Multiple crystal forms of endoglucanase CelD: Signal peptide residues modulate lattice formation

Abstract

The crystal structure of Clostridium thermocellum endoglucanase Cell) revealed an extended NH 2-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 (LyAbstract8-Asp201). Enzymatic digestion of Cell) with various proteases emphasized the flexibility of the NH 2-segment in solution. Proteolytic removal of LyAbstract8 or the substitution of bridge-forming residues by site-directed mutagenesis promoted crystal packing arrangements that differ from that of wild type Cell). Crystals of wild-type CeID ( a = 99.3 Å c =191.8 Å) are trigonal, space group P3,21, with one molecule in the asymmetric unit (form A), whereas crystals of papain-treated CeID ( a = 100.4 Å, c = 248.7 Å), of CelD K38M ( a = 100.1 A, c = 248.4 A) and of papain-treated CeID D201A (a = 99.9 Å, c = 250.0 Å) are trigonal, space group P3,21, with two crystallographically independent molecules (form B), and crystals of chymotrypsin-treated Cell) ( a = 100.0 Å, c = 254.3 Å) and of CelD D201A ( a = 99.8 Å, c = 254.7 Å) are hexagonal, space group P6,22, with one molecule in the asymmetric unit (form C). Only chymotrypsin-treated Cell) (which preserves both LyAbstract8 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 NH 2- and COOH-terminal peptide extensions were found to influence crystal nucleation, but not crystal growth. The crystal structures of papain-treated Cell) and chymotrypsin-treated CeID, determined at 3.5 Å resolution by molecular replacement techniques, demonstrate that a small change in molecular orientation promoted by LyAbstract8 account for the differences between crystal forms B and C.