I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[1226]
Open Access
Abstract: A 2-keto-3-deoxygluconate aldolase from the hyperthermophile Sulfolobus solfataricus catalyzes the nonstereoselective aldol reaction of pyruvate and d-glyceraldehyde to produce 2-keto-3-deoxygluconate (d-KDGlc) and 2-keto-3-deoxy-d-galactonate (d-KDGal). Previous investigations into curing the stereochemical promiscuity of this hyperstable aldolase used high-resolution structures of the aldolase bound to d-KDGlc or d-KDGal to identify critical amino acids involved in substrate binding for mutation. This structure-guided approach enabled mutant variants to be created that could stereoselectively catalyze the aldol reaction of pyruvate and natural d-glyceraldehyde to selectively afford d-KDGlc or d-KDGal. Here we describe the creation of two further mutants of this Sulfolobus aldolase that can be used to catalyze aldol reactions between pyruvate and non-natural l-glyceraldehyde to enable the diastereoselective synthesis of l-KDGlc and l-KDGal. High-resolution crystal structures of all four variant aldolases have been determined (both unliganded and liganded), including Variant 1 with d-KDGlc, Variant 2 with pyruvate, Variant 3 with l-KDGlc, and Variant 4 with l-KDGal. These structures have enabled us to rationalize the observed changes in diastereoselectivities in these variant-catalyzed aldol reactions at a molecular level. Interestingly, the active site of Variant 4 was found to be sufficiently flexible to enable catalytically important amino acids to be replaced while still retaining sufficient enzymic activity to enable production of l-KDGal.
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Sep 2022
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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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I04-Macromolecular Crystallography
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Ayla A.
Wahid
,
Rhys W.
Dunphy
,
Alex
Macpherson
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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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I04-Macromolecular Crystallography
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Abstract: The thermoacidophilic archaea Picrophilus torridus and Sulfolobus solfataricus catabolise glucose via a non-phosphorylative Entner-Doudoroff pathway and a branched Entner-Doudoroff pathway, respectively. Key enzymes for these Entner-Doudoroff pathways are the aldolases, 2-keto-3-deoxygluconate aldolase (KDG-aldolase) and 2-keto-3-deoxy-6-phosphogluconate aldolase (KD(P)G-aldolase). KDG-aldolase from P. torridus (Pt-KDG-aldolase) is highly specific for the non-phosphorylated substrate, 2-keto-3-deoxygluconate (KDG), whereas KD(P)G-aldolase from S. solfataricus (Ss-KD(P)G-aldolase) is an enzyme catalyzing the cleavage of both KDG and 2-keto-3-deoxy-6-phosphogluconate (KDPG), with a preference for KDPG. The structural basis for the high specificity of Pt-KDG-aldolase for KDG as compared to the more promiscuous Ss-KD(P)G-aldolase has not been analysed before. In the current paper we report the elucidation of the structure of Ss-KD(P)G-aldolase in complex with KDPG at 2.35Å and that of KDG-aldolase from P. torridus at 2.50 Å resolution. By superimposition of the active sites of the two enzymes, and subsequent site-directed mutagenesis studies, a network of four amino acids, namely Arg106, Tyr132, Arg237 and Ser241, was identified in Ss-KD(P)G-aldolase that interact with the negatively-charged phosphate group of KDPG, thereby raising the affinity of the enzyme for KDPG. This KDPG-binding network is absent in Pt-KDG-aldolase, which explains the low catalytic efficiency of KDPG cleavage.
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May 2018
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[7131]
Abstract: Acetylating aldehyde dehydrogenases (AcAldDH) catalyse the acetylation of Coenzyme-A (CoA), or in reverse generate acetaldehyde from Acetyl-CoA using NADH as a co-factor. This paper reports the expression, purification, enzyme assay and X-ray crystal structures of an AcAldDH from Geobacillus thermoglucosidasius (GtAcAldDH) to 2.1Å and in complex with CoA and NAD(+) to 4.0Å. In the structure, the AcAldDH forms a close-knit dimer, similar to that seen in other Alcohol Dehydrogenase (ADH) structures. In GtAcAldDH these dimers associate via their N-termini to form weakly-interacting tetramers. This mode of tetrameric association is also seen in an unpublished AcAldDH deposited in the PDB, but is in contrast to all other ADH structures, (including the one other published AcAldDH found in a bacterial microcompartment), in which the dimers bury a large surface area including the C-termini. This novel mode of association sequesters the active sites and potentially-reactive acyl-enzyme intermediates in the centre of the tetramer. In other respects the structure is very similar to the other AcAldDH, binding the cofactors in a corresponding fashion. This similarity enabled the identification of a shortened substrate cavity in G. thermoglucosidasius AcAldDH, explaining the limitations on the length of substrate accepted by the enzyme.
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Aug 2016
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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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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[1226]
Abstract: Bifunctional alcohol/aldehyde dehydrogenase (ADHE) enzymes are found within many fermentative microorganisms. They catalyse the conversion of an acyl-coenzyme A to an alcohol via an aldehyde intermediate; this is coupled to the oxidation of two NADH molecules to maintain the NAD+ pool during fermentative metabolism. The structure of the alcohol dehydrogenase (ADH) domain of an ADHE protein from the ethanol-producing thermophile Geobacillus thermoglucosidasius has been determined to 2.5 Å resolution. This is the first structure to be reported for such a domain. In silico modelling has been carried out to generate a homology model of the aldehyde dehydrogenase domain, and this was subsequently docked with the ADH-domain structure to model the structure of the complete ADHE protein. This model suggests, for the first time, a structural mechanism for the formation of the large multimeric assemblies or `spirosomes' that are observed for this ADHE protein and which have previously been reported for ADHEs from other organisms.
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Oct 2013
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I02-Macromolecular Crystallography
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Abstract: Lipoylation, the covalent attachment of lipoic acid to 2-oxoacid dehydrogenase multi-enzyme complexes, is essential for metabolism in aerobic bacteria and eukarya. In Escherichia coli, lipoylation is catalysed by LplA (lipoate protein ligase) or by LipA (lipoic acid synthetase) and LipB [lipoyl(octanoyl) transferase] combined. Whereas bacterial and eukaryotic LplAs comprise a single two-domain protein, archaeal LplA function typically involves two proteins, LplA-N and LplA-C. In the thermophilic archaeon Thermoplasma acidophilum, LplA-N and LplA-C are encoded by overlapping genes in inverted orientation (lpla-c is upstream of lpla-n). The T. acidophilum LplA-N structure is known, but the LplA-C structure is unknown and LplA-C's role in lipoylation is unclear. In the present study, we have determined the structures of the substrate-free LplA-N–LplA-C complex and E2lipD (dihydrolipoyl acyltransferase lipoyl domain) that is lipoylated by LplA-N–LplA-C, and carried out biochemical analyses of this archaeal lipoylation system. Our data reveal the following: (i) LplA-C is disordered but folds upon association with LplA-N; (ii) LplA-C induces a conformational change in LplA-N involving substantial shortening of a loop that could repress catalytic activity of isolated LplA-N; (iii) the adenylate-binding region of LplA-N–LplA-C includes two helices rather than the purely loop structure of varying order observed in other LplA structures; (iv) LplAN–LplA-C and E2lipD do not interact in the absence of substrate; (v) LplA-N–LplA-C undergoes a conformational change (the details of which are currently undetermined) during lipoylation; and (vi) LplA-N–LplA-C can utilize octanoic acid as well as lipoic acid as substrate. The elucidated functional inter-dependence of LplA-N and LplA-C is consistent with their evolutionary co-retention in archaeal genomes.
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Jan 2013
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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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