I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[33300, 31440]
Open Access
Abstract: α-Methylacyl-CoA racemase in M. tuberculosis (MCR) has an essential role in fatty acid metabolism and cholesterol utilization, contributing to the bacterium’s survival and persistence. Understanding the enzymatic activity and structural features of MCR provides insights into its physiological and pathological significance and potential as a therapeutic target. Here, we report high-resolution crystal structures for wild-type MCR in a new crystal form (at 1.65 Å resolution) and for three active-site mutants, H126A, D156A and E241A, at 2.45, 1.64 and 1.85 Å resolutions, respectively. Our analysis of the new wild-type structure revealed a similar dimeric arrangement of MCR molecules to that previously reported and details of the catalytic site. The determination of the structures of these H126A, D156A and E241A mutants, along with their detailed kinetic analysis, has now allowed for a rigorous assessment of their catalytic properties. No significant change outside the enzymatic active site was observed in the three mutants, establishing that the diminution of catalytic activity is mainly attributable to disruption of the catalytic apparatus involving key hydrogen bonding and water-mediated interactions. The wild-type structure, together with detailed mutational and biochemical data, provide a basis for understanding the catalytic properties of this enzyme, which is important for the design of future anti-tuberculosis drug molecules.
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Mar 2024
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23773]
Open Access
Abstract: Tyrosinases belong to the type-III copper enzyme family, which is involved in melanin production in a wide range of organisms. Despite similar overall characteristics and functions, their structures, activities, substrate specificities and regulation vary. The tyrosinase from the bacterium Verrucomicrobium spinosum (vsTyr) is produced as a pre-pro-enzyme in which a C-terminal extension serves as an inactivation domain. It does not require a caddie protein for copper ion incorporation, which makes it similar to eukaryotic tyrosinases. To gain an understanding of the catalytic machinery and regulation of vsTyr activity, we determined the structure of the catalytically active “core domain” of vsTyr by X-ray crystallography. The analysis showed that vsTyr is an atypical bacterial tyrosinase not only because it is independent of a caddie protein but also because it shows the highest structural (and sequence) similarity to plant-derived members of the type-III copper enzyme family and is more closely related to fungal tyrosinases regarding active site features. By modelling the structure of the pre-pro-enzyme using AlphaFold, we observed that Phe453, located in the C-terminal extension, is appropriately positioned to function as a “gatekeeper” residue. Our findings raise questions concerning the evolutionary origin of vsTyr.
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Sep 2023
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[26617]
Open Access
Abstract: Functional changes in chaperone systems play a major role in the decline of cognition and contribute to neurological pathologies, such as Alzheimer’s disease (AD). While such a decline may occur naturally with age or with stress or trauma, the mechanisms involved have remained elusive. The current models suggest that amyloid-β (Aβ) plaque formation leads to the hyperphosphorylation of tau by a Hsp90-dependent process that triggers tau neurofibrillary tangle formation and neurotoxicity. Several co-chaperones of Hsp90 can influence the phosphorylation of tau, including FKBP51, FKBP52 and PP5. In particular, elevated levels of FKBP51 occur with age and stress and are further elevated in AD. Recently, the dihydropyridine LA1011 was shown to reduce tau pathology and amyloid plaque formation in transgenic AD mice, probably through its interaction with Hsp90, although the precise mode of action is currently unknown. Here, we present a co-crystal structure of LA1011 in complex with a fragment of Hsp90. We show that LA1011 can disrupt the binding of FKBP51, which might help to rebalance the Hsp90-FKBP51 chaperone machinery and provide a favourable prognosis towards AD. However, without direct evidence, we cannot completely rule out effects on other Hsp90-co-chaprone complexes and the mechanisms they are involved in, including effects on Hsp90 client proteins. Nonetheless, it is highly significant that LA1011 showed promise in our previous AD mouse models, as AD is generally a disease affecting older patients, where slowing of disease progression could result in AD no longer being life limiting. The clinical value of LA1011 and its possible derivatives thereof remains to be seen.
