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Massively parallel, computationally guided design of a proenzyme
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Massively parallel, computationally guided design of a proenzyme
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Journal Article
Massively parallel, computationally guided design of a proenzyme
2022
Overview
Confining the activity of a designed protein to a specific microenvironment would have broad-ranging applications, such as enabling cell type-specific therapeutic action by enzymes while avoiding off-target effects. While many natural enzymes are synthesized as inactive zymogens that can be activated by proteolysis, it has been challenging to redesign any chosen enzyme to be similarly stimulus responsive. Here, we develop a massively parallel computational design, screening, and next-generation sequencing-based approach for proenzyme design. For a model system, we employ carboxypeptidase G2 (CPG2), a clinically approved enzyme that has applications in both the treatment of cancer and controlling drug toxicity. Detailed kinetic characterization of the most effectively designed variants shows that they are inhibited by ∼80% compared to the unmodified protein, and their activity is fully restored following incubation with site-specific proteases. Introducing disulfide bonds between the pro- and catalytic domains based on the design models increases the degree of inhibition to 98% but decreases the degree of restoration of activity by proteolysis. A selected disulfide-containing proenzyme exhibits significantly lower activity relative to the fully activated enzyme when evaluated in cell culture. Structural and thermodynamic characterization provides detailed insights into the prodomain binding and inhibition mechanisms. The described methodology is general and could enable the design of a variety of proproteins with precise spatial regulation.
Publisher
National Academy of Sciences
Subject
/ Biophysics and Computational Biology
/ Design
/ Enzyme Precursors - chemistry
/ Enzyme Precursors - pharmacology
/ Enzymes
/ gamma-Glutamyl Hydrolase - chemistry
/ gamma-Glutamyl Hydrolase - pharmacology
/ Humans
/ Protein Engineering - methods
/ Proteins
/ Redesign
/ Rosetta
/ Toxicity
/ zymogens
