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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I04-Macromolecular Crystallography
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Filip
Mihalič
,
Leandro
Simonetti
,
Girolamo
Giudice
,
Marie Rubin
Sander
,
Richard
Lindqvist
,
Marie Berit Akpiroro
Peters
,
Caroline
Benz
,
Eszter
Kassa
,
Dilip
Badgujar
,
Raviteja
Inturi
,
Muhammad
Ali
,
Izabella
Krystkowiak
,
Ahmed
Sayadi
,
Eva
Andersson
,
Hanna
Aronsson
,
Ola
Söderberg
,
Doreen
Dobritzsch
,
Evangelia
Petsalaki
,
Anna K.
Överby
,
Per
Jemth
,
Norman E.
Davey
,
Ylva
Ivarsson
Diamond Proposal Number(s):
[23773]
Open Access
Abstract: Viruses mimic host short linear motifs (SLiMs) to hijack and deregulate cellular functions. Studies of motif-mediated interactions therefore provide insight into virus-host dependencies, and reveal targets for therapeutic intervention. Here, we describe the pan-viral discovery of 1712 SLiM-based virus-host interactions using a phage peptidome tiling the intrinsically disordered protein regions of 229 RNA viruses. We find mimicry of host SLiMs to be a ubiquitous viral strategy, reveal novel host proteins hijacked by viruses, and identify cellular pathways frequently deregulated by viral motif mimicry. Using structural and biophysical analyses, we show that viral mimicry-based interactions have similar binding strength and bound conformations as endogenous interactions. Finally, we establish polyadenylate-binding protein 1 as a potential target for broad-spectrum antiviral agent development. Our platform enables rapid discovery of mechanisms of viral interference and the identification of potential therapeutic targets which can aid in combating future epidemics and pandemics.
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Apr 2023
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I03-Macromolecular Crystallography
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Changrong
Ge
,
Sylvia
Weisse
,
Bingze
Xu
,
Doreen
Dobritzsch
,
Johan
Viljanen
,
Jan
Kihlberg
,
Nhu-Nguyen
Do
,
Nadine
Schneider
,
Harald
Lanig
,
Rikard
Holmdahl
,
Harald
Burkhardt
Open Access
Abstract: Objectives: Rheumatoid arthritis (RA) is an autoimmune disease strongly associated with the major histocompatibility complex (MHC) class II allele DRB1*04:01, which encodes a protein that binds self-peptides for presentation to T cells. This study characterises the autoantigen-presenting function of DRB1*04:01 (HLA-DRA*01:01/HLA-DRB1*04:01) at a molecular level for prototypic T-cell determinants, focusing on a post-translationally modified collagen type II (Col2)-derived peptide.Methods: The crystal structures of DRB1*04:01 molecules in complex with the peptides HSP70289-306, citrullinated CILP982-996 and galactosylated Col2259-273 were determined on cocrystallisation. T cells specific for Col2259-273 were investigated in peripheral blood mononuclear cells from patients with DRB1*04:01-positive RA by cytofluorometric detection of the activation marker CD154 on peptide stimulation and binding of fluorescent DRB1*0401/Col2259-273 tetramer complexes. The cDNAs encoding the T-cell receptor (TCR) α-chains and β-chains were cloned from single-cell sorted tetramer-positive T cells and transferred via a lentiviral vector into TCR-deficient Jurkat 76 cells. Results: The crystal structures identified peptide binding to DRB1*04:01 and potential side chain exposure to T cells. The main TCR recognition sites in Col2259-273 were lysine residues that can be galactosylated. RA T-cell responses to DRB1*04:01-presented Col2259-273 were dependent on peptide galactosylation at lysine 264. Dynamic molecular modelling of a functionally characterised Col2259-273-specific TCR complexed with DRB1*04:01/Col2259-273 provided evidence for differential allosteric T-cell recognition of glycosylated lysine 264. Conclusions: The MHC-peptide-TCR interactions elucidated in our study provide new molecular insights into recognition of a post-translationally modified RA T-cell determinant with a known dominant role in arthritogenic and tolerogenic responses in murine Col2-induced arthritis.
