טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentHandelsman Tal
SubjectProtein-Protein Interactions in the Cellulosome Complex of
Clostridium thermocellum
DepartmentDepartment of Biotechnology and Food Engineering
Supervisors Professor Yuval Shoham
Professor Edward Bayer


Abstract

The cellulosome is a macromolecular machine, whose components interact in a synergistic manner to catalyze the efficient degradation of cellulose.  Each of the cellulosomal subunits consists of a multiple set of modules, two classes of which (dockerin domains on the enzymes and cohesin domains on the scaffolding protein, termed scaffoldin) govern the incorporation of the enzymatic subunits into the cellulosome complex.  This work was focused on the molecular basis of the high affinity cohesin -dockerin interaction, which dictates both the assembly of the cellulosome and its attachment to the cells. 

The dockerin is comprised of two duplicated segments.  Recognition residues were found to occupy positions 10, 11 and 18 of the second duplicated segment and position 22 of the first.  Replacement of these residues in a C. cellulolyticum dockerin with the matching residues from C. thermocellum resulted in a mutant dockerin that binds the C. thermocellum cohesin with high affinity (KA of 1.3 109 M-1), and the affinity towards its cognate cohesin was reduced (from Ka of 1.2 109 M-1 of the wild-type to KA of 4.0 108 M-1 of the mutant). It was also demonstrated that opposite orientation of binding is indeed possible. Prediction of binding and recognition residues of the cohesin was a more challenging taskNonetheless, several binding residues were pinpointed.  Among those, Asp34 plays a central role in the hydrogen-bonding network at the cohesin-dockerin interface.  A single substitution of Asp-to-Asn disrupted normal binding, reducing the affinity by at least three orders of magnitude.  The results underscore the fragile nature of this high affinity interaction.

To facilitate the examination of cohesin-dockerin binding, an enzyme-linked interaction assay (“ELIA”) was also developed in this work.