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

Alon Karpol, Yoav Barak, Raphael Lamed, Yuval Shoham, Edward A. Bayer

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)331-338
Number of pages8
JournalBiochemical Journal
Volume410
Issue number2
DOIs
StatePublished - 1 Mar 2008

Keywords

  • Alanine scanning
  • Cellulosome
  • Clostridium thermocellum
  • Deletion study
  • Multi-enzyme complex
  • Protein-protein interaction

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