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abstracts
X-ray irradiation on a protein crystal can cause some subtle structural modification on the protein structure even if the radiation dose is much smaller than a dose used for a common crystal structure determination. In some case such structural modification increases ambiguity of structural inspection, and eventually could be an obstacle on the elucidation of structure basis of protein function. Bovine heart cytochrome c oxidase (CcO) is one of such proteins having some problem caused by the radiation damage. The proton pumping of CcO is coupled with O2 reduction at the O2 reduction site, thus accurate structure determination of bound ligand as well as CcO itself is very important. Whereas accurate structure determination was impeded by the immediate photolysis of the peroxide ligand upon X-ray irradiation even at a cryogenic temperature[1]. We developed a goniometer based data collection system for the femtosecond crystallography at SACLA (SPring-8 Angstrom Compact free-electron LAser). The femtosecond crystallography is expected to have an advantage in high-resolution and radiation damage free structure determination of very large protein by combined usage of large crystal and femtosecond intense X-ray pulse. In this presentation we are going to show the result of the femtosecond crystallography on the crystal of CcO having large unit cell dimensions. The close inspection of the electron density map calculated at 1.9 Å resolution showed the femtosecond crystallography worked fine for the high resolution and radiation damage free crystal structure determination of CcO.
abstracts
Leukotriene (LT) C4 synthase (LTC4S) is a key enzyme for the production of cysteinyl leukotrienes, LTC4, LTD4 and LTE4, which are relevance to asthma and allergy. LTC4S catalyzes the conjugation of glutathione (GSH) to LTA4. The crystal structure of LTC4S complex with GSH revealed the active sites locate at the interfaces of adjacent monomer in trans-membrane homo-trimer [1]. The unique U-shaped GSH binds the inner hydrophilic interface cavity and the amphiphilic LTA4 was proposed to bind in the hydrophobic V-shaped crevasse on the interface of the trimer hydrophobic surface. Two essential arginine residues was proved to exert as acid-base catalysis at the both sides of two substrates in highly regio- and stereo-selective manner enzymatically and crystallographically [2]. The architecture of the catalysis with Arg104 and Arg31 residues is the unique among various Glutathione-S-transferases. Arg104 activates and stabilizes the thiorate anaion of the bound GSH and Arg31 stabilize and activate epoxy of the other substrate LTA4, and each mutation causes not only reduction of activity but also each substrate binding affinity substantially. Furthermore, the putative LTA4 binding position was confirmed using the anomalous signal of selenium of the bound seleno-dodecylmaltoside (Se-DDM) at the V-shaped crevasse. To identify leads for novel therapeutics, we attempted to search competitive inhibitors against the unique shaped GSH binding site to discriminate the GSH binding sites of other GSTs that accommodate only its extending backbone conformer [3]. Hierarchical in silico screenings of 6 million compounds provided 300,000 dataset for docking, and after energy minimization based on the crystal structure of LTC4S, 111 compounds were selected as candidates for a competitive inhibitor to glutathione. 5-(5-Methylene-4-oxo-4,5-dihydrothiazol-2-ylamino) isophthalic acid moiety was identified to inhibit LTC4 formation both an enzyme assay and a whole-cell assay. Finally, 5-((Z)-5-((E)-2-methyl-3-phenylallylidene)-4-oxo-4,5-dihydrothiazol-2-ylamino) isophthalic acid was found to be the most potent inhibitor with 1.9 µM of IC50, and in the whole-cell assay to inhibit LTC4 synthesis with cell permeability in a concentration dependent manner.
