Overexpression and purification of SmDPP7 WT and mutants.
A synthetic gene coding for full-length SmDPP7 (residues 1-720), codon-optimized for expression in E. coli, was purchased from Genscript (Piscataway, USA). The target sequence corresponding to mature SmDPP7 containing the signal peptide of DAP BIII39 from Pseudoxanthomonas mexicana WO24 was cloned into the pET22b expression plasmid (Merck, Darmstadt, Germany). The mature SmDPP7 was composed of 698 amino acids (residues 23 to 720), with a theoretical molecular weight of 75720.95 and an isoelectric point of 8.08. Plasmids for expression of mutants, K206A, R218A, R218Q, R218K, T220A, and F671A, were obtained with overlap extension PCR using wild type expression plasmid as a template (Table S5). E. coli BL21 Gold (DE3) cells (Agilent Technologies, Santa Clara, USA) transformed with the pET22b-SmDPP7 WT and mutants expression plasmid were grown in TB media at 298 K to an OD600 of 0.6. Overproductions of SmDPP7 WT and mutants were carried out by adding 0.1 mM Isopropyl-β-D-thiogalactopyranoside for 15 h at 298 K. Thereafter, the cells were harvested by centrifugation at 6,000 x g. Cells were disrupted using sonication and the cell extract was obtained by centrifuging the lysate at 20,000 x g for 30 min. The SmDPP7 WT and mutants were purified by precipitation with 35 to 70% ammonium sulfate and hydrophobic column chromatography using a HiPrep 16/10 Butyl column (Cytiva, Marlborough, MA, USA). The eluate was desalted using a HiPrep 26/10 desalting column (Cytiva) and finally subjected to anion-exchange column chromatography using a Mono Q 5/50 GL column (Cytiva). The fractions containing SmDPP7 WT and mutants were pooled, buffer-exchanged to 80 mM Tris/HCl pH 8.5 and concentrated to 5 mg/ml using Vivaspin 20 concentrator (Cytiva). Protein concentration was determined by Bradford assay, and a linear calibration curve was obtained using bovine gamma-globulin ranging from 0 to 0.25 mg/ml.
Overexpression and purification of PgDPP7, SmDPP11 and PgDPP11
Synthetic genes coding for full-length PgDPP7 (residues 1-712, UniProt accession number Q7MWU6) and SmDPP11 (residues 1-715, UniProt accession number B4SNQ8), codon-optimized for expression in E. coli, were purchased from Genscript (NJ, USA). The target sequences corresponding to mature PgDPP7 (Asp25-Ile712) and mature SmDPP11 (Asp24-Gln715), both containing the signal peptide of DAP BIII39 from P. mexicana WO24 were amplified using PCR and cloned into the pET22b expression plasmid (Merck, Darmstadt, Germany). Overproduction and purification of PgDPP7 and SmDPP11 were performed in a way similar to those for SmDPP7 described above. Overproduction and purification of PgDPP11 has been described in the literature17.
Determination of kinetic parameters toward dipeptidyl MCA
Kinetic parameters were determined by fitting the experimental data to the Michaelis–Menten equation using Excel Solver (Microsoft, USA) by nonlinear least-squares fitting with different concentrations of glycyl-l-tyrosine-4-methylcoumaryl-7-amide (1.56, 3.13, 6.25, 12.5, 25, 50, and 100 µM; Gly-Tyr-MCA; scrum) and l-tyrosine-l- tyrosine-4-methylcoumaryl-7-amide (3.13, 6.25, 12.5, 25, 50, 100, and 200 µM; Tyr-Tyr-MCA; scrum) as a substrate. The enzyme reaction was performed in a reaction buffer consisting of 50 mM sodium phosphate buffer pH 7.0, 5 mM EDTA and 0.005% Tween 20 at 298 K for 20 min and standard deviations were calculated from three independent experiments. The fluorescence intensity of the released MCA was measured with excitation at 355 nm and emission at 460 nm using an Infinite 200 PRO microplate reader (Tecan, Switzerland).
