5-80: The unique binding mode of cellulosomal CBM4s from Clostridium thermocellum

Here we report observations from two X-ray structures of Clostridium thermocellum family 4 carbohydrate binding modules from cellobiohydrolase A and cellulase K (PDB codes 3K4Z and 3P6B). These new structures have a unique feature - a binding site peptide loop with a tryptophan (Trp118) residing midway in the loop. Based on sequence alignment, this structural feature might be common in all cellulosomal Clostridial CBM4 modules. Molecular dynamics simulations and experimental binding studies with the Trp118Ala mutant suggest that Trp118 contributes to the binding and possibly the orientation of the module to soluble cellodextrins. Furthermore, the binding cleft aromatic residues, Trp68 and Tyr110, play a crucial role in binding to bacterial microcrystalline cellulose (BMCC), amorphous cellulose and soluble oligodextrins. The binding to BMCC is in disagreement with the structural features of the binding pocket, which does not support binding to the flat surface of crystalline cellulose, suggesting that it binds the amorphous part or the cellulose "whiskers" of BMCC.  We propose that Clostridial CBM4s have possibly evolved to bind the free chain ends of crystalline cellulose in addition to their ability to bind soluble cellodextrins.