knob domain co-crystal structures and, by solution methods, observed allosteric effects propagating >50 Å from the binding sites. This study expands the therapeutic scope of C5, presents new inhibitors and introduces knob domains as new, low molecular weight antibody fragments, with therapeutic potential.
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Feb 2021
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I22-Small angle scattering & Diffraction
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A.
Doekhie
,
R.
Dattani
,
Y-C.
Chen
,
Y.
Yang
,
A.
Smith
,
A. P.
Silve
,
F.
Koumanov
,
S. A.
Wells
,
K. J.
Edler
,
K. J.
Marchbank
,
J. M. H.
Van Den Elsen
,
A.
Sartbaeva
Diamond Proposal Number(s):
[14148]
Open Access
Abstract: Our recently developed ensilication approach can physically stabilize proteins in silica without use of a pre-formed particle matrix. Stabilisation is done by tailor fitting individual proteins with a silica coat using a modified sol-gel process. Biopharmaceuticals, e.g. liquid-formulated vaccines with adjuvants, frequently have poor thermal stability; heating and/or freezing impairs their potency. As a result, there is an increase in the prevalence of vaccine-preventable diseases in low-income countries even when there are means to combat them. One of the root causes lies in the problematic vaccine ‘cold chain’ distribution. We believe that ensilication can improve vaccine availability by enabling transportation without refrigeration. Here, we show that ensilication stabilizes tetanus toxin C fragment (TTCF), a component of the tetanus toxoid present in the diphtheria, tetanus and pertussis (DTP) vaccine. Experimental in vivo immunization data show that the ensilicated material can be stored, transported at ambient temperatures, and even heat-treated without compromising the immunogenic properties of TTCF. To further our understanding of the ensilication process and its protective effect on proteins, we have also studied the formation of TTCF-silica nanoparticles via time-resolved Small Angle X-ray Scattering (SAXS). Our results reveal ensilication to be a staged diffusion-limited cluster aggregation (DLCA) type reaction. An early stage (tens of seconds) in which individual proteins are coated with silica is followed by a subsequent stage (several minutes) in which the protein-containing silica nanoparticles aggregate into larger clusters. Our results suggest that we could utilize this technology for vaccines, therapeutics or other biopharmaceuticals that are not compatible with lyophilization.
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Jun 2020
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B23-Circular Dichroism
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Yun-Chu
Chen
,
Tristan
Smith
,
Robert
Hicks
,
Aswin
Doekhie
,
Francoise
Koumanov
,
Stephen A.
Wells
,
Karen J.
Edler
,
Jean
Van Den Elsen
,
Geoffrey D.
Holman
,
Kevin J.
Marchbank
,
Asel
Sartbaeva
Diamond Proposal Number(s):
[10415]
Open Access
Abstract: Biological substances based on proteins, including vaccines, antibodies, and enzymes, typically degrade at room temperature over time due to denaturation, as proteins unfold with loss of secondary and tertiary structure. Their storage and distribution therefore relies on a “cold chain” of continuous refrigeration; this is costly and not always effective, as any break in the chain leads to rapid loss of effectiveness and potency. Efforts have been made to make vaccines thermally stable using treatments including freeze-drying (lyophilisation), biomineralisation, and encapsulation in sugar glass and organic polymers. Here for the first time we show that proteins can be enclosed in a deposited silica “cage”, rendering them stable against denaturing thermal treatment and long-term ambient-temperature storage, and subsequently released into solution with their structure and function intact. This “ensilication” method produces a storable solid protein-loaded material without the need for desiccation or freeze-drying. Ensilication offers the prospect of a solution to the “cold chain” problem for biological materials, in particular for vaccines.
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Apr 2017
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[7131]
Abstract: The four-component polypeptides of the 2-oxoacid dehydrogenase complex from the thermophilic archaeon Thermoplasma acidophilum assemble to give an active multienzyme complex possessing activity with the branched-chain 2-oxoacids derived from leucine, isoleucine and valine, and with pyruvate. The dihydrolipoyl acyl-transferase (E2) core of the complex is composed of identical trimer-forming units that assemble into a novel 42-mer structure comprising octahedral and icosahedral geometric aspects. From our previously determined structure of this catalytic core, the inter-trimer interactions involve a tyrosine residue near the C-terminus secured in a hydrophobic pocket of an adjacent trimer like a ball-and-socket joint. In the present study, we have deleted the five C-terminal amino acids of the E2 polypeptide (IIYEI) and shown by equilibrium centrifugation that it now only assembles into a trimeric enzyme. This was confirmed by SAXS analysis, although this technique showed the presence of approximately 20% hexamers. The crystal structure of the trimeric truncated E2 core has been determined and shown to be virtually identical with the ones observed in the 42-mer, demonstrating that removal of the C-terminal anchor does not significantly affect the individual monomer or trimer structures. The truncated E2 is still able to bind both 2-oxoacid decarboxylase (E1) and dihydrolipoamide dehydrogenase (E3) components to give an active complex with catalytic activity similar to the native multienzyme complex. This is the first report of an active mini-complex for this enzyme, and raises the question of why all 2-oxoacid dehydrogenase complexes assemble into such large structures.
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May 2014
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Abstract: The dihydrolipoyl acyl-transferase (E2) enzyme forms the structural and catalytic core of the tripartite 2-oxoacid dehydrogenase multienzyme complexes of the central metabolic pathways. Although this family of multienzyme complexes shares a common architecture, their E2 cores form homo-trimers that, depending on the source, further associate into either octahedral (24-mer) or icosahedral (60-mer) assemblies, as predicted by the principles of quasi-equivalence. In the crystal structure of the E2 core from Thermoplasma acidophilum, a thermophilic archaeon, the homo-trimers assemble into a unique 42-mer oblate spheroid. Analytical equilibrium centrifugation and small-angle X-ray scattering analyses confirm that this catalytically active 1.08 MDa assembly exists as a single species in solution, forming a hollow spheroid with a maximum diameter of 220 Å. In this paper we show that a monodisperse macromolecular assembly, built from identical subunits in non-identical environments, forms an irregular protein shell via non-equivalent interactions. This unusually irregular protein shell, combining cubic and dodecahedral geometrical elements, expands on the concept of quasi-equivalence as a basis for understanding macromolecular assemblies by showing that cubic point group symmetry is not a physical requirement in multienzyme assembly. These results extend our basic knowledge of protein assembly and greatly expand the number of possibilities to manipulate self-assembling biological complexes to be utilized in innovative nanotechnology applications.
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Dec 2011
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I04-Macromolecular Crystallography
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Abstract: The interaction of complement receptor 2 (CR2)—which is present on B cells and follicular dendritic cells—with its antigen-bound ligand C3d results in an enhanced antibody response, thus providing an important link between the innate and adaptive immune systems. Although a cocrystal structure of a complex between C3d and the ligand-binding domains of CR2 has been published, several aspects of this structure, including the position in C3d of the binding interface, remained controversial because of disagreement with biochemical data. We now report a cocrystal structure of a CR2(SCR1-2):C3d complex at 3.2 angstrom resolution in which the interaction interfaces differ markedly from the previously published structure and are consistent with the biochemical data. It is likely that, in the previous structure, the interaction was influenced by the presence of zinc acetate additive in the crystallization buffer, leading to a nonphysiological complex. Detailed knowledge of the binding interface now at hand gives the potential to exploit the interaction in vaccine design or in therapeutics directed against autoreactive B cells.
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Apr 2011
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