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1.
ChemMedChem ; : e202400025, 2024 Apr 06.
Article in English | MEDLINE | ID: mdl-38581280

ABSTRACT

Identification and assessment of novel targets is essential to combat drug resistance in the treatment of HIV/AIDS. HIV Capsid (HIV-CA), the protein playing a major role in both the early and late stages of the viral life cycle, has emerged as an important target. We have applied an NMR fragment screening platform and identified molecules that bind to the N-terminal domain (NTD) of HIV-CA at a site close to the interface with the C-terminal domain (CTD). Using X-ray crystallography, we have been able to obtain crystal structures to identify the binding mode of these compounds. This allowed for rapid progression of the initial, weak binding, fragment starting points to compounds 37 and 38, which have 19F-pKi values of 5.3 and 5.4 respectively.

2.
Nat Commun ; 15(1): 2071, 2024 Mar 07.
Article in English | MEDLINE | ID: mdl-38453915

ABSTRACT

IL-11 and IL-6 activate signalling via assembly of the cell surface receptor gp130; however, it is unclear how signals are transmitted across the membrane to instruct cellular responses. Here we solve the cryoEM structure of the IL-11 receptor recognition complex to discover how differences in gp130-binding interfaces may drive signalling outcomes. We explore how mutations in the IL6ST gene encoding for gp130, which cause severe immune deficiencies in humans, impair signalling without blocking cytokine binding. We use cryoEM to solve structures of both IL-11 and IL-6 complexes with a mutant form of gp130 associated with human disease. Together with molecular dynamics simulations, we show that the disease-associated variant led to an increase in flexibility including motion within the cytokine-binding core and increased distance between extracellular domains. However, these distances are minimized as the transmembrane helix exits the membrane, suggesting a stringency in geometry for signalling and dimmer switch mode of action.


Subject(s)
Interleukin-11 , Interleukin-6 , Humans , Interleukin-11/genetics , Interleukin-6/metabolism , Cytokine Receptor gp130/genetics , Cytokine Receptor gp130/metabolism , Signal Transduction , Receptors, Interleukin-6/genetics
3.
Sci Immunol ; 7(78): eade5686, 2022 12 09.
Article in English | MEDLINE | ID: mdl-36459543

ABSTRACT

Cytokines interact with their receptors in the extracellular space to control immune responses. How the physicochemical properties of the extracellular space influence cytokine signaling is incompletely elucidated. Here, we show that the activity of interleukin-2 (IL-2), a cytokine critical to T cell immunity, is profoundly affected by pH, limiting IL-2 signaling within the acidic environment of tumors. Generation of lactic acid by tumors limits STAT5 activation, effector differentiation, and antitumor immunity by CD8+ T cells and renders high-dose IL-2 therapy poorly effective. Directed evolution enabled selection of a pH-selective IL-2 mutein (Switch-2). Switch-2 binds the IL-2 receptor subunit IL-2Rα with higher affinity, triggers STAT5 activation, and drives CD8+ T cell effector function more potently at acidic pH than at neutral pH. Consequently, high-dose Switch-2 therapy induces potent immune activation and tumor rejection with reduced on-target toxicity in normal tissues. Last, we show that sensitivity to pH is a generalizable property of a diverse range of cytokines with broad relevance to immunity and immunotherapy in healthy and diseased tissues.


Subject(s)
Interleukin-2 , Neoplasms , Humans , STAT5 Transcription Factor , CD8-Positive T-Lymphocytes , Cytokines , Hydrogen-Ion Concentration
4.
EMBO Rep ; 23(10): e55450, 2022 10 06.
Article in English | MEDLINE | ID: mdl-35920255

ABSTRACT

Interleukin 27 (IL-27) is a heterodimeric cytokine that elicits potent immunosuppressive responses. Comprised of EBI3 and p28 subunits, IL-27 binds GP130 and IL-27Rα receptor chains to activate the JAK/STAT signaling cascade. However, how these receptors recognize IL-27 and form a complex capable of phosphorylating JAK proteins remains unclear. Here, we used cryo electron microscopy (cryoEM) and AlphaFold modeling to solve the structure of the IL-27 receptor recognition complex. Our data show how IL-27 serves as a bridge connecting IL-27Rα (domains 1-2) with GP130 (domains 1-3) to initiate signaling. While both receptors contact the p28 component of the heterodimeric cytokine, EBI3 stabilizes the complex by binding a positively charged surface of IL-27Rα and Domain 1 of GP130. We find that assembly of the IL-27 receptor recognition complex is distinct from both IL-12 and IL-6 cytokine families and provides a mechanistic blueprint for tuning IL-27 pleiotropic actions.