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Jun 2023
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B21-High Throughput SAXS
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Open Access
Abstract: The translation factor IF5A is a highly conserved protein playing a well-recognized and well-characterized role in protein synthesis; nevertheless, some of its features as well as its abundance in the cell suggest that it may perform additional functions related to RNA metabolism. Here, we have undertaken a structural and functional characterization of aIF5A from the crenarchaeal Sulfolobus solfataricus model organism. We confirm the association of aIF5A with several RNA molecules in vivo and demonstrate that the protein is endowed with a ribonuclease activity which is specific for long and structured RNA. By means of biochemical and structural approaches we show that aIF5A can exist in both monomeric and dimeric conformations and the monomer formation is favored by the association with RNA. Finally, modelling of the three-dimensional structure of S. solfataricus aIF5A shows an extended positively charged surface which may explain its strong tendency to associate to RNA in vivo.
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Oct 2022
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B21-High Throughput SAXS
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Mette Ahrensback
Roesgaard
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Jeppe E.
Lundsgaard
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Estella A.
Newcombe
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Nina L.
Jacobsen
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Francesco
Pesce
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Emil E.
Tranchant
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Søren
Lindemose
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Andreas
Prestel
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Rasmus
Hartmann-Petersen
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Kresten
Lindorff-Larsen
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Birthe B.
Kragelund
Open Access
Abstract: Compared to folded proteins, the sequences of intrinsically disordered proteins (IDPs) are enriched in polar and charged amino acids. Glutamate is one of the most enriched amino acids in IDPs, while the chemically similar amino acid aspartate is less enriched. So far, the underlying functional differences between glutamates and aspartates in IDPs remain poorly understood. In this study, we examine the differential effects of aspartate and glutamates in IDPs by comparing the function and conformational ensemble of glutamate and aspartate variants of the disordered protein Dss1, using a range of assays, including interaction studies, nuclear magnetic resonance spectroscopy, small-angle X-ray scattering and molecular dynamics simulation. First, we analyze the sequences of the rapidly growing database of experimentally verified IDPs (DisProt) and show that glutamate enrichment is not caused by a taxonomy bias in IDPs. From analyses of local and global structural properties as well as cell growth and protein-protein interactions using a model acidic IDP from yeast and three Glu/Asp variants, we find that while the Glu/Asp variants support similar function and global dimensions, the variants differ in their binding affinities and population of local transient structural elements. We speculate that these local structural differences may play roles in functional diversity, where glutamates can support increased helicity, important for folding and binding, while aspartates support extended structures and form helical caps, as well as playing more relevant roles in, e.g., transactivation domains and ion-binding.
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Oct 2022
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B21-High Throughput SAXS
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Open Access
Abstract: Protein aggregation is a well-recognized problem in industrial preparation, including biotherapeutics. These low-energy states constantly compete with a native-like conformation, which is more pronounced in the case of macromolecules of low stability in the solution. A better understanding of the structure and function of such aggregates is generally required for the more rational development of therapeutic proteins, including single-chain fusion cytotoxins to target specific receptors on cancer cells. Here, we identified and purified such particles as side products of the renaturation process of the single-chain fusion cytotoxin, composed of two diphtheria toxin (DT) domains and interleukin 13 (IL-13), and applied various experimental techniques to comprehensively understand their molecular architecture and function. Importantly, we distinguished soluble purified dimeric and fractionated oligomeric particles from aggregates. The oligomers are polydisperse and multimodal, with a distribution favoring lower and even stoichiometries, suggesting they are composed of dimeric building units. Importantly, all these oligomeric particles and the monomer are cystine-dependent as their innate disulfide bonds have structural and functional roles. Their reduction triggers aggregation. Presumably the dimer and lower oligomers represent the metastable state, retaining the native disulfide bond. Although significantly reduced in contrast to the monomer, they preserve some fraction of bioactivity, manifested by their IL-13RA2 receptor affinity and selective cytotoxic potency towards the U-251 glioblastoma cell line. These molecular assemblies probably preserve structural integrity and native-like fold, at least to some extent. As our study demonstrated, the dimeric and oligomeric cytotoxin may be an exciting model protein, introducing a new understanding of its monomeric counterpart’s molecular characteristics.
