Generation of N-TIMP2-DOPA and N-TIMP2-HqAla variants. To choose the positions for the incorporation of L-DOPA or HqAla within N-TIMP2, the crystal structures of TIMP-2-MMP-14 (PDB 1BUV) and TIMP-2-MMP-10 (PDB 4ILW) complexes were analyzed in PyMol (The PyMOL Molecular Graphics System, Version 1.1 Schrödinger, LLC.). The gene encoding for N-TIMP2 (positions 1-127) was cloned into a pMECS expression vector (a kind gift from Dr. Serge Muyldermans, Vrije University Brussels, Brussels, Belgium) using restriction free PCR (RF-PCR)46, which served as a template for introducing the TAG point mutation in the selected positions (S2, Y36, S69, A70 and L100) of N-TIMP2. All plasmid sequences were verified by Sanger sequencing (Genetics Unit, NIBN, Ben-Gurion University of the Negev, Israel).
The following primers were used in the RF-PCR to generate the gene for each clone:
Position | Primers |
S2 | FWD: 5'-GCCGGCCATGGCCTGCTAGTGCTCCCCGGTGCACC-3' REV: 5'-GGTGCACCGGGGAGCACTAGCAGGCCATGGCCGGC-3' |
Y36 | FWD: 5'-CTCTGGAAACGACATTTAGGGCAACCCTATCAAG-3' REV: 5'-CTTGATAGGGTTGCCCTAAATGTCGTTTCCAGAG-3' |
S69 | FWD: 5'-GTTTATCTACACGGCCCCCTCCTAGGCAGTGTGTGGGGTC-3' REV: 5'-GACCCCACACACTGCCTAGGAGGGGGCCGTGTAGATAAAC-3' |
A70 | FWD: 5'- GTTTATCTACACGGCCCCCTCCTCGTAGGTGTGTGGGGTCTC-3' REV: 5'- GAGACCCCACACACCTACGAGGAGGGGGCCGTGTAGATAAAC-3' |
L100 | FWD: 5'- CAAGATGCACATCACCTAGTGTGACTTCATCGTG-3' REV: 5'- CACGATGAAGTCACACTAGGTGATGTGCATCTTG-3' |
Production and purification of N-TIMP2-DOPA and N-TIMP2-HqAla variants. pMECS plasmids encoding the different clones of N-TIMP2 were co-transformed into E. coli strain WK6, with either one of the following plasmids: (i) pAC-DHPheRS6TRN plasmid, containing the DHPheRS/Mj-tRNACUA genes for L-DOPA incorporation47 or (ii) pEVOL-HqAlaRS plasmid, containing the HqAlaRS/Mj-tRNACUATyr genes for HqAla incorporation32, both for TAG suppression. The bacteria were grown with stirring at 200 rpm at 37°C in TB medium (17 mM KH2PO4, 94 mM K2HPO4, 12 g/L peptone, 24 g/L yeast extract, 0.4% glycerol) containing: 100 µg/ml ampicillin for N-TIMP2; 100 µg/ml ampicillin, 10 µg/ml tetracycline and 5 mM L-DOPA (Sigma-Aldrich, Israel, added at OD600 = 0.4) for N-TIMP2-DOPA clones; or 100 µg/ml ampicillin, 50 µg/ml chloramphenicol and 3 mM HqAla (BLD Pharmatech Ltd., China) for N-TIMP2-HqAla clones. At an OD600 of 0.4, 0.2% arabinose (Mercury, Rosh Ha’ayin, Israel) was added to N-TIMP2-HqAla clones (for PylRS induction), and at an OD600 of 0.6–0.9 the expression of all proteins (N-TIMP2, N-TIMP2-DOPA and N-TIMP2-HqAla variants) was induced by addition of 1 mM IPTG (Sigma-Aldrich, Israel) to the medium and temperature adjustment to 28°C (for N-TIMP2 and N-TIMP2-HqAla) or 22°C (and under anaerobic conditions for N-TIMP2-DOPA) and overnight incubation. The cell pellet obtained by centrifugation at 4800 g for 30 min of 500 mL of bacterial cell culture (with a final OD600 < 20)] was subjected to osmotic shock using 9 mL of TES buffer (500 mM sucrose, 200 mM Tris-HCl, 0.5 mM EDTA, pH 8) for 2 h at 4°C and 200 rpm, followed by incubation overnight in 18 mL of TES buffer (diluted 1:4 in doubly distilled water) to yield soluble proteins (i.e., periplasmic extracts). The proteins were further purified using affinity chromatography on Ni-NTA gravitational beads (Invitrogen, CA, USA) and eluted with 0.5 M imidazole in phosphate buffered saline (PBS). The eluted fraction was dialyzed against PBS, and the size and purity of the proteins were evaluated by using SDS − PAGE gel electrophoresis and mass spectrometry (MALDI-TOF Reflex-IV, Ilse Katz Institute for Nanoscale Science and Technology, BGU, Israel). For LC-MS/MS analysis of N-TIMP2-DOPA and N-TIMP2-HqAla variants, excised SDS-PAGE gel bands were denatured, reduced, alkylated and digested by trypsin. Digested peptides were then subjected to tandem mass spectrometry analysis by the LTQ-Orbitrap XL ETD system (Ilse Katz Institute for Nanoscale Science and Technology Shared Resource Facility, BGU, Israel). Protein concentrations were determined by UV-Vis absorbance at 280 nm, using a NanoDrop Spectrophotometer (Thermo Fisher Scientific), with an extinction coefficient (ε280) of 13,500 M− 1·cm− 1 for all N-TIMP2 proteins.
