Utilizing genetic code expansion to modify N-TIMP2 specificity towards MMP-2, MMP-9, and MMP-14

Matrix metalloproteinases (MMPs) regulate the degradation of extracellular matrix (ECM) components in biological processes. MMP activity is controlled by natural tissue inhibitors of metalloproteinases (TIMPs) that non-selectively inhibit the function of multiple MMPs via interaction with the MMPs' Zn2+-containing catalytic pocket. Recent studies suggest that TIMPs engineered to confer MMP specificity could be exploited for therapeutic purposes, but obtaining specific TIMP-2 inhibitors has proved to be challenging. Here, in an effort to improve MMP specificity, we incorporated the metal-binding non-canonical amino acids (NCAAs), 3,4-dihydroxyphenylalanine (L-DOPA) and (8-hydroxyquinolin-3-yl)alanine (HqAla), into the MMP-inhibitory N-terminal domain of TIMP2 (N-TIMP2) at selected positions that interact with the catalytic Zn2+ ion (S2, S69, A70, L100) or with a structural Ca2+ ion (Y36). Evaluation of the inhibitory potency of the NCAA-containing variants towards MMP-2, MMP-9 and MMP-14 in vitro revealed that most showed a significant loss of inhibitory activity towards MMP-14, but not towards MMP-2 and MMP-9, resulting in increased specificity towards the latter proteases. Substitutions at S69 conferred the best improvement in selectivity for both L-DOPA and HqAla variants. Molecular modeling provided an indication of how MMP-2 and MMP-9 are better able to accommodate the bulky NCAA substituents at the intermolecular interface with N-TIMP2. The models also showed that, rather than coordinating to Zn2+, the NCAA side chains formed stabilizing polar interactions at the intermolecular interface with MMP-2 and MMP-9. Our findings illustrate how incorporation of NCAAs can be used to probe—and possibly exploit—differential tolerance for substitution within closely related protein–protein complexes as a means to improve specificity.

www.nature.com/scientificreports/ the steric factor that comes into play when natural amino acids are replaced with bulky NCAAs. In applying the chosen strategy, we targeted several positions in N-TIMP2 that are not only located close to the MMP's Zn 2+ and Ca 2+ ions when the two proteins interact (to potentially take advantage of the metal-binding capability of the NCAA) but also interact with MMP subsites that are not highly conserved. The mutated N-TIMP2 subsites included residues S2, Y36, S69, A70 and L100 (Fig. 1B). To incorporate the NCAAs into N-TIMP2, suppression of the amber codon was performed by introducing (using PCR) a TAG codon at each of the selected positions in N-TIMP2 (one in each clone).

Incorporation of NCAAs into N-TIMP2. For amber suppression and incorporation of L-DOPA or
HqAla into different positions in N-TIMP2, we co-transformed Escherichia coli strain WK6 with two plasmids, as described in the Methods section. Wild-type N-TIMP2, N-TIMP2-DOPA and N-TIMP2-HqAla variants were produced in bacteria and purified using affinity chromatography ( Fig. 2 and Supplementary  Fig. S1). The production yields were as follows: N-TIMP2-4.

