Expression and crystallization ofDrosophilaEcR/USP

Abstract

Poster Sessions 4 1 binding protein (LBP), which binds insulin and 4-kDa hormone-likepeptide (leginsulin). LBP shows protein kinase activity in vitro and the activity is stimulated by binding of leginsulin. Carrot EDGP and soybean LBP share about 33% sequence homology. Carrot EDGP binds also insulin and leginsulin from soybean in vitro and localizes in the plasma membrane and middle lamellae of cell walls, EDGP also shows protein kinase activity. However, detail about function of EDGP is still unclear. Thus, we work on structural study of carrot EDGP. The structure will provide to a clue to understand the function of EDGP and pave a way for further analyses, which expected new solution for plant defense systems and in signal transduction. EDGP was purified from culture medium of carrot callus by ion exchange chromatography. Crystals of EDGP were obtained by conventional hanging drop vapor diffusion method. The crystal belongs to hexagonal system with cell dimensions of a = b = 129.8, c = 44.4 Å, and γ = 120 . Structure determination of EDGP is now in progress. Clostridium botulinum produces seven immunologically distinct neurotoxin (BoNT; 150 kDa) serotypes, classified as A-G. In culture fluid and naturally contaminated foods, BoNT exists as part of large toxin complexes (TCs) through association with non-toxic nonhemagglutinin (NTNHA; 130 kDa) and three hemagglutinin (HA) subcomponents, 33 and 17 kDa, respectively). Serotype A-D strains produce M-TC (BoNT/NTNHA complex; 280 kDa) and L-TC (BoNT/NTNHA/HAs complex; 750 kDa), while serotypes E and F strains produce only M-TC. The M-TC is formed first by assembly of a single BoNT and a single NTNHA molecule, and is subsequently converted to the complete L-TC. However, M-TC containing nicked NTNHA at unique site could no longer convert to the L-TC with HA subcomponents. Although the crystal structures of several serotype BoNTs and serotype D HA-33/ HA-17 complex[1] have been determined, no structure of BoNT complex forms (M-TC and L-TC) has been demonstrated. In this study, highly purified M-TC was obtained from the supernatant of C. botulinum type D strain 4947 through several chromatographic runs. The nicked form of M-TC was prepared by limited trypsin treatment, and was crystallized using the hanging-drop vapor-diffusion technique. The drops consisted of 6 μl protein solution (2.8 mg ml -1 ) and 4 μl reservoir solution (0.1 M MES pH 6.5, 0.18 M cesium and 15% PEG 6000) derived from condition number 24 of the Hampton Crystal Screen 2. The crystals grew at 293 K and reached dimension of 0.2 0.1 0.05 mm in 7 days. X-ray data were collected on a Rigaku R-AXIS VII imaging-plate system, using CuKα radiation from a Rigaku FR-E rotating-anode generator. The crystals diffracted to approx. 8Å resolution.[1]Hasegawa K. et al., J. Biol. Chem. 2007, 282, 24777 Keywords: crystallization, botulinum toxin complex, protein interactions P04.01.30 Acta Cryst. (2008). A64, C239Expression and crystallization of Drosophila EcR/USP Saori Kamachi 1 , Masami Isogai 1 , Asako Yamaguch...