Single-molecule dissection of the high-affinity cohesin-dockerin complex.

Abstract

Cellulose-degrading enzyme systems are of significant interest from both a scientific and technological perspective due to the diversity of cellulase families, their unique assembly and substrate binding mechanisms, and their potential applications in several key industrial sectors, notably cellulose hydrolysis for second-generation biofuel production. Particularly fascinating are cellulosomes, the multimodular extracellular complexes produced by numerous anaerobic bacteria. Using single-molecule force spectroscopy, we analyzed the mechanical stability of the intermolecular interfaces between the cohesin and the dockerin modules responsible for self-assembly of the cellulosomal components into the multienzyme complex. The observed cohesin-dockerin rupture forces (>120 pN) are among the highest reported for a receptor-ligand system to date. Using an atomic force microscope protocol that quantified single-molecule binding activity, we observed force-induced dissociation of calcium ions from the duplicated loop-helix F-hand motif located within the dockerin module, which in the presence of EDTA resulted in loss of affinity to the cohesin partner. A cohesin amino acid mutation (D39A) that eliminated hydrogen bonding with the dockerin's critically conserved serine residues reduced the observed rupture forces. Consequently, no calcium loss occurred and dockerin activity was maintained throughout multiple forced dissociation events. These results offer insights at the single-molecule level into the stability and folding of an exquisite class of high-affinity protein-protein interactions that dictate fabrication and architecture of cellulose-degrading molecular machines.

• Authors: Stefan W Stahl, Michael A Nash, Daniel B Fried, Michal Slutzki, Yoav Barak, Edward A Bayer, Hermann E Gaub
• Journal: Proceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad Sci U S A) Vol. 109 Issue 50 Pg. 20431-6 (Dec 11 2012) ISSN: 1091-6490 [Electronic] United States
• PMID: 23188794 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Bacterial Proteins
• Cell Cycle Proteins
• Chromosomal Proteins, Non-Histone
• Multiprotein Complexes
• Recombinant Fusion Proteins
• cohesins
• Calcium

Topics

• Amino Acid Substitution
• Bacterial Proteins (chemistry, genetics, metabolism)
• Biophysics
• Calcium (metabolism)
• Cell Cycle Proteins (chemistry, genetics, metabolism)
• Chromosomal Proteins, Non-Histone (chemistry, genetics, metabolism)
• Clostridium thermocellum (genetics, metabolism)
• Crystallography, X-Ray
• Hydrogen Bonding
• Microscopy, Atomic Force
• Models, Molecular
• Multiprotein Complexes (chemistry)
• Mutagenesis, Site-Directed
• Protein Interaction Domains and Motifs
• Protein Multimerization
• Protein Stability
• Recombinant Fusion Proteins (chemistry, genetics, metabolism)
• Thermodynamics
• Unfolded Protein Response