Subject(s)
Cytokine Receptor gp130 , Interleukin-27 , Receptors, Interleukin , Cytokine Receptor gp130/chemistry , Humans , Interleukin-12 , Interleukin-27/chemistry , Interleukin-6 , Interleukins , Receptors, Interleukin/chemistry
5.
Elife ; 102021 04 19.
Article in English | MEDLINE | ID: mdl-33871355

ABSTRACT

Cytokines elicit pleiotropic and non-redundant activities despite strong overlap in their usage of receptors, JAKs and STATs molecules. We use IL-6 and IL-27 to ask how two cytokines activating the same signaling pathway have different biological roles. We found that IL-27 induces more sustained STAT1 phosphorylation than IL-6, with the two cytokines inducing comparable levels of STAT3 phosphorylation. Mathematical and statistical modeling of IL-6 and IL-27 signaling identified STAT3 binding to GP130, and STAT1 binding to IL-27Rα, as the main dynamical processes contributing to sustained pSTAT1 levels by IL-27. Mutation of Tyr613 on IL-27Rα decreased IL-27-induced STAT1 phosphorylation by 80% but had limited effect on STAT3 phosphorgylation. Strong receptor/STAT coupling by IL-27 initiated a unique gene expression program, which required sustained STAT1 phosphorylation and IRF1 expression and was enriched in classical Interferon Stimulated Genes. Interestingly, the STAT/receptor coupling exhibited by IL-6/IL-27 was altered in patients with systemic lupus erythematosus (SLE). IL-6/IL-27 induced a more potent STAT1 activation in SLE patients than in healthy controls, which correlated with higher STAT1 expression in these patients. Partial inhibition of JAK activation by sub-saturating doses of Tofacitinib specifically lowered the levels of STAT1 activation by IL-6. Our data show that receptor and STATs concentrations critically contribute to shape cytokine responses and generate functional pleiotropy in health and disease.


Subject(s)
Cytokine Receptor gp130/agonists , Interleukin-27/pharmacology , Interleukin-6/pharmacology , Receptors, Interleukin/agonists , STAT1 Transcription Factor/metabolism , STAT3 Transcription Factor/metabolism , Th1 Cells/drug effects , Amino Acid Motifs , Binding, Competitive , Case-Control Studies , Cells, Cultured , Cytokine Receptor gp130/genetics , Cytokine Receptor gp130/metabolism , Humans , Interferon Regulatory Factor-1/metabolism , Interleukin-27/metabolism , Interleukin-6/metabolism , Kinetics , Lupus Erythematosus, Systemic/immunology , Lupus Erythematosus, Systemic/metabolism , Models, Biological , Mutation , Phosphorylation , Protein Binding , Protein Interaction Domains and Motifs , Receptors, Interleukin/genetics , Receptors, Interleukin/metabolism , Signal Transduction , Th1 Cells/immunology , Th1 Cells/metabolism
6.
Sci Signal ; 13(649)2020 09 15.
Article in English | MEDLINE | ID: mdl-32934073

