Functional asymmetry in cohesin binding belies inherent symmetry of the dockerin module: insight into cellulosome assembly revealed by systematic mutagenesis

• Published in: Biochemical journal Vol. 410; no. 2; p. 331
• Main Authors: Karpol, Alon, Barak, Yoav, Lamed, Raphael, Shoham, Yuval, Bayer, Edward A
• Format: Journal Article
• Language: English
• Published: England 01.03.2008
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding Sites; Calcium; Cell Cycle Proteins - chemistry; Cell Cycle Proteins - genetics; Cell Cycle Proteins - metabolism; Cellulose - biosynthesis; Chromosomal Proteins, Non-Histone - chemistry; Chromosomal Proteins, Non-Histone - genetics; Chromosomal Proteins, Non-Histone - metabolism; Clostridium thermocellum - genetics; Clostridium thermocellum - metabolism; Cohesins; Escherichia coli - genetics; Kinetics; Models, Molecular; Multienzyme Complexes - metabolism; Mutagenesis, Site-Directed; Nuclear Proteins - chemistry; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Polymorphism, Single Nucleotide; Protein Conformation; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Sequence Deletion
• ISSN: 1470-8728
• DOI: 10.1042/BJ20071193

The cellulosome is an intricate multi-enzyme complex, known for its efficient degradation of recalcitrant cellulosic substrates. Its supramolecular architecture is determined by the high-affinity intermodular cohesin-dockerin interaction. The dockerin module comprises a calcium-binding, duplicated 'F-hand' loop-helix motif that bears striking similarity to the EF-hand loop-helix-loop motif of eukaryotic calcium-binding proteins. In the present study, we demonstrate by progressive truncation and alanine scanning of a representative type-I dockerin module from Clostridium thermocellum, that only one of the repeated motifs is critical for high-affinity cohesin binding. The results suggest that the near-symmetry in sequence and structure of the repeated elements of the dockerin is not essential to cohesin binding. The first calcium-binding loop can be deleted entirely, with almost full retention of binding. Likewise, significant deletion of the second repeated segment can be achieved, provided that its calcium-binding loop remains intact. Essentially the same conclusion was verified by systematically mutating the highly conserved residues in the calcium-binding loop. Mutations in one of the calcium-binding loops failed to disrupt cohesin recognition and binding, whereas a single mutation in both loops served to reduce the affinity significantly. The results are mutually compatible with recent crystal structures of the type-I cohesin-dockerin heterodimer, which demonstrate that the dockerin can bind in an equivalent manner to its cohesin counterpart through either its first or second repeated motif. The observed plasticity in cohesin-dockerin binding may facilitate cellulosome assembly in vivo or, alternatively, provide a conformational switch that promotes access of the tethered cellulosomal enzymes to their polysaccharide substrates.