| Abstrakt: |
The crystal structure ofClostridium thermocellumendoglucanase CelD revealed an extended NH2-terminal segment (involving residues from the putative leader peptide) sticking out from the enzyme core to interact with a symmetry related molecule through an intermolecular salt bridge (Lys38-Asp201). Enzymatic digestion of CelD with various proteases emphasized the flexibility of the NH2-segment in solution. Proteolytic removal of Lys38 or the substitution of bridge-forming residues by site-directed mutagenesis promoted crystal packing arrangements that differ from that of wild type CelD. Crystals of wild-type CelD (a=99.3 Åc=191.8 Å) are trigonal, space groupP3121, with one molecule in the asymmetric unit (form A), whereas crystals of papain-treated CelD (a=100.4 Å,c=248.7 Å), of CelDK38M(a=100.1 Å,c=248.4 Å) and of papain-treated CelDD201A(a=99.9 Å,c=250.0 Å) are trigonal, space groupP3121, with two crystallographically independent molecules (form B), and crystals of chymotrypsin-treated CelD (a=100.0 Å,c=254.3 Å) and of CelDD201A(a=99.8 Å,c=254.7 Å) are hexagonal, space groupP6122, with one molecule in the asymmetric unit (form C). Only chymotrypsin-treated CelD (which preserves both Lys38 and Asp201) can grow in crystal form A upon macroseeding, indicating that formation of the intermolecular salt bridge is critical for stability of this crystal form. Flexible NH2- and COOH-terminal peptide extensions were found to influence crystal nucleation, but not crystal growth. The crystal structures of papain-treated CelD and chymotrypsin-treated CelD, determined at 3.5 Å resolution by molecular replacement techniques, demonstrate that a small change in molecular orientation promoted by Lys38 account for the differences between crystal forms B and C. |
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