ABSTRACT

Interleukin-10 (IL-10) is a dimeric cytokine with both immunosuppressive and immunostimulatory activities; however, IL-10-based therapies have shown only marginal clinical benefits. Here, we explored whether the stability of the IL-10 receptor complex contributes to the immunomodulatory potency of IL-10. We generated an IL-10 mutant with enhanced affinity for its IL-10Rß receptor using yeast surface display. Compared to the wild-type cytokine, the affinity-enhanced IL-10 variants recruited IL-10Rß more efficiently into active cell surface signaling complexes and triggered greater STAT1 and STAT3 activation in human monocytes and CD8+ T cells. These effects, in turn, led to more robust induction of IL-10-mediated gene expression programs at low ligand concentrations in both human cell subsets. IL-10-regulated genes are involved in monocyte energy homeostasis, migration, and trafficking and in CD8+ T cell exhaustion. At nonsaturating doses, IL-10 did not induce key components of its gene expression program, which may explain its lack of efficacy in clinical settings. Our engineered IL-10 variant showed a more robust bioactivity profile than that of wild-type IL-10 at low doses in monocytes and CD8+ T cells. Moreover, CAR-modified T cells expanded with the engineered IL-10 variant displayed superior cytolytic activity than those expanded with wild-type IL-10. Our study provides insights into how IL-10 receptor complex stability fine-tunes IL-10 biology and opens new opportunities to revitalize failed IL-10 therapies.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Interleukin-10/immunology , Monocytes/immunology , Mutation/immunology , Signal Transduction/immunology , Animals , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/metabolism , Cell Movement/genetics , Cell Movement/immunology , Cells, Cultured , Energy Metabolism/genetics , Energy Metabolism/immunology , Gene Expression Profiling/methods , Humans , Interleukin-10/genetics , Interleukin-10/metabolism , Ligands , Monocytes/cytology , Monocytes/metabolism , Protein Binding , Receptors, Interleukin-10/genetics , Receptors, Interleukin-10/immunology , Receptors, Interleukin-10/metabolism , STAT1 Transcription Factor/genetics , STAT1 Transcription Factor/immunology , STAT1 Transcription Factor/metabolism , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/immunology , STAT3 Transcription Factor/metabolism , Sf9 Cells , Signal Transduction/genetics , Spodoptera
7.
Elife ; 82019 11 27.
Article in English | MEDLINE | ID: mdl-31774398

ABSTRACT

Cytokines activate signaling via assembly of cell surface receptors, but it is unclear whether modulation of cytokine-receptor binding parameters can modify biological outcomes. We have engineered IL-6 variants with different affinities to gp130 to investigate how cytokine receptor binding dwell-times influence functional selectivity. Engineered IL-6 variants showed a range of signaling amplitudes and induced biased signaling, with changes in receptor binding dwell-times affecting more profoundly STAT1 than STAT3 phosphorylation. We show that this differential signaling arises from defective translocation of ligand-gp130 complexes to the endosomal compartment and competitive STAT1/STAT3 binding to phospho-tyrosines in gp130, and results in unique patterns of STAT3 binding to chromatin. This leads to a graded gene expression response and differences in ex vivo differentiation of Th17, Th1 and Treg cells. These results provide a molecular understanding of signaling biased by cytokine receptors, and demonstrate that manipulation of signaling thresholds is a useful strategy to decouple cytokine functional pleiotropy.


Subject(s)
Cytokine Receptor gp130/chemistry , Interleukin-6/chemistry , STAT1 Transcription Factor/metabolism , T-Lymphocytes, Regulatory/metabolism , Th1 Cells/metabolism , Th17 Cells/metabolism , Binding Sites , Cell Differentiation , Cloning, Molecular , Cytokine Receptor gp130/genetics , Cytokine Receptor gp130/metabolism , Endosomes/chemistry , Endosomes/metabolism , Gene Expression , HeLa Cells , Humans , Interleukin-6/genetics , Interleukin-6/metabolism , Kinetics , Models, Molecular , Phosphorylation , Primary Cell Culture , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Engineering/methods , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , STAT1 Transcription Factor/genetics , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/metabolism , Signal Transduction , T-Lymphocytes, Regulatory/cytology , T-Lymphocytes, Regulatory/immunology , Th1 Cells/cytology , Th1 Cells/immunology , Th17 Cells/cytology , Th17 Cells/immunology
8.
Mol Microbiol ; 110(6): 897-913, 2018 12.
Article in English | MEDLINE | ID: mdl-29802781

ABSTRACT

Bacterial biofilms are communities of microbial cells encased within a self-produced polymeric matrix. In the Bacillus subtilis biofilm matrix, the extracellular fibres of TasA are essential. Here, a recombinant expression system allows interrogation of TasA, revealing that monomeric and fibre forms of TasA have identical secondary structure, suggesting that fibrous TasA is a linear assembly of globular units. Recombinant TasA fibres form spontaneously, and share the biological activity of TasA fibres extracted from B. subtilis, whereas a TasA variant restricted to a monomeric form is inactive and subjected to extracellular proteolysis. The biophysical properties of both native and recombinant TasA fibres indicate that they are not functional amyloid-like fibres. A gel formed by TasA fibres can recover after physical shear force, suggesting that the biofilm matrix is not static and that these properties may enable B. subtilis to remodel its local environment in response to external cues. Using recombinant fibres formed by TasA orthologues we uncover species variability in the ability of heterologous fibres to cross-complement the B. subtilis tasA deletion. These findings are indicative of specificity in the biophysical requirements of the TasA fibres across different species and/or reflect the precise molecular interactions needed for biofilm matrix assembly.