Competitive inhibition assay using dipeptide library
The dipeptide library was purchased from Funakoshi Co., Ltd. (Tokyo, Japan). Different concentrations (3.13, 6.25, 12.5, 25, 50, 100, and 200 µM) of dipeptides as the inhibitor and 200 µM Tyr-Tyr-MCA (PEPTIDE INSTITUTE, Osaka, Japan) as the substrate for DPP7 were added into a reaction buffer consisting of 50 mM sodium phosphate buffer pH 7.0, 5 mM EDTA and 0.005% Tween 20. For DPP11s, different concentrations (1.56, 3.13, 6.25, 12.5, 25, 50, and 100 µM) of dipeptides as the inhibitor and 100 µM Leu-Asp-MCA (Peptide Institute, Japan) as the substrate were used. The enzyme reaction was performed at 298 K for 20 min and standard deviations were calculated from three independent experiments. Half-maximal inhibitory concentration (IC50) values were determined by fitting to a sigmoid curve (4-parameter logistic curve) using ImageJ software40 and inhibition constants (Ki) were calculated using the Cheng-Prusoff equation41. The fluorescence intensity of the released MCA was measured with the same method of determination of kinetics parameters toward dipeptidyl MCA.
Crystallisation of SmDPP7
Crystals of the SmDPP7/dipeptide complexes were prepared as follows: Asn-Tyr and Val-Tyr-Pro were purchased from GenScript (Piscataway, NJ, USA). Phe-Tyr and Tyr-Tyr-Tyr were purchased from Sigma-Aldrich (St Louis, MO, USA) and Santa Cruz Biotech (Dallas, TX, SUA) respectively. The tripeptides, Val-Tyr-Pro and Tyr-Tyr-Tyr (tripeptides were used as a substrate, and the N-terminal dipeptide was observed as the bound product as described below), were dissolved in 80 mM Tris-HCl, pH 8.5 to concentrations of 30.0 mM and 20.0 mM, respectively. The 5-mg/ml SmDPP7 solution was mixed with aliquots of the respective ligand solutions at a volume ratio of 9:1 for the tripeptides Val-Tyr-Pro and Tyr-Tyr-Tyr, with final ligand concentrations of 3.0 mM and 2.0 mM, respectively. The samples were crystallised using the hanging-drop method; 1 μl of protein solution was mixed with the same volume of reservoir solution (20%(w/v) PEG8000 and 0.2 M ammonium acetate) and incubated at 293 K. The drops were suspended over 200 μl of reservoir solution in 48-well plates.
Asn-Tyr and Phe-Tyr were dissolved in purified water to concentrations of 100 mM. The 5-mg/ml SmDPP7 solution was mixed with aliquots of the respective ligand solutions at a volume ratio of 25:1 for the dipeptides Asn-Tyr and Phe-Tyr, with a final ligand concentration of 4.0 mM. The samples were crystallised using the hanging-drop method; 0.95 μl of protein solution was mixed with the same volume of reservoir solution (20%(w/v) PEG8000 and 0.2 M Calcium acetate) and incubated at 293 K. The drops were suspended over 200 μl of reservoir solution in 48-well plates.
A dipeptide complex, Tyr-Tyr complex, was obtained by co-crystallisation of SmDPP7 with a tripeptide Tyr-Tyr-Tyr, because the tripeptide Tyr-Tyr-Tyr was commercially available at a lower cost as compared with custom peptide synthesis of the dipeptide Tyr-Tyr. For the Tyr-Tyr complex, clear continuous electron density was observed for the first two residues of the tripeptide (Figure S2a), and no clear electron density was observed for the last residue. Because the SmDPP7 enzyme reaction occurred in the solution used for crystallisation, the Tyr-Tyr-Tyr (P2-P1-P1’) tripeptide acted as the substrate, and the reaction products were the N-terminal Tyr-Tyr (P2-P1) dipeptide and the C-terminal Tyr (P1’). While the N-terminal product Tyr-Tyr remained at the active site, the C-terminal product Tyr dissociated from the active site. The asymmetric unit of the Tyr-Tyr complex was composed of two independent SmDPP7 subunits; in both subunits (Figure 3a), the hydrolysed dipeptide product (NH2-Tyr-Tyr-COOH), rather than a reaction intermediate, was observed (Figure S2a). Similarly, another dipeptide complex, Val-Tyr complex, was obtained by co-crystallisation of SmDPP7 with a tripeptide Val-Tyr-Pro and the hydrolysed dipeptide product (NH2-Val-Tyr-COOH) was observed (Figure S2b). In this case, co-crystallisation with Val-Tyr-Pro was performed to confirm the ability of imino-bond (X-Pro) hydrolysis by SmDPP7, though the dipeptide Val-Tyr was commercially available at a reasonable cost. The other dipeptide complexes Phe-Tyr and Asn-Tyr complexes, were obtained by co-crystallisation of SmDPP7 with dipeptides Phe-Tyr and Asn Tyr, respectively (Figures S2c and S2d).