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Mar 2022
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I03-Macromolecular Crystallography
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Changrong
Ge
,
Dongmei
Tong
,
Erik
Lönnblom
,
Bibo
Liang
,
Weiwei
Cai
,
Cecilia
Fahlquist-Hagert
,
Taotao
Li
,
Alf
Kastbom
,
Inger
Gjertsson
,
Doreen
Dobritzsch
,
Rikard
Holmdahl
Diamond Proposal Number(s):
[8492]
Open Access
Abstract: Objectives: Cartilage oligomeric matrix protein (COMP) is an autoantigen in rheumatoid arthritis and experimental arthritis models. We aimed to investigate the structure, function and relevance of anti-COMP antibodies. Methods: The pathogenicity of monoclonal anti-COMP antibodies in mice was investigated by passive transfer experiments and interaction with cartilage by immunohistochemical staining. The interaction of the monoclonal antibody 15A11 in complex with its specific COMP epitope P6 was determined by X-ray crystallography. The modulation of the binding to 15A11 by calcium ions was studied by ELISA and the surface plasma resonance technique. The clinical relevance and value of serum IgG specific for the COMP P6 epitope and its citrullinated variants were evaluated in a large Swedish cohort of RA patients. Results: The murine monoclonal anti-COMP antibody 15A11 induced arthritis in naïve mice. The crystal structure of 15A11-P6 complex explained how the antibody could bind to COMP, which can be modulated by calcium ions. Moreover, serum IgGs specific for the COMP P6 peptide and its citrullinated variants were detectable at significantly higher levels in RA patients compared to the healthy controls and correlated with a higher disease activity score. Conclusions: We provide the structural basis for binding a pathogenic anti-COMP antibody to cartilage. The recognized epitope can be citrullinated and levels of antibodies to this epitope are elevated in RA patients and correlate with higher disease activity, implicating a pathogenic role of anti-COMP antibodies in a subset of RA patients.
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Jan 2022
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I03-Macromolecular Crystallography
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Jie
Yang
,
Vladimir O.
Talibov
,
Stefan
Peintner
,
Claire
Rhee
,
Vasanthanathan
Poongavanam
,
Matthis
Geitmann
,
Matteo Rossi
Sebastiano
,
Bernd
Simon
,
Janosch
Hennig
,
Doreen
Dobritzsch
,
U. Helena
Danielson
,
Jan
Kihlberg
Diamond Proposal Number(s):
[15868]
Open Access
Abstract: Lysine-specific demethylase 1 (LSD1) is an epigenetic enzyme which regulates the methylation of Lys4 of histone 3 (H3) and is overexpressed in certain cancers. We used structures of H3 substrate analogues bound to LSD1 to design macrocyclic peptide inhibitors of LSD1. A linear, Lys4 to Met-substituted, 11-mer (4) was identified as the shortest peptide distinctly interacting with LSD1. It was evolved into macrocycle 31, which was >40 fold more potent (Ki = 2.3 μM) than 4. Linear and macrocyclic peptides exhibited unexpected differences in structure–activity relationships for interactions with LSD1, indicating that they bind LSD1 differently. This was confirmed by the crystal structure of 31 in complex with LSD1-CoREST1, which revealed a novel binding mode at the outer rim of the LSD1 active site and without a direct interaction with FAD. NMR spectroscopy of 31 suggests that macrocyclization restricts its solution ensemble to conformations that include the one in the crystalline complex. Our results provide a solid basis for the design of optimized reversible LSD1 inhibitors.
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Feb 2020
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Changrong
Ge
,
Bingze
Xu
,
Bibo
Liang
,
Erik
Lönnblom
,
Susanna L.
Lundström
,
Roman A.
Zubarev
,
Burcu
Ayoglu
,
Peter
Nilsson
,
Thomas
Skogh
,
Alf
Kastbom
,
Vivianne
Malmström
,
Lars
Klareskog
,
René E. M.
Toes
,
Theo
Rispens
,
Doreen
Dobritzsch
,
Rikard
Holmdahl
Diamond Proposal Number(s):
[8492, 11265]
Abstract: OBJECTIVES: Anti-citrullinated protein antibodies (ACPAs) develop many years before the clinical onset of rheumatoid arthritis. Here we addressed the molecular basis of the specificity and cross-reactivity of ACPAs from patients with rheumatoid arthritis (RA). METHODS: Antibodies isolated from RA patients were expressed as monoclonal chimeric antibodies with mouse Fc and characterized for glycosylation using mass-spectrometry and cross-reactivity using Biacore and Luminex immunoassay. Crystal structures of the antigen-binding fragment (Fab) of the monoclonal ACPA E4 in complex with three different citrullinated peptides were solved using x-ray crystallography. The prevalence of autoantibodies reactive against three of the citrullinated peptides that also interact with E4, was investigated by Luminex immunoassay in two Swedish cohorts of RA patients. RESULTS: Analysis of the crystal structures of a monoclonal ACPA in complex with citrullinated peptides revealed key residues of several complementarity-determining regions (CDR) that recognize the citrulline as well as the neighboring peptide backbone, but with limited contact with the side chains of the peptides. The same citrullinated peptides were recognized by high titers of autoantibodies in two large cohorts of RA patients. CONCLUSIONS: The data show for the first time how ACPA, derived from human RA, recognize citrulline. The specific citrulline recognition and backbone-mediated interactions provide a structural explanation of the promiscuous recognition of citrullinated peptides by RA-specific ACPAs.