Subject(s)
Amyloidogenic Proteins/metabolism , Bacillus subtilis/physiology , Bacterial Proteins/metabolism , Biofilms , Amyloidogenic Proteins/chemistry , Amyloidogenic Proteins/genetics , Bacillus subtilis/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
9.
Biochem J ; 475(2): 415-428, 2018 01 23.
Article in English | MEDLINE | ID: mdl-29229757

ABSTRACT

The Gram-negative bacterium Serratia marcescens secretes many proteins that are involved in extracellular chitin degradation. This so-called chitinolytic machinery includes three types of chitinase enzymes and a lytic polysaccharide monooxygenase. An operon has been identified in S. marcescens, chiWXYZ, that is thought to be involved in the secretion of the chitinolytic machinery. Genetic evidence points to the ChiX protein being a key player in the secretion mechanism, since deletion of the chiX gene in S. marcescens led to a mutant strain blocked for secretion of all members of the chitinolytic machinery. In this work, a detailed structural and biochemical characterisation of ChiX is presented. The high-resolution crystal structure of ChiX reveals the protein to be a member of the LAS family of peptidases. ChiX is shown to be a zinc-containing metalloenzyme, and in vitro assays demonstrate that ChiX is an l-Ala d-Glu endopeptidase that cleaves the cross-links in bacterial peptidoglycan. This catalytic activity is shown to be intimately linked with the secretion of the chitinolytic machinery, since substitution of the ChiX Asp-120 residue results in a variant protein that is both unable to digest peptidoglycan and cannot rescue the phenoytype of a chiX mutant strain.


Subject(s)
Bacterial Proteins/chemistry , Chitinases/metabolism , Gene Expression Regulation, Bacterial , N-Acetylmuramoyl-L-alanine Amidase/chemistry , Peptidoglycan/chemistry , Serratia marcescens/enzymology , Zinc/chemistry , Amino Acid Motifs , Aspartic Acid/chemistry , Aspartic Acid/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Chitin/metabolism , Chitinases/genetics , Cloning, Molecular , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Hydrolysis , Models, Molecular , N-Acetylmuramoyl-L-alanine Amidase/genetics , N-Acetylmuramoyl-L-alanine Amidase/metabolism , Operon , Peptidoglycan/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Serratia marcescens/genetics , Substrate Specificity , Zinc/metabolism
10.
Biochim Biophys Acta ; 1857(12): 1925-1934, 2016 12.
Article in English | MEDLINE | ID: mdl-27687473

ABSTRACT

Upon photoexcitation, the reaction center (RC) pigment-proteins that facilitate natural photosynthesis achieve a metastable separation of electrical charge among the embedded cofactors. Because of the high quantum efficiency of this process, there is a growing interest in their incorporation into biohybrid materials for solar energy conversion, bioelectronics and biosensing. Multiple bioelectrochemical studies have shown that reaction centers from various photosynthetic organisms can be interfaced with diverse electrode materials for the generation of photocurrents, but many mechanistic aspects of native protein functionality in a non-native environment is unknown. In vivo, RC's catalyse ubiquinone-10 reduction, protonation and exchange with other lipid phase ubiquinone-10s via protein-controlled spatial orientation and protein rearrangement. In contrast, the mechanism of ubiquinone-0 reduction, used to facilitate fast RC turnover in an aqueous photoelectrochemical cell (PEC), may not proceed via the same pathway as the native cofactor. In this report we show truncation of the native isoprene tail results in larger RC turnover rates in a PEC despite the removal of the tail's purported role of ubiquinone headgroup orientation and binding. Through the use of reaction centers with single or double mutations, we also show the extent to which two-electron/two-proton ubiquinone chemistry that operates in vivo also underpins the ubiquinone-0 reduction by surface-adsorbed RCs in a PEC. This reveals that only the ubiquinone headgroup is critical to the fast turnover of the RC in a PEC and provides insight into design principles for the development of new biophotovoltaic cells and biosensors.


