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.

Keywords: data mining; ligase-activity determinant; peptide ligase.

Fig. 1.

Enzymatic activity of recombinant VyPAL1–3 and VyAEP1. (A) Reaction scheme of ligase-mediated cyclization of GN14-SL. (B) Analytical HPLC and MALDI-TOF mass spectrometry data of VyPAL2-mediated cyclization under different reaction pH values. The asterisk indicates racemized synthetic GN14-SL. Note that MALDI-TOF MS was more sensitive against cyclic cGN14 than the linear species. (C) Quantitative summary of product ratio and reaction yield of each enzyme analyzed using RP-HPLC. For each reaction, a molar ratio of purified active enzyme:GN14-SL of 1:500 was mixed and reacted at 37 °C for 10 min. Average yield and SDs were calculated from experiments performed in triplicate.

Fig. 2.

Crystal structure of VyPAL2. (A) The VyPAL2 monomer: Its cap region is colored in green, core in purple, and linker in orange. N-glycosylation sites are displayed as cyan sticks. (A, Insets) Active-site residues (Right) and one representative glycosylation site (Left). The electronic density map (with Fourier coefficients 2Fo − Fc and phases from the model) is overlaid and contoured at 1-sigma level. (B) Diagram of VyPAL2 protein domains. Blue arrowheads indicate glycosylation sites. Cleavage sites as determined by LC-MS/MS are indicated. Active-site residues (His172 and Cys214) as well as succinimide 171 are labeled.

Fig. 3.

Modeling of substrate LKVIHNSL binding in the core domain of VyPAL2. The electrostatic surface of the VyPAL2 core domain is represented with positive charges in blue and negative charges in red. Protein regions important for activity are labeled. Active-site residues His172 and Cys214 are represented as green sticks. (A) The linker region (orange), α6-helix (green), and substrate-binding pocket S1 are indicated. (B) Substrate residues (LKVIHNSL, yellow) are shown as sticks. The distance between the sulfur atom of Cys214 and the carbon of the carbonyl group of substrate–Asn is 3.5 Å. (C) Residues lining the substrate-binding pockets are depicted as sticks. The VyPAL2 core domain is represented as a gray surface. The S1 pocket (magenta) comprises residues R69, H70, Snn171, H172, G173, E212, A213, C214, S242, and D263. The S2 pocket (red) is shaped by W243 and I244. The S3 pocket (yellow) is lined by C247 and C260 that form a disulfide bond. The S4 pocket (lime) is shaped by T245, Y246, and L264. On the other side of the active site, the S1′ pocket is lined (cyan) by H172, G173, and A174, and the S2′ pocket (green) is open and comprises I178, G179, M180, and Y185. VyPAL polymorphism sites around the S1 pocket are highlighted (Insets). VyPAL1 residues are colored and labeled in green, VyPAL2 in blue, VyPAL3 in magenta, and VyAEP1 in orange. The catalytic residues Cys214 and His172 of VyPAL2 are shown as red and cyan sticks, respectively.

Fig. 4.

Retroengineering in the S1′ pocket of VyPAL3. All reactions were performed in the pH range 4.5–8.0. MS peaks of the hydrolysis product GN14 are marked with a red dashed line. (A, Left) MS analysis of reactions catalyzed by the VyPAL3 wild type. (B, Left) MS spectra of reactions catalyzed by VyPAL3-Y175G. (A and B, Right) HPLC profiles of reactions catalyzed by VyPAL3 and VyPAL3-Y175G. (C) Quantitative summary of product ratio and reaction yield analyzed using RP-HPLC for VyPAL3-Y175G. Average yield and SDs were calculated from experiments performed in triplicate.

Fig. 5.

Activity of VcAEP and the VcAEP-Y168A mutant (LAD2). MS analysis and HPLC-based quantitative summary of the reaction catalyzed by (A) VcAEP wild type and (B) VcAEP-Y168A mutant targeting the LAD2 region. All reactions were performed at pH values ranging from 4.5 to 8.0. MS peaks of the hydrolysis product GN14, cyclization product cGN14, and sodium ion adduct of cGN14 are marked with red, blue, or gray dashed lines. A dramatic improvement of ligase activity for the VcAEP-Y168A mutant is clearly visible. Average yield and SDs were calculated from experiments performed in triplicate.

Fig. 6.

Ligase-activity determinants of PALs and the proposed catalytic mechanism. (A) Sequence alignment of PALs and AEPs studied in this work. The catalytic triad Asn–His–Cys is shaded in black. Residues belonging to the S1 pocket are shaded in blue. Proposed LAD residues are boxed in red. Residues of LAD1 and LAD2, that favor ligase activity, are highlighted in blue, and those favoring protease activity are highlighted in red. The conserved disulfide bond near LAD1 is highlighted in orange. The PPL is in a green box and the MLA loop is in purple. The nomenclature of secondary structures was adapted from ref. with alterations according to the crystal structure of VyPAL2 (this work). Residues and motifs crucial for activity are labeled with the same color codes as used in the sequence alignment. Residues below the dashed line correspond to the oxyanion hole, and those above the dashed line correspond to the proposed activity determinants. (B) Schemes proposed for ligation and hydrolysis by VyPAL2 and the role of LAD1 and LAD2. The first step of the mechanism is identical for hydrolysis and ligation, leading to formation of the S-acyl enzyme intermediate, and is the rate-limiting step. Its main determinant is LAD1. LAD2 controls the nature of activity to either favor the nucleophilic attack by a peptide (ligation) or a water molecule (hydrolysis).