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Aug 2018
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[11171, 15868]
Abstract: ADH-A from Rhodococcus ruber DSM 44541 catalyzes the oxidation of (S)-1-phenylethanol 3,000-fold more efficiently as compared to the 2-hydroxylated derivative (R)-phenylethane-1,2-diol. The enzyme is also highly selective for sec-alcohols with comparably low activities with the corresponding primary alcohols. When challenged with a substrate containing two secondary alcohols, such as 1-phenylpropane-(1R,2S)-diol, ADH-A favors the oxidation of the benzylic carbon of this alcohol. The catalytic efficiency, however, is modest in comparison to the activity with (S)-1-phenylethanol. To investigate the structural requirements for improved oxidation of vicinal diols we conducted iterative saturation mutagenesis combined with activity screening. A first-generation variant, B1 (Y54G, L119Y) displays a two-fold higher kcat value with 1-phenylpropane-(1R,2S)-diol and a shift in the cooperative behavior in alcohol binding, from negative in the wild type, to positive in B1, suggesting a shift from a less active enzyme form (T) in the wild type to a more active form (R) in the B1 variant. Also, the regiopreference changed to favor oxidation of C-2. A second-generation variant, B1F4 (F43T, Y54G, L119Y, F282W), shows further improvement in the turnover and regioselectivity in oxidation of 1-phenylpropane-(1R,2S)-diol. The crystal structures of the B1 and B1F4 variants describe the structural alterations to the active site the most significant of which is a re-positioning of a Tyr side-chain located distal to the coenzyme and the catalytic zinc ion. The links between the changes in structures and stereoselectivities are rationalized by molecular dynamics simulations of substrate binding at the respective active sites.
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Jul 2018
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[15868]
Abstract: β-Ureidopropionase catalyzes the third step of the reductive pyrimidine catabolic pathway responsible for breakdown of uracil, thymine and pyrimidine-based antimetabolites such as 5-fluorouracil. Nitrilase-like β-ureidopropionases use a tetrad of conserved residues (Cys233, Lys196, Glu119 and Glu207) for catalysis and occur in a variety of oligomeric states. Positive cooperativity towards the substrate N-carbamoyl-β-alanine and an oligomerization-dependent mechanism of substrate activation and product inhibition have been reported for the enzymes from some species but not others. Here, the activity of recombinant human β-ureidopropionase is shown to be similarly regulated by substrate and product, but in a pH-dependent manner. Existing as homodimer at pH 9, the enzyme increasingly associates to octamers and larger oligomers with decreasing pH. Only at physiological pH it is responsive to effector binding, with N-carbamoyl-β-alanine causing association to more active higher molecular mass species, and β-alanine dissociation to inactive dimers. The parallel between the pH and ligand-induced effects suggests protonation state changes to play a crucial role in the allosteric regulation mechanism. Disruption of dimer-dimer interfaces by site-directed mutagenesis generated dimeric, inactive enzyme variants. The crystal structure of the T299C variant refined to 2.08 Å resolution revealed high structural conservation between human and fruit fly β-ureidopropionase, and supports the hypothesis that enzyme activation by oligomer assembly involves ordering of loop regions forming the entrance to the active site at the dimer-dimer interface, effectively positioning the catalytically important Glu207 in the active site.
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Jul 2018
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[11171]
Abstract: Laboratory evolution of alcohol dehydrogenase produced enzyme variants with improved turnover numbers with a vicinal 1,2-diol and its corresponding hydroxyketone. Crystal structure and transient kinetics analysis aids in rationalizing the new functions of these variants.
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Feb 2018
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[11171]
Abstract: Alcohol dehydrogenase A (ADH-A) from Rhodococcus ruber DSM 44541 is a promising biocatalyst for redox transformations of arylsubstituted sec-alcohols and ketones. The enzyme is stereoselective in the oxidation of 1-phenylethanol with a 300-fold preference for the (S)-enantiomer. The low catalytic efficiency with (R)-1-phenylethanol has been attributed to nonproductive binding of this substrate at the active site. Aiming to modify the enantioselectivity, to rather favor the (R)-alcohol, and also test the possible involvement of nonproductive substrate binding as a mechanism in substrate discrimination, we performed directed laboratory evolution of ADH-A. Three targeted sites that contribute to the active-site cavity were exposed to saturation mutagenesis in a stepwise manner and the generated variants were selected for improved catalytic activity with (R)-1-phenylethanol. After three subsequent rounds of mutagenesis, selection and structure-function analysis of isolated ADH-A variants, we conclude: (1) W295 has a key role as a structural determinant in the discrimination between (R)- and (S)-1-phenylethanol and a W295A substitution fundamentally changes the stereoselectivity of the protein. One observable effect is a faster rate of NADH release, which changes the rate-limiting step of the catalytic cycle from coenzyme release to hydride transfer. (2) The obtained change in enantiopreference, from the (S)- to the (R)-alcohol, can be partly explained by a shift in the nonproductive substrate binding modes.
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Sep 2017
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