Subject(s)
Electrochemistry/methods , Light , Photosynthesis/radiation effects , Photosynthetic Reaction Center Complex Proteins/radiation effects , Rhodobacter sphaeroides/radiation effects , Ubiquinone/radiation effects , Biosensing Techniques , Electrochemistry/instrumentation , Electrodes , Electron Transport , Kinetics , Models, Biological , Mutation , Oxidation-Reduction , Photosynthetic Reaction Center Complex Proteins/chemistry , Photosynthetic Reaction Center Complex Proteins/genetics , Photosynthetic Reaction Center Complex Proteins/metabolism , Protein Conformation , Rhodobacter sphaeroides/genetics , Rhodobacter sphaeroides/metabolism , Solar Energy , Structure-Activity Relationship , Ubiquinone/metabolism
11.
Biochem J ; 473(13): 1941-52, 2016 07 01.
Article in English | MEDLINE | ID: mdl-27130157

ABSTRACT

The membrane-bound protein EssC is an integral component of the bacterial Type VII secretion system (T7SS), which is a determinant of virulence in important Gram-positive pathogens. The protein is predicted to consist of an intracellular repeat of forkhead-associated (FHA) domains at the N-terminus, two transmembrane helices and three P-loop-containing ATPase-type domains, D1-D3, forming the C-terminal intracellular segment. We present crystal structures of the N-terminal FHA domains (EssC-N) and a C-terminal fragment EssC-C from Geobacillus thermodenitrificans, encompassing two of the ATPase-type modules, D2 and D3. Module D2 binds ATP with high affinity whereas D3 does not. The EssC-N and EssC-C constructs are monomeric in solution, but the full-length recombinant protein, with a molecular mass of approximately 169 kDa, forms a multimer of approximately 1 MDa. The observation of protomer contacts in the crystal structure of EssC-C together with similarity to the DNA translocase FtsK, suggests a model for a hexameric EssC assembly. Such an observation potentially identifies the key, and to date elusive, component of pore formation required for secretion by this recently discovered secretion system. The juxtaposition of the FHA domains suggests potential for interacting with other components of the secretion system. The structural data were used to guide an analysis of which domains are required for the T7SS machine to function in pathogenic Staphylococcus aureus The extreme C-terminal ATPase domain appears to be essential for EssC activity as a key part of the T7SS, whereas D2 and FHA domains are required for the production of a stable and functional protein.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Type VII Secretion Systems/metabolism , Bacterial Proteins/genetics , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Proteins/genetics , Staphylococcus aureus/metabolism
12.
Mol Biochem Parasitol ; 201(1): 26-30, 2015 May.
Article in English | MEDLINE | ID: mdl-25982270

ABSTRACT

Tubulin-binding cofactor C stimulates GTPase activity and contributes to the release of the heterodimeric α/ß-tubulin from a super-complex of tubulin monomers and two ancillary cofactors. We have determined the 2.2 Å resolution crystal structure of the C-terminal domain of tubulin-binding cofactor C from Leishmania major based on single wavelength anomalous dispersion measurements targeting a selenomethionine derivative. Although previously predicted to consist of two domains the structure is best described as a single domain dominated by a right-handed ß-helix of five turns that form a triangular prism. One face of the prism is covered by the C-terminal residues leaving another face solvent exposed. Comparisons with an orthologous human GTPase activating protein match key residues involved in binding nucleotide and identify the face of the ß-helix fold likely involved in interacting with the ß-tubulin:GTP complex.


Subject(s)
Leishmania major/chemistry , Molecular Chaperones/chemistry , Amino Acid Sequence , Crystallography, X-Ray , Humans , Models, Molecular , Molecular Sequence Data , Protein Conformation , Sequence Homology
13.
Acta Crystallogr F Struct Biol Commun ; 71(Pt 5): 539-46, 2015 May.
Article in English | MEDLINE | ID: mdl-25945706

ABSTRACT

Tubulin-binding cofactor A (TBCA) participates in microtubule formation, a key process in eukaryotic biology to create the cytoskeleton. There is little information on how TBCA might interact with ß-tubulin en route to microtubule biogenesis. To address this, the protozoan Leishmania major was targeted as a model system. The crystal structure of TBCA and comparisons with three orthologous proteins are presented. The presence of conserved features infers that electrostatic interactions that are likely to involve the C-terminal tail of ß-tubulin are key to association. This study provides a reagent and template to support further work in this area.


