Structural biology contributions to the discovery of drugs to treat chronic myelogenous leukaemia

A case study showing how the determination of multiple cocrystal structures of the protein tyrosine kinase c-Abl was used to support drug discovery, resulting in a compound effective in the treatment of chronic myelogenous leukaemia

Low-resolution detergent tracing in protein crystals using xenon or krypton to enhance X-ray contrast

Xenon and krypton show different solubilities in polar versus apolar solvents. Therefore, these noble gases should accumulate in apolar regions of protein crystals. Specifically, they should accumulate in lipid and detergent solvent regions within crystals of membrane proteins, which can be used as a basis for contrast-variation experiments to distinguish such apolar solvent regions from the aqueous phase by a low-resolution X-ray diffraction experiment. This possibility was explored with the OmpF porin, one of the general diffusion pores of the Escherichia coli outer membrane. Trigonal crystals were exposed to elevated pressures of the two noble gases (up to 10(7) Pa) for several minutes and subsequently flash-cooled to liquid-nitrogen temperatures. Both rare gases bind to a number of 'specific' sites, which can be classified as 'typical' noble-gas binding sites. Compared with a representative water-soluble protein, they are however much more abundant in OmpF. In addition, a very large number of weakly populated sites are observed which accumulate in the region of the 'detergent belt' for crystals exposed to xenon. After application of a Fourier-filtering protocol, low-resolution images of the detergent belt can be obtained. The resulting maps are similar to maps obtained from low-resolution neutron diffraction experiments on contrast-matched crystals

Crystal structure of osmoporin OmpC from E. coli at 2.0 A

Porins form transmembrane pores in the outer membrane of Gram-negative bacteria with matrix porin OmpF and osmoporin OmpC from Escherichia coli being differentially expressed depending on environmental conditions. The three-dimensional structure of OmpC has been determined to 2.0 A resolution by X-ray crystallography. As expected from the high sequence similarity, OmpC adopts the OmpF-like 16-stranded hollow beta-barrel fold with three beta-barrels associated to form a tight trimer. Unlike in OmpF, the extracellular loops form a continuous wall at the perimeter of the vestibule common to the three pores, due to a 14-residues insertion in loop L4. The pore constriction and the periplasmic outlet are very similar to OmpF with 74% of the pore lining residues being conserved. Overall, only few ionizable residues are exchanged at the pore lining. The OmpC structure suggests that not pore size, but electrostatic pore potential and particular atomic details of the pore linings are the critical parameters that physiologically distinguish OmpC from OmpF

nanoDSF: label-free thermal unfolding assay of G-protein-coupled receptors for compound screening and buffer composition optimization

A thermal unfolding based assay using low volume differential intrinsic tryptophan scanning fluorimetry (nanoDSF) was applied to study the stabilizing effects of ligands on G-protein-coupled receptors (GPCRs). GPCRs are the fourth largest superfamily in the human genome and are the largest class of targets for drug discovery. The system has been validated using human adenosine A2A receptor (A2AR). A2AR binds natural (adenosine and caffeine) and synthetic ligands with different affinities to mediate a variety of physiological and pharmacological responses. Several well-characterized ligands were used for the unfolding experiments. The ΔTm shift values obtained from nanoDSF analysis and traditional ligand binding studies correlate well with each other. We further characterized a second human GPCR target (test-GPCR) for which traditional cysteine-reactive DSF has been problematic. nanoDSF demonstrated that small molecule ligands can stabilize the detergent-solubilized receptor, thus showing the target GPCR is active in a selected detergent and lipid-free environment. In addition, we report a buffer composition screen to further stabilize the receptor in its detergent environment for biophysical assays. Based on our results, we show that the nanoDSF technology will allow the development of an automated screening platform in a label-free environment to evaluate a large number of compounds for lead discovery and to improve receptor stability for biophysical assays by screening buffer conditions