MMP inhibition by N-TIMP2-DOPA and N-TIMP2-HqAla variants.
To assess the potency of N-TIMP2-DOPA and N-TIMP2-HqAla variants in inhibiting MMP activity, an MMP activity assay was performed, in which activated MMP-2 (designated MMP-2 ACT ) and the catalytic domains of MMP-9 and MMP-14 (designated MMP-9 CAT and MMP-14 CAT , respectively) were incubated with various concentrations of N-TIMP2 variants (0-25 nM) and an MMP chromogenic substrate, and the cleavage of the substrate as a function of time was monitored. To determine the inhibition constants (K i ), the slope of each catalytic reaction was calculated and fitted to Morrison's tight binding equation (Fig. 5, Table 1). None of the variants containing NCAAs showed improved inhibition toward any of the MMPs tested. However, as intended, the substitutions diminished the inhibitory activity toward the different MMPs (although to widely varying extents), resulting in an enhancement of specificity. Whereas N-TIMP2 bound MMP-14 CAT with a K i of 0.71 nM (Table 1), a finding consistent with previous studies 35,36 , nearly all the N-TIMP2-DOPA and N-TIMP2-HqAla mutants lost their inhibitory activity towards MMP-14 CAT by more than one order of magnitude compared to N-TIMP2. In contrast, most of the N-TIMP2-DOPA and N-TIMP2-HqAla mutants retained their inhibition potency towards MMP-2 ACT and MMP-9 CAT . Notably, N-TIMP2-Y36HqAla, N-TIMP2-S69HqAla and N-TIMP2-S69DOPA exhibited the best inhibition of MMP-2 ACT and MMP-9 CAT , with only a one-to twofold diminishment of potency compared to N-TIMP2. The MMP inhibition assays suggested that the selected positions within N-TIMP2 exhibit different degrees of tolerance for mutagenesis-induced by either L-DOPA or HqAla-that may differentially impact their specificity toward the different MMPs.
Incorporation of L-DOPA and HqAla into N-TIMP2 increases its specificity towards MMP-9 CAT and MMP-2 CAT . Our MMP inhibition studies revealed different degrees of tolerance for mutations of the selected N-TIMP2 positions in terms of retention of inhibition specificity for different MMPs. We observed that the inhibitory activity of most N-TIMP2-DOPA and N-TIMP2-HqAla variants was retained for MMP-2 ACT and MMP-9 CAT but lost for MMP-14 CAT . To compare the degree of preference of each N-TIMP2 variant for MMP-2 ACT or MMP-9 CAT relative to MMP-14 CAT , we calculated an inhibition specificity ratio as the ratio between the affinity (K i ) of each N-TIMP2 variant for MMP-14 CAT divided by the Ki for MMP-2 ACT or MMP-9 CAT ( Table 2). All N-TIMP2 variants, except for N-TIMP2-S2HqAla and N-TIMP2-A70HqAla, showed increased specificity for both MMP-2 ACT and MMP-9 CAT vs. MMP-14 CAT , in comparison to N-TIMP2. Notably, N-TIMP2-S69HqAla and N-TIMP2-S69DOPA showed the strongest specificity for MMP-2 ACT , with inhibition specificity ratios of 18.96 and 46.61, respectively, and for MMP-9 CAT , with inhibition specificity ratios of 25.59 and 45.85, respectively.

Molecular modeling of MMPs bound to N-TIMP2-DOPA and N-TIMP2-HqAla variants.
To investigate the structural basis for the selectivity enhancements of N-TIMP2-S69HqAla and N-TIMP2-S69DOPA variants toward MMP-2 and MMP-9 in preference to MMP-14, we used molecular modeling approaches. The crystal structure of MMP-14 CAT bound to TIMP2 (1BUV) 37 was used as a template. Superposition on the template of experimental structures for MMP-2 CAT and MMP-9 CAT showed minimal global differences in the MMP catalytic domain and facile accommodation of TIMP2 with few or no clashes. The TIMP2 chain in each complex was truncated to include only the residues of N-TIMP2, and then Ser69 was mutated in silico to HqAla or L-DOPA. Next, all nine model complexes (N-TIMP2, N-TIMP2-S69HqAla, or N-TIMP2-S69DOPA bound to       www.nature.com/scientificreports/ and potentially forming an intramolecular H-bond with Ser75 of N-TIMP2-S69DOPA (Fig. 8D). Overall, substitution of L-DOPA69 conferred novel strong interactions with either MMP-2 or MMP-9 but did not stabilize the interaction with MMP-14.