Subject(s)
Leishmania major/chemistry , Microtubules/chemistry , Molecular Chaperones/chemistry , Protozoan Proteins/chemistry , Tubulin/chemistry , Amino Acid Sequence , Leishmania major/genetics , Microtubules/genetics , Molecular Chaperones/genetics , Molecular Sequence Data , Protein Structure, Secondary , Protein Structure, Tertiary , Protozoan Proteins/genetics , Tubulin/genetics
14.
Biochem J ; 458(3): 449-58, 2014 Mar 15.
Article in English | MEDLINE | ID: mdl-24428762

ABSTRACT

Salmonella enterica is an opportunistic pathogen that produces a [NiFe]-hydrogenase under aerobic conditions. In the present study, genetic engineering approaches were used to facilitate isolation of this enzyme, termed Hyd-5. The crystal structure was determined to a resolution of 3.2 Å and the hydro-genase was observed to comprise associated large and small subunits. The structure indicated that His229 from the large subunit was close to the proximal [4Fe-3S] cluster in the small subunit. In addition, His229 was observed to lie close to a buried glutamic acid (Glu73), which is conserved in oxygen-tolerant hydrogenases. His229 and Glu73 of the Hyd-5 large subunit were found to be important in both hydrogen oxidation activity and the oxygen-tolerance mechanism. Substitution of His229 or Glu73 with alanine led to a loss in the ability of Hyd-5 to oxidize hydrogen in air. Furthermore, the H229A variant was found to have lost the overpotential requirement for activity that is always observed with oxygen-tolerant [NiFe]-hydrogenases. It is possible that His229 has a role in stabilizing the super-oxidized form of the proximal cluster in the presence of oxygen, and it is proposed that Glu73could play a supporting role in fine-tuning the chemistry of His229 to enable this function.


Subject(s)
Bacterial Proteins/chemistry , Hydrogenase/chemistry , Oxygen/metabolism , Salmonella enterica/enzymology , Bacterial Proteins/genetics , Catalysis , Crystallography, X-Ray , Genetic Engineering , Glutamic Acid/genetics , Histidine/genetics , Hydrogen/chemistry , Hydrogen/metabolism , Hydrogenase/genetics , Hydrogenase/metabolism , Oxygen/chemistry , Protein Conformation , Protein Subunits/chemistry , Salmonella enterica/genetics
15.
BMC Struct Biol ; 14: 1, 2014 Jan 10.
Article in English | MEDLINE | ID: mdl-24410837

ABSTRACT

BACKGROUND: 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (IspF) catalyzes the conversion of 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate to 2C-methyl-D-erythritol-2,4-cyclodiphosphate and cytidine monophosphate in production of isoprenoid-precursors via the methylerythritol phosphate biosynthetic pathway. IspF is found in the protozoan Plasmodium falciparum, a parasite that causes cerebral malaria, as well as in many Gram-negative bacteria such as Burkholderia cenocepacia. IspF represents a potential target for development of broad-spectrum antimicrobial drugs since it is proven or inferred as essential in these pathogens and absent from mammals. Structural studies of IspF from these two important yet distinct pathogens, and comparisons with orthologues have been carried out to generate reagents, to support and inform a structure-based approach to early stage drug discovery. RESULTS: Efficient recombinant protein production and crystallization protocols were developed, and high-resolution crystal structures of IspF from P. falciparum (Emphasis/Emphasis>IspF) and B. cenocepacia (BcIspF) in complex with cytidine nucleotides determined. Comparisons with orthologues, indicate a high degree of order and conservation in parts of the active site where Zn2+ is bound and where recognition of the cytidine moiety of substrate occurs. However, conformational flexibility is noted in that area of the active site responsible for binding the methylerythritol component of substrate. Unexpectedly, one structure of BcIspF revealed two molecules of cytidine monophosphate in the active site, and another identified citrate coordinating to the catalytic Zn2+. In both cases interactions with ligands appear to help order a flexible loop at one side of the active site. Difficulties were encountered when attempting to derive complex structures with other ligands. CONCLUSIONS: High-resolution crystal structures of IspF from two important human pathogens have been obtained and compared to orthologues. The studies reveal new data on ligand binding, with citrate coordinating to the active site Zn2+ and when present in high concentrations cytidine monophosphate displays two binding modes in the active site. Ligand binding appears to order a part of the active site involved in substrate recognition. The high degree of structural conservation in and around the IspF active site suggests that any structural model might be suitable to support a program of structure-based drug discovery.


