Structural insights into IL-11-mediated signalling and human IL6ST variant-associated immunodeficiency.

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.

IL-2 is inactivated by the acidic pH environment of tumors enabling engineering of a pH-selective mutein.

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.

Structural insights into the assembly and activation of the IL-27 signaling complex.

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.

Competitive binding of STATs to receptor phospho-Tyr motifs accounts for altered cytokine responses.

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.

Engineered IL-10 variants elicit potent immunomodulatory effects at low ligand doses.

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-10Rβ receptor using yeast surface display.

Kinetics of cytokine receptor trafficking determine signaling and functional selectivity.

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.

Formation of functional, non-amyloidogenic fibres by recombinant Bacillus subtilis TasA.

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.

Structure and activity of ChiX: a peptidoglycan hydrolase required for chitinase secretion by .

The Gram-negative bacterium 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 , , that is thought to be involved in the secretion of the chitinolytic machinery.

On the mechanism of ubiquinone mediated photocurrent generation by a reaction center based photocathode.

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.

EssC: domain structures inform on the elusive translocation channel in the Type VII secretion system.

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.

Crystal structure of the C-terminal domain of tubulin-binding cofactor C from Leishmania major.

Tubulin-binding cofactor C stimulates GTPase activity and contributes to the release of the heterodimeric α/β-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.

The structure of tubulin-binding cofactor A from Leishmania major infers a mode of association during the early stages of microtubule assembly.

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 β-tubulin en route to microtubule biogenesis. To address this, the protozoan Leishmania major was targeted as a model system.

How the structure of the large subunit controls function in an oxygen-tolerant [NiFe]-hydrogenase.

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.

Crystal structures of IspF from Plasmodium falciparum and Burkholderia cenocepacia: comparisons inform antimicrobial drug target assessment.

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.

The architecture of Trypanosoma brucei tubulin-binding cofactor B and implications for function.

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.

The architecture of EssB, an integral membrane component of the type VII secretion system.

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.

Characterization of Staphylococcus aureus EssB, an integral membrane component of the Type VII secretion system: atomic resolution crystal structure of the cytoplasmic segment.

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.

Leishmania TDR1 structure, a unique trimeric glutathione transferase capable of deglutathionylation and antimonial prodrug activation.

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.

Structure of Leishmania major cysteine synthase.

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.

Mechanism of recombination of the P+H(A)- radical pair in mutant Rhodobacter sphaeroides reaction centers with modified free energy gaps between P+B(A)- and P+H(A)-.

The kinetics of recombination of the P(+)H(A)(-) radical pair were compared in wild-type reaction centers from Rhodobacter sphaeroides and in seven mutants in which the free energy gap, ΔG, between the charge separated states P(+)B(A)(-) and P(+)H(A)(-) was either increased or decreased. Five of the mutant RCs had been described previously, and X-ray crystal structures of two newly constructed complexes were determined by X-ray crystallography. The charge recombination reaction was accelerated in all mutants with a smaller ΔG than in the wild-type, and was slowed in a mutant having a larger ΔG.

Pseudomonas aeruginosa 4-amino-4-deoxychorismate lyase: spatial conservation of an active site tyrosine and classification of two types of enzyme.

4-Amino-4-deoxychorismate lyase (PabC) catalyzes the formation of 4-aminobenzoate, and release of pyruvate, during folate biosynthesis. This is an essential activity for the growth of gram-negative bacteria, including important pathogens such as Pseudomonas aeruginosa. A high-resolution (1.

Exploiting the high-resolution crystal structure of Staphylococcus aureus MenH to gain insight into enzyme activity.

Background: MenH (2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase) is a key enzyme in the biosynthesis of menaquinone, catalyzing an unusual 2,5-elimination of pyruvate from 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate.

Results: The crystal structure of Staphylococcus aureus MenH has been determined at 2 Å resolution. In the absence of a complex to inform on aspects of specificity a model of the enzyme-substrate complex has been used in conjunction with previously published kinetic analyses, site-directed mutagenesis studies and comparisons with orthologues to investigate the structure and reactivity of MenH.

Structure of recombinant Leishmania donovani pteridine reductase reveals a disordered active site.

Pteridine reductase (PTR1) is a potential target for drug development against parasitic Trypanosoma and Leishmania species, protozoa that are responsible for a range of serious diseases found in tropical and subtropical parts of the world. As part of a structure-based approach to inhibitor development, specifically targeting Leishmania species, well ordered crystals of L. donovani PTR1 were sought to support the characterization of complexes formed with inhibitors.

Structure of Staphylococcus aureus adenylosuccinate lyase (PurB) and assessment of its potential as a target for structure-based inhibitor discovery.

The medium-resolution structure of adenylosuccinate lyase (PurB) from the bacterial pathogen Staphylococcus aureus in complex with AMP is presented. Oxalate, which is likely to be an artifact of crystallization, has been modelled in the active site and occupies a position close to that where succinate is observed in orthologous structures. PurB catalyzes reactions that support the provision of purines and the control of AMP/fumarate levels.