Discussion
This study presents a strategy for improving inhibitor specificity for individual enzymes within a homologous family, via mutagenesis of selected residues that participate in inhibitor-enzyme interactions. MMP family members share a common multi-domain structure, but exhibit differences in the subsites of their catalytic domains that lead to a variety of specificities for different substrates and inhibitors. As MMPs are zinc-and calcium-dependent enzymes, a potential strategy for inhibiting MMPs could be to target these cations to disrupt their coordination by MMP residues. Since MMP subsites differ in their size and shape, the size and volume of amino acids within ligands and potential inhibitors, such as TIMP family members, will affect their affinity and selectivity towards different MMPs. In the current study, two approaches were combined to manipulate the affinity and selectivity of TIMP-2 for different MMPs. In the first, the NCAAs L-DOPA and HqAla, which possess metal-binding capacity and have large side-chains, were incorporated into N-TIMP2 at selected positions with the potential to interact strongly with the Zn 2+ and Ca 2+ ions in the MMP catalytic domain. In the second approach, L-DOPA and HqAla were strategically placed to interact differently with distinctive subsites of different MMPs and hence to confer selectivity in binding and inhibition potencies. Incorporation of the bulky, polar, metal-binding NCAAs L-DOPA and HqAla at selected positions in N-TIMP2 did not improve its inhibition potency towards the examined MMPs-observations that highlight the important role played by the selected HqAla is represented in magenta, lime, and orange, respectively, with potential H-bonds indicated with black dashed lines. HqAla appears to interact more closely with MMP-2 and MMP-9, whereas a steric clash with Phe204 precludes close interaction with MMP-14. www.nature.com/scientificreports/ N-TIMP2 positions in TIMP/MMP interactions. Nevertheless, the findings that the inhibitory activity towards MMP-14 CAT was significantly impaired for all N-TIMP2-DOPA and N-TIMP2-HqAla variants, but was retained for MMP-2 ACT and MMP-9 CAT for most variants, emphasize the potential of these positions to alter N-TIMP2 selectivity towards different MMPs. An examination of the different substitution positions within N-TIMP-2 provides explanations for our findings. Position Ser2 of N-TIMP2, which is located in the N-terminal segment (residues Cys1-Pro5), is involved in the direct interaction between N-TIMP2 and the MMP catalytic pocket 13 and would therefore not be tolerant to mutation to a bulkier residue, as we observed for the substitutions with either L-DOPA or HqAla and as was previously shown for other substitutions, such as S2E 38 and S2D 39 , at this position. Our results are thus in line with previous findings, as Ser2 substitutions with both NCAAs led to significantly decreased affinities towards all tested MMPs, with a K i fold > 13, except for the retention of MMP-9 CAT inhibition by N-TIMP2-S2DOPA. Notably, N-TIMP2-S2HqAla showed a decrease of > 2 orders of magnitude in affinity towards all three MMPs, suggesting that the bulky side chain of HqAla, compared to Ser, interferes with MMP binding, probably due to steric hindrance.
Position Y36 of N-TIMP2 is located on the tip of the AB loop (residues D30-K41) and interacts with MMP-14 at a site that is distant from the catalytic pocket 40,41 . It was previously found that different mutations (Y36F, Y36G and Y36W) led to decreased affinities towards MMP-14, exhibiting significant higher inhibition constants (K i -fold www.nature.com/scientificreports/ of 15-103) and lower association rate constants (K on -fold of 36-180), compared to N-TIMP2, whereas their affinities towards MMP-2 were maintained 42 . Our results extend these previous findings, as N-TIMP2-Y36HqAla showed a 6.64-fold decreased affinity towards MMP-14 CAT , whereas it maintained its inhibitory potency towards both MMP-2 ACT and MMP-9 CAT , compared to N-TIMP2. Positions S69 and A70 are located on the surface-exposed C-connector loop of N-TIMP2 (residues Ser68-Cys72 41 ) that interacts with the MMP catalytic domain. The crystal structures of the TIMP2/MMP-13 and TIMP2/MMP-14 complexes