Subject(s)
Burkholderia cenocepacia/enzymology , Phosphorus-Oxygen Lyases/chemistry , Phosphorus-Oxygen Lyases/metabolism , Plasmodium falciparum/enzymology , Amino Acid Sequence , Burkholderia cenocepacia/chemistry , Catalytic Domain , Citric Acid/metabolism , Crystallography, X-Ray , Erythritol/analogs & derivatives , Erythritol/metabolism , Models, Molecular , Plasmodium falciparum/chemistry , Protein Conformation , Protein Structure, Secondary , Recombinant Proteins/metabolism , Sequence Alignment , Substrate Specificity , Sugar Phosphates/metabolism , Zinc/metabolism
16.
FEBS J ; 280(14): 3270-80, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23627368

ABSTRACT

Tubulin-binding cofactor (TBC)-B is implicated in the presentation of α-tubulin ready to polymerize, and at the correct levels to form microtubules. Bioinformatics analyses, including secondary structure prediction, CD, and crystallography, were combined to characterize the molecular architecture of Trypanosoma brucei TBC-B. An efficient recombinant expression system was prepared, material-purified, and characterized by CD. Extensive crystallization screening, allied with the use of limited proteolysis, led to structures of the N-terminal ubiquitin-like and C-terminal cytoskeleton-associated protein with glycine-rich segment domains at 2.35-Å and 1.6-Å resolution, respectively. These are compact globular domains that appear to be linked by a flexible segment. The ubiquitin-like domain contains two lysines that are spatially conserved with residues known to participate in ubiquitinylation, and so may represent a module that, through covalent attachment, regulates the signalling and/or protein degradation associated with the control of microtubule assembly, catastrophe, or function. The TBC-B C-terminal cytoskeleton-associated protein with glycine-rich segment domain, a known tubulin-binding structure, is the only such domain encoded by the T. brucei genome. Interestingly, in the crystal structure, the peptide-binding groove of this domain forms intermolecular contacts with the C-terminus of a symmetry-related molecule, an association that may mimic interactions with the C-terminus of α-tubulin or other physiologically relevant partners. The interaction of TBC-B with the α-tubulin C-terminus may, in particular, protect from post-translational modifications, or simply assist in the shepherding of the protein into polymerization.


Subject(s)
Microtubule-Associated Proteins/chemistry , Protozoan Proteins/chemistry , Amino Acid Sequence , Circular Dichroism , Conserved Sequence , Crystallography, X-Ray , Hydrogen Bonding , Models, Molecular , Molecular Sequence Data , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Structural Homology, Protein , Trypanosoma brucei brucei , Tubulin/chemistry
17.
Structure ; 21(4): 595-603, 2013 Apr 02.
Article in English | MEDLINE | ID: mdl-23499020

ABSTRACT

The membrane-bound EssB is an integral and essential component of the bacterial type VII secretion system that can contribute to pathogenicity. The architecture of Geobacillus thermodenitrificans EssB has been investigated by combining crystallographic and EPR spectroscopic methods. The protein forms a dimer that straddles the cytoplasmic membrane. A helical fold is observed for the C-terminal segment, which is positioned on the exterior of the membrane. This segment contributes most to dimer formation. The N-terminal segment displays a structure related to the pseudokinase fold and may contribute to function by recognizing substrates or secretion system partners. The remaining part of EssB may serve as an anchor point for the secretion apparatus, which is embedded in the cytoplasmic membrane with the C-terminal domain protruding out to interact with partner proteins or components of peptidoglycan.


Subject(s)
Bacterial Outer Membrane Proteins/chemistry , Bacterial Secretion Systems , Geobacillus/chemistry , Models, Molecular , Protein Conformation , Bacterial Outer Membrane Proteins/metabolism , Chromatography, Gel , Cloning, Molecular , Crystallization , Crystallography, X-Ray , Dimerization , Electron Spin Resonance Spectroscopy , Mutagenesis , Recombinant Proteins/chemistry
18.
Biochem J ; 449(2): 469-77, 2013 Jan 15.
Article in English | MEDLINE | ID: mdl-23098276

