Massively parallel, computationally-guided design of a pro-enzyme


The ability to localize a protein’s activity to a specific microenvironment by design 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 pro-enzymes that can be activated by proteolysis, it has been a difficult challenge to effectively re-design any chosen enzyme to be similarly stimulus-responsive. Here, we develop a massively parallel computational design, screening, and next-generation sequencing-based approach for pro-enzyme design. As a model system, we employ CPG2, a clinically approved enzyme that has applications in both the treatment of cancer and controlling methotrexate toxicity. Our designed pro-enzymes are inhibited up to 5-fold in vitro, and their activity is restored following incubation with specific proteases expressed by various tumor cell types. Pro-enzymes exhibit significantly lower activity relative to the fully activated enzyme when evaluated in cell culture. Structural and thermodynamic characterization of CPG2 provides insights into the mechanisms associated with pro-domain inhibition. The described approach is general and should enable the design of a variety of pro-proteins for precise spatial regulation of their functions.

Submission Details

ID: BhKCzXma

Submitter: Brahm Yachnin

Submission Date: March 27, 2021, 2:52 p.m.

Version: 1

Publication Details
Yachnin BJ, Azouz LR, White RE 3rd, Minetti CASA, Remeta DP, Tan VM, Drake JM, Khare SD,Proc Natl Acad Sci U S A (2022) Massively parallel, computationally guided design of a proenzyme PMID:35377786
Additional Information

The data provided here are the next-generation sequencing data for the 7500 sequences in the "massively parallel screening" assay, as well as the accompanying Rosetta-derived metrics for each design.

Study Summary

Number of data points 562500
Proteins Carboxypeptidase G2 (CPG2) circular permutation-N89 (CPG2-CP-N89), K177A mutant
Unique complexes 7362
Assays/Quantities/Protocols Experimental Assay: SeqCountPCRProduct-18 ; Experimental Assay: SeqCountPCRProduct-17 ; Experimental Assay: SeqCountFullLibrary-16 ; Experimental Assay: SeqCountFullLibrary-15 ; Experimental Assay: SeqCount100uM_MTX-14 ; Experimental Assay: SeqCount100uM_MTX-13 ; Experimental Assay: SeqCount100uM_MTX-12 ; Experimental Assay: SeqCount50uM_MTX-11 ; Experimental Assay: SeqCount50uM_MTX-10 ; Experimental Assay: SeqCount50uM_MTX-9 ; Experimental Assay: SeqCount30uM_MTX-7 ; Experimental Assay: SeqCount30uM_MTX-5 ; Experimental Assay: SeqCount0uM_MTX-4 ; Experimental Assay: SeqCount0uM_MTX-3 ; Experimental Assay: SeqCount0uM_MTX-2 ; Experimental Assay: SeqCount0uM_MTX-1 ; Derived Quantity: pcterror_PCRProduct ; Derived Quantity: stdev_PCRProduct ; Derived Quantity: avr_PCRProduct ; Derived Quantity: pcterror_FullLibrary ; Derived Quantity: stdev_FullLibrary ; Derived Quantity: avr_FullLibrary ; Derived Quantity: pcterror_MTX100uM ; Derived Quantity: stdev_MTX100uM ; Derived Quantity: avr_MTX100uM ; Derived Quantity: pcterror_MTX50uM ; Derived Quantity: stdev_MTX50uM ; Derived Quantity: avr_MTX50uM ; Derived Quantity: pcterror_MTX30uM ; Derived Quantity: stdev_MTX30uM ; Derived Quantity: avr_MTX30uM ; Derived Quantity: pcterror_MTX0uM ; Derived Quantity: stdev_MTX0uM ; Derived Quantity: avr_MTX0uM ; Computational Protocol: total_score ; Computational Protocol: angle_constraint ; Computational Protocol: atom_pair_constraint ; Computational Protocol: chainbreak ; Computational Protocol: coordinate_constraint ; Computational Protocol: dihedral_constraint ; Computational Protocol: dslf_fa13 ; Computational Protocol: fa_atr ; Computational Protocol: fa_dun ; Computational Protocol: fa_elec ; Computational Protocol: fa_intra_rep ; Computational Protocol: fa_intra_sol_xover4 ; Computational Protocol: fa_rep ; Computational Protocol: fa_sol ; Computational Protocol: hbond_bb_sc ; Computational Protocol: hbond_lr_bb ; Computational Protocol: hbond_sc ; Computational Protocol: hbond_sr_bb ; Computational Protocol: lk_ball_wtd ; Computational Protocol: omega ; Computational Protocol: p_aa_pp ; Computational Protocol: pro_close ; Computational Protocol: rama_prepro ; Computational Protocol: ref ; Computational Protocol: res_type_constraint ; Computational Protocol: yhh_planarity ; Computational Protocol: buriedhbonds ; Computational Protocol: cc_all ; Computational Protocol: cc_catbot_28 ; Computational Protocol: cc_catterm_10 ; Computational Protocol: cc_cattop_33 ; Computational Protocol: cc_core_9 ; Computational Protocol: cc_dimbeta_10 ; Computational Protocol: cstE ; Computational Protocol: num_muts ; Computational Protocol: rescount ; Computational Protocol: rescountB ; Computational Protocol: shapecomp ; Computational Protocol: series ; Computational Protocol: design ; Computational Protocol: DNA Sequence
Libraries NGS and Rosetta-derived data

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
94.3 Carboxypeptidase G2 (CPG2) circular permutation-N89 (CPG2-CP-N89), K177A mutant P06621 CBPG_PSES6