suggest that this loop may have different affinities towards each MMP, as MMP-14 forms favorable contacts with it and MMP-13 repulses it, via residues A66 and V71 in TIMP2 and the bulky Y176 residue in MMP-13 compared to the smaller T190 in MMP-14 43 . Our structural modeling corroborates the distinct nature of the interactions occurring between this loop and the different MMPs examined here, due, in particular, to the bulkier Phe204 residue of MMP-14, at a position where MMP-2 and MMP-9 possess the smaller residues Ala196 and Pro193, respectively. This difference appears to explain much of the decreased affinity towards MMP-14 CAT for both the bulky L-DOPA and HqAla substitutions at position 69, in comparison to Ser at position 69 of N-TIMP2. Previous computational analysis of binding landscapes for the interactions between N-TIMP2 with MMP-9 CAT , in which selected positions in N-TIMP2 were randomly mutated, has shown position S69 to be tolerant to randomization 44 , which may further explain the retention of inhibition potency towards MMP-9 CAT upon substitution with either L-DOPA or HqAla at this position.
Position L100 on N-TIMP2 is located on the EF loop between two beta-strands, sE and sF 37 . In this loop -previously identified as one of the N-TIMP2 binding sites for MMP-3 45 and MMP-14-L100 is in close proximity to the MMP-14 catalytic Zn 2+ ion 44 , which may explain the observed reduction (by ~ 20-fold) in affinity towards MMP-14 CAT upon substitution of L100 with L-DOPA.
Our molecular modeling provides an indication of how local sequence differences between the MMPs are likely to lead to differential susceptibility to inhibition by N-TIMP2 variants with insertion of the bulky HqAla or L-DOPA in position 69. Specifically, MMP-14 is much less susceptible to inhibition by the variants probably as a consequence of steric hindrance between HqAla or L-DOPA and Phe204 of MMP-14, a position occupied by smaller residues in MMP-2 and MMP-9. Overall, this work suggests that different TIMP/MMP complexes have differential ability to tolerate the introduction of bulky residues within interface positions. In the absence of crystal structures, molecular dynamic simulations can be used to elucidate the molecular basis for these differences in selectivity.
In summary, in this study, the properties of HqAla and L-DOPA that shaped their differential interactions with the different MMPs can be attributed to their bulkiness and ability to form polar interactions, rather than to their known metal-binding capability. In the future, however, our approach might be extended to take advantage of metal coordination by metal-binding NCAAs at the interface of N-TIMP2 with its MMP targets (perhaps by choosing other metal-binding NCAAs and other positions within N-TIMP2) in order to improve N-TIMP2 potency towards different MMPs. This approach may also be used to optimize and modulate binding interactions of other protein complexes involving other metalloproteins.  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 (Sequencing Unit, NIBN, Ben-Gurion University of the Negev, Israel).

Materials and methods
The following primers were used in the RF-PCR to generate the gene for each clone: where V i -enzyme velocity in the presence of inhibitor, V 0 -enzyme velocity in the absence of inhibitor, Eenzyme concentration, I-inhibitor concentration, and K i app -the apparent inhibition constant, which is given by Eq. (2): where K i -inhibition constant, S-substrate concentration, and K M -Michaelis-Menten constant.

Position Primers
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.pdb 48 or the MMP-9 chain of 4JIJ.pdb 49 onto the MMP-14 chain of 1BUV 37 . The C-terminal domain of TIMP-2 was deleted and modified catalytic residues were backmutated to the wild-type sequence.
Mutations with L-DOPA or HqAla and incorporation sites were chosen using PyMOL 50 and a database of NCAAs SwissSidechain 51 . 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 YASARA 52 , i.e., 500 ps of energy minimization with the YASARA2 forcefield under explicit solvation in a cubic simulation box extending 10 Å 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.

Data availability
The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.