7M6U

Crystal structure of a circular permutation and computationally designed pro-enzyme of carboxypeptidase G2

Summary for 7M6U

• Entry DOI: 10.2210/pdb7m6u/pdb
• Related: 1CG2 6XJ5
• Descriptor: Carboxypeptidase G2 circular permuation pro-domain fusion, ZINC ION, SULFATE ION, ... (4 entities in total)
• Functional Keywords: pro-enzyme, enzyme design, directed enzyme-prodrug therapy, methotrexate, circular permutation, hydrolase
• Biological source: Pseudomonas sp. (strain RS-16); More
• Total number of polymer chains: 4
• Total formula weight: 190273.35

Authors

Yachnin, B.J.,Khare, S.D. (deposition date: 2021-03-26, release date: 2021-04-14, Last modification date: 2023-10-18)

Primary citation

Yachnin, B.J.,Azouz, L.R.,White 3rd, R.E.,Minetti, C.A.S.A.,Remeta, D.P.,Tan, V.M.,Drake, J.M.,Khare, S.D.
Massively parallel, computationally guided design of a proenzyme.
Proc.Natl.Acad.Sci.USA, 119:e2116097119-e2116097119, 2022

PubMed Abstract: 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.

PubMed: 35377786
DOI: 10.1073/pnas.2116097119

Experimental method

X-RAY DIFFRACTION (2.59 Å)