X-ray data collection
For data collection under cryogenic conditions, dipeptide-complex crystals in a droplet were directly transferred to harvesting solutions [16%(w/v) PEG8000, 0.16 M ammonium acetate and 20%(v/v) glycerol] and [16%(w/v) PEG8000, 0.16 M calcium acetate and 20%(v/v) glycerol] respectively for 10 seconds. Crystals were mounted in nylon loops or MicroMountsTM (MiTeGen, Ithaca, NY, USA) and flash-cooled in a cold nitrogen gas stream at 100 K immediately before data collection. Data were collected by the rotation method at 100 K using a MAR300HE CCD detector or EIGER 16M detector with synchrotron radiation source on the beamline BL44XU at SPring-8. Laue group and unit-cell parameters were determined using the xia2/DIALS software package42 with XDS43 or MOSFLM44. The cell parameters and data-collection statistics are summarised in Table S1.
The initial phase determination was performed for the Val-Tyr complex of SmDPP7 using the molecular replacement method. One protomer of PmDAP BII18 (PDB code: 3WOL), which has approximately 78% amino-acid sequence identity to SmDPP7, was used as a search model. Cross-rotation and translation functions were calculated using the MOLREP program45 from CCP4 suite46. Structure refinement was carried out with the program REFMAC547, and further iterative manual model building and refinement were performed using the programs Coot48 and REFMAC547. The stereochemistry of the model was verified using RAMPAGE49 and PROCHECK50 programs. The refined structure of the Val-Tyr complex was then used for the structural determination of the Tyr-Tyr complex by the difference Fourier method The refined structure of the Tyr-Tyr complex was used for the initial phase determination of Asn-Tyr and Phe-Tyr complexes. Cross-rotation and translation functions were calculated using the program PHASER51 from the CCP4 suite46. After the final round of refinement, the bound dipeptide molecules were removed from the model. Then, the amplitude |Fc| and phase angles calculated from the partial structure were used to calculate a weighted m|Fo|–D|Fc| omit map47, where ‘m’ is the figure of merit (approximately equal to the cosine of the phase error) and ‘D’ is the estimate of the coordinate error in the partial structure (Figure S2). The refinement statistics are summarised in Table S2.
Isothermal Titration Calorimetry
The bindings were analysed using a MICROCAL PEAQ-ITC microcalorimeter (Malvern, UK). The binding reactions were performed in 50 mM sodium phosphate pH 7.0 and 2.5% dimethyl sulfoxide at 25 °C and were stirred at 750 rpm. A single injection of 0.4 μl and 19 times injections of 2.0 μl of the dipeptide solution were injected into 350 μl of enzyme solution (wild-type SmDPP7). The wild-type SmDPP7 concentration was 25 µM and the concentration of each dipeptide solution was 250 µM in reaction buffer. Each injection was performed for 4 seconds with an interval of 150 sec between injections. The dissociation constant (Kd) and heats of binding (ΔH) were obtained using MICROCAL PEAQ-ITC Analysis software (Malvern, UK). Gibbs free energy (ΔG) values were calculated according to ΔG = −RT ln Ka = RT ln Kd (Ka = 1/ Kd, association constants).
Figures 3 to 6, S2 and S4 were produced using the program UCSF Chimera52. Figure S3 was created using Adobe Illustrator (Adobe Systems Inc., San Jose, USA).