MMP inhibition studies. The human MMP-9 catalytic domain (MMP-9CAT, residues 107–215, 391– 443) and the human MMP-14 catalytic domain (MMP-14CAT, residues 112–292) were purified as described previously35. The inhibition constants (Ki) of N-TIMP2 proteins against pre-activated MMP-2 (MMP-2ACT; pre-activated in vitro using 4-aminophenylmercuric acetate (APMA), Sigma-Aldrich, Israel), MMP-9CAT and MMP-14CAT were determined as previously described35. The inhibition of the catalytic activity of MMP-2ACT (0.6 nM), MMP-9CAT (3 nM) and MMP-14CAT (1 nM) was measured against the chromogenic MMP substrate, Ac-Pro-Leu-Gly-[2-mercapto-4-methyl-pentanoyl]-Leu-Gly-OC2H5 (ENZO Life Sciences, USA). MMPs were incubated with 0–25 nM of N-TIMP2, N-TIMP2-DOPA and N-TIMP2-HqAla variants in assay buffer (50 mM HEPES, 10 mM CaCl2, 0.05% Brij-35, 1 mM DTNB, pH 7.5) for 1 h at 37°C. Thereafter, the chromogenic substrate, at a final concentration of 100 µM, was added to the reaction, and the absorbance was monitored at 412 nm using a Synergy 2 plate reader (BioTek, USA) at 37°C for 30–60 min at 1-min intervals. Data analysis was performed according to the manufacturer's instructions and fitted by multiple regressions to Morrison’s tight binding inhibition equation (Eq. 1), the classic competitive inhibition equation for tight binding, by using Prism (GraphPad Software). Mean values of Ki ± standard error of the mean (SEM) were obtained from three independent experiments. Statistical analysis was performed using Student’s t-test.
$$\frac{{V}_{i}}{{V}_{0}}=1-\frac{\left(\left[E\right]+\left[I\right]+{K}_{i}^{app}\right)-\sqrt{(\left[E\right]+\left[I\right]+{K}_{i}^{app}{)}^{2}-4[E\left]\right[I]}}{2\left[E\right]} (Eq.1)$$
where Vi - enzyme velocity in the presence of inhibitor, V0 - enzyme velocity in the absence of inhibitor, E - enzyme concentration, I - inhibitor concentration, S - substrate concentration, KM - Michaelis-Menten constant, and Kiapp - the apparent inhibition constant, which is given by Eq. 2:
$${K}_{i}^{app}={K}_{i}\left(1+\frac{\left[S\right]}{{K}_{M}}\right) (Eq.2)$$
where Ki - inhibition constant.
MMP/TIMP modeling. The models of MMP-2/N-TIMP2 and MMP-9/N-TIMP2 complexes were constructed by superposing the MMP-2 chain of 3AYU.pdb48 or the MMP-9 chain of 4JIJ.pdb49 onto the MMP-14 chain of 1BUV37. The C-terminal domain of TIMP-2 was deleted and modified catalytic residues were back-mutated to the wild-type sequence.
Mutations with L-DOPA or HqAla and incorporation sites were chosen using PyMOL50 and a database of NCAAs SwissSidechain51. The rotamer of the mutated sidechain was chosen so as to minimize clashes. Complexes of the wild type and the variants were then subjected to identical molecular dynamics simulation relaxation protocols using YASARA52, i.e., 500 ps of energy minimization with the YASARA2 forcefield under explicit solvation in a cubic simulation box extending 10Å from the protein. The relaxation was carried out with the following parameters: temperature 298 K, solvent density 0.997 g/L, pH 7.4, timestep 2 fs, frames saved every 25 ps. The global energy of each resulting frame was plotted to ensure a plateau of convergence to verify that relaxation was complete. After relaxation, representative frames were chosen for structural comparisons.