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Structural determinants for peptide-bond formation by asparaginyl ligases.

ABSTRACT: Asparaginyl endopeptidases (AEPs) are cysteine proteases which break Asx (Asn/Asp)-Xaa bonds in acidic conditions. Despite sharing a conserved overall structure with AEPs, certain plant enzymes such as butelase 1 act as a peptide asparaginyl ligase (PAL) and catalyze Asx-Xaa bond formation in near-neutral conditions. PALs also serve as macrocyclases in the biosynthesis of cyclic peptides. Here, we address the question of how a PAL can function as a ligase rather than a protease. Based on sequence homology of butelase 1, we identified AEPs and PALs from the cyclic peptide-producing plants Viola yedoensis (Vy) and Viola canadensis (Vc) of the Violaceae family. Using a crystal structure of a PAL obtained at 2.4-Å resolution coupled to mutagenesis studies, we discovered ligase-activity determinants flanking the S1 site, namely LAD1 and LAD2 located around the S2 and S1' sites, respectively, which modulate ligase activity by controlling the accessibility of water or amine nucleophile to the S-ester intermediate. Recombinantly expressed VyPAL1-3, predicted to be PALs, were confirmed to be ligases by functional studies. In addition, mutagenesis studies on VyPAL1-3, VyAEP1, and VcAEP supported our prediction that LAD1 and LAD2 are important for ligase activity. In particular, mutagenesis targeting LAD2 selectively enhanced the ligase activity of VyPAL3 and converted the protease VcAEP into a ligase. The definition of structural determinants required for ligation activity of the asparaginyl ligases presented here will facilitate genomic identification of PALs and engineering of AEPs into PALs.

SUBMITTER: Hemu X

PROVIDER: S-EPMC6576118 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Structural determinants for peptide-bond formation by asparaginyl ligases.

Hemu Xinya X El Sahili Abbas A Hu Side S Wong Kaho K Chen Yu Y Wong Yee Hwa YH Zhang Xiaohong X Serra Aida A Goh Boon Chong BC Darwis Dina A DA Chen Ming Wei MW Sze Siu Kwan SK Liu Chuan-Fa CF Lescar Julien J Tam James P JP

Proceedings of the National Academy of Sciences of the United States of America 20190523 24

Asparaginyl endopeptidases (AEPs) are cysteine proteases which break Asx (Asn/Asp)-Xaa bonds in acidic conditions. Despite sharing a conserved overall structure with AEPs, certain plant enzymes such as butelase 1 act as a peptide asparaginyl ligase (PAL) and catalyze Asx-Xaa bond formation in near-neutral conditions. PALs also serve as macrocyclases in the biosynthesis of cyclic peptides. Here, we address the question of how a PAL can function as a ligase rather than a protease. Based on sequenc ...[more]

PMID: 31123145

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Project description:Background: Protein theranostics integrate both diagnostic and treatment functions on a single disease-targeting protein. However, the preparation of these multimodal agents remains a major challenge. Ideally, conventional recombinant proteins should be used as starting materials for modification with the desired detection and therapeutic functionalities, but simple chemical strategies that allow the introduction of two different modifications into a protein in a site-specific manner are not currently available. We recently discovered two highly efficient peptide ligases, namely butelase-1 and VyPAL2. Although both ligate at asparaginyl peptide bonds, these two enzymes are bio-orthogonal with distinguishable substrate specificities, which can be exploited to introduce distinct modifications onto a protein. Methods: We quantified substrate specificity differences between butelase-1 and VyPAL2, which provide orthogonality for a tandem ligation method for protein dual modifications. Recombinant proteins or synthetic peptides engineered with the preferred recognition motifs of butelase-1 and VyPAL2 at their respective C- and N-terminal ends could be modified consecutively by the action of the two ligases. Results: Using this method, we modified an EGFR-targeting affibody with a fluorescein tag and a mitochondrion-lytic peptide at its respective N- and C-terminal ends. The dual-labeled protein was found to be a selective bioimaging and cytotoxic agent for EGFR-positive A431 cancer cells. In addition, the method was used to prepare a cyclic form of the affibody conjugated with doxorubicin. Both modified affibodies showed increased cytotoxicity to A431 cells by 10- and 100-fold compared to unconjugated doxorubicin and the free peptide, respectively. Conclusion: Bio-orthogonal tandem ligation using two asparaginyl peptide ligases with differential substrate specificities is a straightforward approach for the preparation of multifunctional protein biologics as potential theranostics.

Dataset Information

Structural determinants for peptide-bond formation by asparaginyl ligases.

Publications

Structural determinants for peptide-bond formation by asparaginyl ligases.

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