ABSTRACT

The Type VII protein translocation/secretion system, unique to Gram-positive bacteria, is a key virulence determinant in Staphylococcus aureus. We aim to characterize the architecture of this secretion machinery and now describe the present study of S. aureus EssB, a 52 kDa bitopic membrane protein essential for secretion of the ESAT-6 (early secretory antigenic target of 6 kDa) family of proteins, the prototypic substrate of Type VII secretion. Full-length EssB was heterologously expressed in Escherichia coli, solubilized from the bacterial membrane, purified to homogeneity and shown to be dimeric. A C-terminal truncation, EssB∆C, and two soluble fragments termed EssB-N and EssB-C, predicted to occur on either side of the cytoplasmic membrane, have been successfully purified in a recombinant form, characterized and, together with the full-length protein, used in crystallization trials. EssB-N, the 25 kDa N-terminal cytoplasmic fragment, gave well-ordered crystals and we report the structure, determined by SAD (single-wavelength anomalous diffraction) targeting an SeMet (selenomethionine) derivative, refined to atomic (1.05 Å; 1 Å=0.1 nm) resolution. EssB-N is dimeric in solution, but crystallizes as a monomer and displays a fold comprised of two globular domains separated by a cleft. The structure is related to that of serine/threonine protein kinases and the present study identifies that the Type VII secretion system exploits and re-uses a stable modular entity and fold that has evolved to participate in protein-protein interactions in a similar fashion to the catalytically inert pseudokinases.


Subject(s)
Bacterial Proteins/metabolism , Membrane Proteins/metabolism , Staphylococcus aureus/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Crystallography, X-Ray , Cytoplasm/metabolism , Escherichia coli/genetics , Membrane Proteins/chemistry , Membrane Proteins/genetics , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Conformation , Protein Multimerization , Protein Structure, Tertiary , Protein Transport , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Spectrometry, Mass, Electrospray Ionization , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Staphylococcus aureus/genetics , Surface Plasmon Resonance , Two-Hybrid System Techniques
19.
Proc Natl Acad Sci U S A ; 109(29): 11693-8, 2012 Jul 17.
Article in English | MEDLINE | ID: mdl-22753509

ABSTRACT

Thiol-dependent reductase I (TDR1), an enzyme found in parasitic Leishmania species and Trypanosoma cruzi, is implicated in deglutathionylation and activation of antimonial prodrugs used to treat leishmaniasis. The 2.3 Å resolution structure of TDR1 reveals a unique trimer of subunits each containing two glutathione-S-transferase (GST) domains. The similarities of individual domains and comparisons with GST classes suggest that TDR1 evolved by gene duplication, diversification, and gene fusion; a combination of events previously unknown in the GST protein superfamily and potentially explaining the distinctive enzyme properties of TDR1. The deglutathionylation activity of TDR1 implies that glutathione itself has regulatory intracellular roles in addition to being a precursor for trypanothione, the major low mass thiol present in trypanosomatids. We propose that activation of antiparasite Sb(V)-drugs is a legacy of the deglutathionylation activity of TDR1 and involves processing glutathione adducts with concomitant reduction of the metalloid to active Sb(III) species.


Subject(s)
Evolution, Molecular , Glutathione/chemistry , Leishmania/enzymology , Models, Molecular , Oxidoreductases/chemistry , Prodrugs/metabolism , Protein Conformation , Amino Acid Sequence , Antimony/chemistry , Base Sequence , Crystallography , Genes, Duplicate/genetics , Glutathione Transferase/chemistry , Molecular Sequence Data , Oxidoreductases/metabolism , Polymers/chemistry , Protein Structure, Tertiary , Sequence Alignment , Sequence Analysis, DNA
20.
Article in English | MEDLINE | ID: mdl-22750854

ABSTRACT

Cysteine biosynthesis is a potential target for drug development against parasitic Leishmania species; these protozoa are responsible for a range of serious diseases. To improve understanding of this aspect of Leishmania biology, a crystallographic and biochemical study of L. major cysteine synthase has been undertaken, seeking to understand its structure, enzyme activity and modes of inhibition. Active enzyme was purified, assayed and crystallized in an orthorhombic form with a dimer in the asymmetric unit. Diffraction data extending to 1.8 Šresolution were measured and the structure was solved by molecular replacement. A fragment of γ-poly-D-glutamic acid, a constituent of the crystallization mixture, was bound in the enzyme active site. Although a D-glutamate tetrapeptide had insignificant inhibitory activity, the enzyme was competitively inhibited (K(i) = 4 µM) by DYVI, a peptide based on the C-terminus of the partner serine acetyltransferase with which the enzyme forms a complex. The structure surprisingly revealed that the cofactor pyridoxal phosphate had been lost during crystallization.


Subject(s)
Cysteine Synthase/chemistry , Leishmania major/enzymology , Cysteine Synthase/antagonists & inhibitors , Cysteine Synthase/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Glutamic Acid/chemistry , Glutamic Acid/metabolism , Models, Molecular , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Quaternary , Structural Homology, Protein
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