Lanthipeptides are an important group of natural products with diverse biological functions, and their biosynthesis requires the removal of N-terminal leader peptides (LPs) by designated proteases. LanPM1 enzymes, a subgroup of M1 zinc-metallopeptidases, have been recently identified as bifunctional proteases with both endo- and aminopeptidase activities to remove LPs of class III and class IV lanthipeptides. Herein, we report the biochemical and structural characterization of EryP as the LanPM1 enzyme from the biosynthesis of class III lanthipeptide erythreapeptin. We determined X-ray crystal structures of EryP in three conformational states, the open, intermediate and closed states, and identified a unique interdomain Ca2+ binding site as a regulatory element that modulates its domain dynamics and proteolytic activity. Inspired by this regulatory Ca2+ binding, we developed a strategy to engineer LanPM1 enzymes for enhanced catalytic activities by strengthening interdomain associations and driving the conformational equilibrium toward their closed forms.
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This work is supported by the National Science Foundation of China (grant nos. 21922703, 91953112 and 21861142005 to H.W.; 81871615 and 81670008 to R.B.), the Natural Science Foundation of Jiangsu Province (grant no. BK20190004 and BK20202004 to H.W. and BK20200335 to W.W.), the Fundamental Research Funds for the Central Universities (grant no. 14380131 to H.W.), the Jiangsu Innovation & Entrepreneurship Talents Plan to H.W., National Key R&D Program of China (grant no. 2019YFA0905800 to H.W.), Ministry of Science and Technology of the People’s Republic of China (grant no. 2018ZX09201018–005 to R.B.) and National Mega-project for Innovative Drugs (grant no.2019ZX09721001–001–001 to R.B.). We thank National Center for Protein Sciences Shanghai (NCPSS) beamlines BL18U and BL19U allowance. We thank the staffs of NCPSS beamlines BL18U and BL19U and SSRF BL17U, Shanghai, People’s Republic of China, for assistance during data collection. We thank the High-Performance Computing Center of Nanjing University for the numerical calculations on its blade cluster system.
The authors declare no competing interests.
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Structural comparison between EryAcyc and AplAcyc.
Extended Data Fig. 2 EryP cleaves AplAcyc peptide as an endopeptidase at multiple sites, as determined by MALDI-TOF analysis.
Assay conditions: 100 μM AplAcyc peptide and 1.0 μM EryP were incubated in 20 mM Tris buffer, pH 8.0, at 37 °C for indicated time.
Extended Data Fig. 3 EryP contains a zinc binding motif that is highly conserved in M1 Zn2+-dependent metallopeptidases.
(a) The overall crystal structure of EryP and the close-up view of the Zn binding residues. (b) Sequence alignment of EryP with M1 Zn2+-dependent metallopeptidases. Catalytic residues are labeled with stars and the conserved HEXXH(X)18E is highlighted. Accession numbers of related proteins: EryP(WP_009950696.1), AplP(AHB63590.1), ePepN(AAA24317.1), ERAP1(NP_001185470.1).
The Fo-Fc density maps (contoured at 3.0 σ) for the Zn2+ (a) and Ca2+ (b) with the ion removed are shown as grey mesh.
Extended Data Fig. 5 Both endopeptidase and aminopeptidase activities of EryPE307Q and EryPY392F were abolished.
(a) EryPE307Q and EryPY392F were inactive toward EryALP peptide. Assay conditions: EryALP peptide (100 μM) and EryP (1.0 μM) were incubated in 20 mM Tris buffer, pH 8.0, at 37 °C for 24 h. (b) The aminopeptidase activity of EryPE307Q and EryPY392F toward amino acid pNA derivatives were almost abolished compared with EryP. * represents the sodium adducts of peptides in MS. Error bars indicate standard deviation of three independent replicates. Data represent the mean ± s.d. from three replicates.
Extended Data Fig. 6 Bestatin and Zn2+-chelating reagent o-phenanthroline significantly decreased both endo- and aminopeptidase activities of EryP.