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Aug 2022
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Open Access
Abstract: Vascular endothelial growth factors (VEGFs) are the key regulators of blood and lymphatic vessels’ formation and function. Each of the proteins from the homologous family VEGFA, VEGFB, VEGFC and VEGFD employs a core cysteine-knot structural domain for the specific interaction with one or more of the cognate tyrosine kinase receptors. Additional diversity is exhibited by the involvement of neuropilins–transmembrane co-receptors, whose b1 domain contains the binding site for the C-terminal sequence of VEGFs. Although all relevant isoforms of VEGFs that interact with neuropilins contain the required C-terminal Arg residue, there is selectivity of neuropilins and VEGF receptors for the VEGF proteins, which is reflected in the physiological roles that they mediate. To decipher the contribution made by the C-terminal sequences of the individual VEGF proteins to that functional differentiation, we determined structures of molecular complexes of neuropilins and VEGF-derived peptides and examined binding interactions for all neuropilin-VEGF pairs experimentally and computationally. While X-ray crystal structures and ligand-binding experiments highlighted similarities between the ligands, the molecular dynamics simulations uncovered conformational preferences of VEGF-derived peptides beyond the C-terminal arginine that contribute to the ligand selectivity of neuropilins. The implications for the design of the selective antagonists of neuropilins’ functions are discussed.
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Feb 2022
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[21741]
Open Access
Abstract: Bile acids (BAs) are hydroxylated steroids derived from cholesterol that act at the intestinal level to facilitate the absorption of several nutrients and also play a role as signaling molecules. In the liver of various vertebrates, the trafficking of BAs is mediated by bile acid-binding proteins (L-BABPs). The ability to host hydrophobic or amphipathic molecules makes BABPs suitable for the distribution of a variety of physiological and exogenous substances. Thus, BABPs have been proposed as drug carriers, and more recently, they have also been employed to develop innovative nanotechnology and biotechnology systems. Here, we report an efficient protocol for the production, purification, and crystallization of chicken liver BABP (cL-BABP). By means of target expression as His6-tag cL-BABP, we obtained a large amount of pure and homogeneous proteins through a simple purification procedure relying on affinity chromatography. The recombinant cL-BABP showed a raised propensity to crystallize, allowing us to obtain its structure at high resolution and, in turn, assess the structural conservation of the recombinant cL-BABP with respect to the liver-extracted protein. The results support the use of recombinant cL-BABP for the development of drug carriers, nanotechnologies, and innovative synthetic photoswitch systems.
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Apr 2021
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B21-High Throughput SAXS
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Raquel R.
Lopes
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Catarina S.
Tomé
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Roberto
Russo
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Roberta
Paterna
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João
Leandro
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Nuno R.
Candeias
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Lídia M. D.
Gonçalves
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Miguel
Teixeira
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Pedro M. F.
Sousa
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Rita C.
Guedes
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João B.
Vicente
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Pedro M. P.
Gois
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Paula
Leandro
Diamond Proposal Number(s):
[20161]
Open Access
Abstract: Phenylketonuria (PKU) is a genetic disease caused by deficient activity of human phenylalanine hydroxylase (hPAH) that, when untreated, can lead to severe psychomotor impairment. Protein misfolding is recognized as the main underlying pathogenic mechanism of PKU. Therefore, the use of stabilizers of protein structure and/or activity is an attractive therapeutic strategy for this condition. Here, we report that 3-hydroxyquinolin-2(1H)-one derivatives can act as protectors of hPAH enzyme activity. Electron paramagnetic resonance spectroscopy demonstrated that the 3-hydroxyquinolin-2(1H)-one compounds affect the coordination of the non-heme ferric center at the enzyme active-site. Moreover, surface plasmon resonance studies showed that these stabilizing compounds can be outcompeted by the natural substrate l-phenylalanine. Two of the designed compounds functionally stabilized hPAH by maintaining protein activity. This effect was observed on the recombinant purified protein and in a cellular model. Besides interacting with the catalytic iron, one of the compounds also binds to the N-terminal regulatory domain, although to a different location from the allosteric l-Phe binding site, as supported by the solution structures obtained by small-angle X-ray scattering.
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Mar 2021
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Krios I-Titan Krios I at Diamond
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Open Access
Abstract: Chemotactic responses in motile bacteria are the result of sophisticated signal transduction by large, highly organized arrays of sensory proteins. Despite tremendous progress in the understanding of chemosensory array structure and function, a structural basis for the heightened sensitivity of networked chemoreceptors is not yet complete. Here, we present cryo-electron tomography visualisations of native-state chemosensory arrays in E. coli minicells. Strikingly, these arrays appear to exhibit a p2-symmetric array architecture that differs markedly from the p6-symmetric architecture previously described in E. coli. Based on this data, we propose molecular models of this alternative architecture and the canonical p6-symmetric assembly. We evaluate our observations and each model in the context of previously published data, assessing the functional implications of an alternative architecture and effects for future studies.
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Mar 2021
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