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 PDB Entries

Structure ID Release Date Resolution Structure Title
1CG2 1996-12-20T00:00:00+0000 2.5 CARBOXYPEPTIDASE G2
6XJ5 2020-06-22T00:00:00+0000 3.11 Carboxypeptidase G2 modified with a versatile bioconjugate for metalloprotein design
6XJ5 2020-06-22T00:00:00+0000 3.11 Carboxypeptidase G2 modified with a versatile bioconjugate for metalloprotein design
7M6U 2021-03-26T00:00:00+0000 2.59 Crystal structure of a circular permutation and computationally designed pro-enzyme of carboxypeptidase G2

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