Enzymatic C-to-C Protein Ligation

Abstract

Transpeptidase-catalyzed protein and peptide modifications have been widely utilized for generating conjugates of interest for biological investigation or therapeutic applications. However, all known transpeptidases are constrained to ligating in the N-to-C orientation, limiting the scope of attainable products. Here, we report that an engineered asparaginyl ligase accepts diverse incoming nucleophile substrate mimetics, particularly when a means of selectively quenching the reactivity of byproducts released from the recognition sequence is employed. In addition to directly catalyzing formation of l-/d- or α-/β-amino acid junctions, we find C-terminal Leu-ethylenediamine (Leu-Eda) motifs to be bona fide mimetics of native N-terminal Gly-Leu sequences. Appending a C-terminal Leu-Eda to synthetic peptides or, via an intein-splicing approach, to recombinant proteins enables direct transpeptidase-catalyzed C-to-C ligations. This work significantly expands the synthetic scope of enzyme-catalyzed protein transpeptidation reactions.

Keywords: Biorthogonal; Enzyme Bioconjugation; Enzyme Catalysis; Protein Engineering; Site-Specificity.

Figure 1

[C247A]OaAEP1‐catalyzed formation of non‐natural ligation junctions. A) Scheme illustrating the Ni²⁺‐mediated nucleophile quenching strategy. Below the scheme are RP‐HPLC chromatograms (280 nm) of the ligation of a model NGLH‐containing peptide substrate (Ac‐RWRGWRNGLH, 100 μM) to the conventional substrate (GLRL, at 2 equiv) with/without NiSO₄ addition (as indicated, 2 mM) as catalyzed by 100 nM [C247A]OaAEP1 in 100 mM Tris‐HCl, pH 7.5, for 1 h. B) Ligation of substrate mimetics to the model NGLH‐containing peptide as in (A), except all shown reactions were supplemented with NiSO₄ and the mimetic peptides were provided at 5 equiv Figures S1 and S2 show RP‐HPLC chromatograms for all Ni²⁺‐free reactions and MALDI‐TOF MS traces for all reactions, respectively. Table S1 lists observed and calculated peptide conjugate masses. C) Ligation of the d‐ and β‐amino acid substrate mimetics (500 μM) to NGLH‐modified protein substrates (a nanobody, VHH_6e, and eGFP, as indicated; 50 μM) as catalyzed by 400 nM [C247A]OaAEP1 in 100 mM HEPES pH 7, 25 °C for 2 h. Shown are reconstructed ESI‐MS spectra with the observed product masses. Table S2 lists observed and calculated protein conjugate masses.

Figure 2

[C247A]OaAEP1‐catalyzed C‐to‐C ligation. A) Ligation of C‐terminal substrate mimetics (Ac‐GLRL‐hydrazide or Ac‐GLRL‐Eda) to the model substrate Ac‐RWRGWRNGLH. Reactions were conducted as in Figure 1A. Shown are RP‐HPLC chromatograms (280 nm) of the crude reaction mixtures and % conversion to product (*) is indicated. B) Assessing the degree of substrate mimicry via by monitoring the ligation of a series of Xaa‐Eda (Ac‐GLRX‐Eda) peptides to Ac‐RWRGWRNGLH as in (A). Figures S4 and S5 show RP‐HPLC chromatograms for all Ni²⁺‐free reactions and MALDI‐TOF MS traces for all reactions, respectively. The chromatogram for the P2′′ Leu‐Eda peptide is the same as shown in Figure 2A. Table S1 lists observed and calculated peptide conjugate masses. C) Ligation of the Leu‐Eda substrate mimetics to NGLH‐modified protein substrates as in Figure 1C. Shown are reconstructed ESI‐MS spectra with the observed product masses. Table S2 lists observed and calculated protein conjugate masses.

Figure 3

Generation of C‐to‐C protein fusions using orientation‐controllable bifunctional linker peptides. A) Ligation of the bifunctional linker peptide (GLHRL‐Eda) to the model substrate Ac‐RWRGWRNGLH with/without Ni²⁺, as indicated. Reactions were conducted as in Figure 1A except that the bifunctional linker was provided at 1 mM (10 mol equiv), enzyme at 200 nM, and NiSO₄ at 5 mM. Reactions containing Ni²⁺ were incubated for 15 min prior to enzyme addtion. Shown are RP‐HPLC chromatograms (280 nm) of the crude reaction mixtures. Figure S6 shows MALDI‐TOF MS traces of the same reactions. Table S1 lists observed and calculated peptide conjugate masses. B) SDS‐PAGE analysis of eGFP‐linker‐eGFP C‐to‐C fusion formation when the initial attachment of the linker to eGFP‐NGLH is controlled via Ni²⁺ addition or uncontrolled. NiSO₄ was removed prior to protein‐linker to protein‐NGLH ligations. C) SDS‐PAGE analysis of the formation of photocleavable eGFP and nanobody (VHH_6e) protein‐linker‐protein C‐to‐C fusions and their cleavage via UV irradiation. Initial linker (GLHG‐Anp‐L‐Eda) attachment was conducted in the presence of Ni²⁺ to control ligation orientation. Reconstructed ESI‐MS spectra of the protein‐linker conjugates are shown in Figure S7. Table S2 lists observed and calculated protein conjugate masses. Anp=3‐amino‐3‐(2‐nitro‐phenyl)propionic acid.

Figure 4

Direct [C247A]OaAEP1‐catalyzed C‐to‐C ligation of recombinant proteins. A) Scheme detailing the intein‐mediated strategy for generating recombinant proteins bearing a C‐terminal Leu‐Eda motif. B) SDS‐PAGE analysis of the direct C‐to‐C ligation of SUMO‐L‐Eda to eGFP‐NGLH with/without NiSO₄, as indicated. C) Generation of a nanobody C‐to‐C heterodimer with reactions and analysis as in (B). D) SDS‐PAGE analysis of the ligation of bifunctional GLH‐SUMO‐L‐Eda to eGFP‐NGLH with/without NiSO₄, as indicated. The addition of Ni²⁺ suppresses formation of the trimeric protein conjugate (lane 3). Reconstructed ESI‐MS spectra of the C‐terminal Leu‐Eda‐modified proteins are shown in Figure S9. POI=protein of interest.