Selectively Modulating Conformational States of USP7 Catalytic Domain for Activation.

Ubiquitin-specific protease 7 (USP7) deubiquitinase activity is controlled by a number of regulatory factors, including stimulation by intramolecular accessory domains. Alone, the USP7 catalytic domain (USP7cd) shows limited activity and apo USP7cd crystal structures reveal a disrupted catalytic triad. By contrast, ubiquitin-conjugated USP7cd structures demonstrate the canonical cysteine protease active-site ...
geometry; however, the structural features of the USP7cd that stabilize the inactive conformation and the mechanism of transition between inactive and active states remain unclear. Here we use comparative structural analyses, molecular dynamics simulations, and in silico sequence re-engineering via directed sampling by RosettaDesign to identify key molecular determinants of USP7cd activation and successfully engineer USP7cd for improved activity. Full kinetic analysis and multiple X-ray crystal structures of our designs indicate that electrostatic interactions in the distal "switching loop" region and local packing in the hydrophobic core mediate subtle but significant conformational changes that modulate USP7cd activation.
Mesh Terms:
Amino Acid Sequence, Amino Acid Substitution, Catalytic Domain, Cloning, Molecular, Crystallography, X-Ray, Enzyme Activation, Enzyme Inhibitors, Escherichia coli, Gene Expression, Genetic Vectors, Humans, Hydrophobic and Hydrophilic Interactions, Kinetics, Molecular Dynamics Simulation, Mutation, Peptidomimetics, Protein Binding, Protein Conformation, alpha-Helical, Protein Conformation, beta-Strand, Protein Interaction Domains and Motifs, Recombinant Proteins, Static Electricity, Substrate Specificity, Thermodynamics, Ubiquitin-Specific Peptidase 7
Structure
Date: Dec. 02, 2017
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