Crystal structure of the EphA4 protein tyrosine kinase domain in the apo- and dasatinib-bound state

AbstractThe Eph family of receptor tyrosine kinases regulates diverse cellular processes while the over-expression of a member of this family, EphA4, has been reported in a variety of malignant carcinomas. To gain insight into molecular mechanisms and to facilitate structure-based inhibitor design, we solved the crystal structure of the native EphA4 kinase domain in both the apo and dasatinib bound forms. Analysis of the two structures provides insight into structural features of inhibitor binding and revealed a hydrophobic back-pocket in the ATP- binding site of EphA4 which was previously unidentified. The structures suggest a route towards development of novel and specific inhibitors

Crystal structure of acetylcholine-binding protein from Bulinus truncatus reveals the conserved structural scaffold and sites of variation in nicotinic acetylcholine receptors

The crystal structure of acetylcholine-binding protein (AChBP) from the mollusk Lymnaea stagnalis is the established model for the ligand binding domains of the ligand-gated ion channel family, which includes nicotinic acetylcholine, 5-hydroxytryptamine (5-H

Crystal structure of the tubulin tyrosine carboxypeptidase complex VASH1–SVBP

The cyclic enzymatic removal and ligation of the C-terminal tyrosine of α-tubulin generates heterogeneous microtubules and affects their functions. Here we describe the crystal and solution structure of the tubulin carboxypeptidase complex between vasohibin (VASH1) and small vasohibin-binding protein (SVBP), which folds in a long helix, which stabilizes the VASH1 catalytic domain. This structure, combined with molecular docking and mutagenesis experiments, reveals which residues are responsible for recognition and cleavage of the tubulin C-terminal tyrosine

A fragment-like approach to PYCR1 inhibition

Pyrroline-5-carboxylate reductase 1 (PYCR1) is the final enzyme involved in the biosynthesis of proline and has been found to be upregulated in various forms of cancer. Due to the role of proline in maintaining the redox balance of cells and preventing apoptosis, PYCR1 is emerging as an attractive oncology target. Previous PYCR1 knockout studies led to a reduction in tumor growth. Accordingly, a small molecule inhibitor of PYCR1 could lead to new treatments for cancer, and a focused screening effort identified pargyline as a fragment-like hit. We report the design and synthesis of the first tool compounds as PYCR1 inhibitors, derived from pargyline, which were assayed to assess their ability to attenuate the production of proline. Structural activity studies have revealed the key determinants of activity, with the most potent compound (4) showing improved activity in vitro in enzyme (IC50 = 8.8 µM) and pathway relevant effects in cell-based assays

Altered peptide ligands revisited: vaccine design through chemically modified HLA-A2-restricted T cell epitopes

Virus or tumor Ag–derived peptides that are displayed by MHC class I molecules are attractive starting points for vaccine development because they induce strong protective and therapeutic cytotoxic T cell responses. In thus study, we show that the MHC binding and consequent T cell reactivity against several HLA-A*02 restricted epitopes can be further improved through the incorporation of nonproteogenic amino acids at primary and secondary anchor positions. We screened more than 90 nonproteogenic, synthetic amino acids through a range of epitopes and tested more than 3000 chemically enhanced altered peptide ligands (CPLs) for binding affinity to HLA-A*0201. With this approach, we designed CPLs of viral epitopes, of melanoma-associated Ags, and of the minor histocompatibility Ag UTA2-1, which is currently being evaluated for its antileukemic activity in clinical dendritic cell vaccination trials. The crystal structure of one of the CPLs in complex with HLA-A*0201 revealed the molecular interactions likely responsible for improved binding. The best CPLs displayed enhanced affinity for MHC, increasing MHC stability and prolonging recognition by Ag-specific T cells and, most importantly, they induced accelerated expansion of antitumor T cell frequencies in vitro and in vivo as compared with the native epitope. Eventually, we were able to construct a toolbox of preferred nonproteogenic residues with which practically any given HLA-A*02 restricted epitope can be readily optimized. These CPLs could improve the therapeutic outcome of vaccination strategies or can be used for ex vivo enrichment and faster expansion of Ag-specific T cells for transfer into patients

A fragment-like approach to PYCR1 inhibition

Pyrroline-5-carboxylate reductase 1 (PYCR1) is the final enzyme involved in the biosynthesis of proline and has been found to be upregulated in various forms of cancer. Due to the role of proline in maintaining the redox balance of cells and preventing apoptosis, PYCR1 is emerging as an attractive oncology target. Previous PYCR1 knockout studies led to a reduction in tumor growth. Accordingly, a small molecule inhibitor of PYCR1 could lead to new treatments for cancer, and a focused screening effort identified pargyline as a fragment-like hit. We report the design and synthesis of the first tool compounds as PYCR1 inhibitors, derived from pargyline, which were assayed to assess their ability to attenuate the production of proline. Structural activity studies have revealed the key determinants of activity, with the most potent compound (4) showing improved activity in vitro in enzyme (IC50 = 8.8 µM) and pathway relevant effects in cell-based assays

Crystal structure of the tubulin tyrosine carboxypeptidase complex VASH1–SVBP

The cyclic enzymatic removal and ligation of the C-terminal tyrosine of α-tubulin generates heterogeneous microtubules and affects their functions. Here we describe the crystal and solution structure of the tubulin carboxypeptidase complex between vasohibin (VASH1) and small vasohibin-binding protein (SVBP), which folds in a long helix, which stabilizes the VASH1 catalytic domain. This structure, combined with molecular docking and mutagenesis experiments, reveals which residues are responsible for recognition and cleavage of the tubulin C-terminal tyrosine

Co-expression of protein complexes in prokaryotic and eukaryotic hosts: Experimental procedures, database tracking and case studies

Structure determination and functional characterization of macromolecular complexes requires the purification of the different subunits in large quantities and their assembly into a functional entity. Although isolation and structure determination of endogenous complexes has been reported, much progress has to be made to make this technology easily accessible. Co-expression of subunits within hosts such as Escherichia coli and insect cells has become more and more amenable, even at the level of high-throughput projects. As part of SPINE (Structural Proteomics In Europe), several laboratories have investigated the use co-expression techniques for their projects, trying to extend from the common binary expression to the more complicated multi-expression systems. A new system for multi-expression in E. coli and a database system dedicated to handle co-expression data are described. Results are also reported from various case studies investigating different methods for performing co-expression in E. coli and insect cells