Bestatin and Zn2+-chelating reagent o-phenanthroline significantly decreased both endo- and aminopeptidase activities of EryP.
Extended Data Fig. 7 The docking model of a dipeptide Leu-Glu into the active site of EryP in the closed state.
The binding pocket (a) and 2D diagram (b) of dipeptide Leu-Glu binding with EryPclosed are shown. Following the convention for naming peptidase sites, the site responsible for accommodating the peptide side chain N-terminal to the cleavage site is named S1, and the subsequent position are named S1′. In the dipeptide docking model, the highly conserved G272AME275N motif in EryP binds to the dipeptide substrate through multiple hydrogen bonds in the docking model. In particular, residue E275 directly binds to the amino group at the N-terminus of the dipeptide with additional binding from residues E132 and E329. This docking model suggests that both S1 and S1′ pockets are spacious to accommodate amino acid side chains of various sizes in peptide substrates, allowing the enzyme to sequentially cleave various residues from EryALP as an aminopeptidase.
Extended Data Fig. 8 Computational docking of a hexapeptide (ELDAPN) as an endopeptidase substrate to be cleaved between residues Asp and Ala in EryPclosed.
(a-b) and structures of ERAP1 (PDB ID: 2YD0) (c) and IRAP (PDB ID: 5MJ6) (d) bound with peptide inhibitors. In the hexapeptide docking model, the hexapeptide adopted a bent conformation with the scissile peptide bond between Asp and Ala of the hexapeptide binding to the catalytic zinc ion with the amide oxygen. The N-terminal segment of the hexapeptide was accommodated in the internal cavity extending from the catalytic site in domain II toward the interdomain Ca2+ binding site. The N-terminal amino group of the hexapeptide is anchored firmly by electrostatic interactions with E132. The carboxylate of the Asp of the hexapeptide forms hydrogen bonds with N328 and Y392 in the S1 pocket as well as the side chain of the Ala forms hydrophobic interactions with residues in the S1′ pocket from the G272AMEN motif. This docking model also reveals that there is space to accommodate additional amino acid extending at both the N- and C-terminus of the hexapeptide, a configuration that would allow the binding of longer peptides as endopeptidase substrates in EryPclosed.
Cutaway views of the binding pocket of EryPclosed (a), docked EryPclosed-(10 mer peptide) complex (b), ERAP1-(10 mer peptide) complex (PDB ID: 6RQX) (c) and APN-substance P complex (PDB ID: 6RQX) (d) are shown, respectively. M1 aminopeptidases are shown in surface mode, the ligands are shown in cartoon mode.
Extended Data Fig. 10 Close views of EryP-(10-mer peptide) MD representative snapshot, ERAP1-(10-mer peptide) complex and Porcine APN-(substance P) complex.
(a) EryP-(10-mer peptide) MD representative snapshot, (b) ERAP1-(10-mer peptide) complex crystal structure and (c) Porcine APN-(substance P) complex crystal structure. Ca2+ ion is shown in red sphere and Zn2+ ion in yellow sphere. As shown in (a), the 10-mer peptide lied in a fit inside the cavity formed around the active site in the space between domains I/ II and domain IV of EryP and showed an extended conformation similar to ERAP1-(10 mer peptide) and porcine APN-(substance P) complexes (b and c). The N-terminus of the 10-mer peptide that bears the phosphinic moiety is bound on the catalytic zinc ion (yellow sphere) in a motif observed previously in crystal structures of homologous aminopeptidases with bound phosphinic groups. Van der Waals interactions and electrostatic interactions with the main chain and side chains of residues of EryP were found to stabilize the substrate (a). In addition, space for additional atoms is available at the N/C-terminus of the 10-mer peptide, a configuration that would allow the accommodation of longer internal peptide and N-terminal peptide substrates in this conformation of EryP.
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Zhao, C., Sheng, W., Wang, Y. et al. Conformational remodeling enhances activity of lanthipeptide zinc-metallopeptidases. Nat Chem Biol (2022). https://doi.org/10.1038/s41589-022-01018-2