journal menu
abstracts
High-throughput protein X-ray crystallography offers an unprecedented opportunity to facilitate drug discovery. The most reliable approach is to determine the three-dimensional (3D) structure of the protein-ligand complex by soaking the ligand in apo-crystals, but many lead compounds are not readily water-soluble. Such lead compounds must be dissolved in concentrated organic solvents such as DMSO. Therefore, to date, it has been impossible to produce crystals of protein-ligand complexes by soaking in apo-crystals, because protein crystals dissolve immediately upon soaking in concentrated organic solvents containing lead compounds. The problem arises from the influence of osmotic shock on crystal packing during soaking. We propose an approach to avoid the damage by growing protein crystals in a high-strength hydrogel(1-3). Interestingly, the hydrogel-grown crystals did not dissolve at all for more than thirty minutes in concentrated organic solvents and ionic-strength solutions such as 60% DMSO, and 5.0M lithium acetate. Their X-ray diffraction data were suitable for structure analysis. Surprisingly, some of the crystals diffracted to the highest resolution reported in the Protein Data Bank. Furthermore, the 3D structure determined from hydrogel-grown apo-avidin crystals which were transferred to a solution containing the ligand revealed a clear electron-density map of the ligand bound to the active site. This result indicates that it is possible to bind ligand compounds into hydrogel-grown apo-crystals.
abstracts
"We present the crystal structure of AcrB in complex with Linezolid[1]. AcrB is an inner-membrane Resistance-Nodulation-Division efflux pump and is part of the AcrAB-TolC multidrug-resistance tripartite efflux system in E. Coli. Crystal structures of AcrB by itself as well as several drug-bound complexes have been structurally characterized. Linezolid is an approved oxazolidinone antibiotic used for the treatment of serious infections caused by Gram-positive bacteria that are resistant to other antibiotics, and has been called a ""reserve antibiotic"", a drug of last resort against potentially intractable infections. This antibiotic inhibits bacterial protein synthesis by specifically binding to the 50S ribosomal subunit. Linezolid has no clinically significant effect on most Gram-negative bacteria. This is thought to be a result of relatively low intracellular concentration of Linezolid due to efflux, but there is no direct evidence yet to support this hypothesis. This membrane protein-drug complex shows that an antibiotic specific to Gram-positive bacteria can also bind an efflux pump from E. coli, a Gram-negative bacterium. The crystal structure of AcrB and Linezolid complex reveals that Linezolid binds to the A385/F386 loops of the symmetric trimers of AcrB in the same fashion as several other antibiotics that are extruded by efflux pumps. A conformational change of a loop in the bottom of the periplasmic cleft is also observed."
