Artificial mini-proteins able to target catalytic sites of matrix metalloproteinases (MMPs) were designed using a functional motif grafting approach. The motif corresponded to the 4 N-terminal residues of TIMP-2, a broad-spectrum natural protein inhibitor of MMPs. Scaffolds able to reproduce the functional topology of this motif as described in the TIMP-2/MMP-14 complex were obtained by exhaustive screening of the Protein Data Bank (PDB) using the STAMPS software (Search for Three-dimensional Atom Motif in Protein Structure). Ten artificial protein binders satisfying all topologic, steric and electrostatic criteria applied for selection were produced for experimental evaluation. These binders targeted catalytic sites of MMPs with affinities ranging from 450 nM and 590 μM prior to optimization. The crystal structures of two artificial binders in complex with the catalytic domain of MMP-12 showed that the intermolecular interactions established by the functional motif in these artificial binders corresponded to those found in the TIMP-2/MMP-14 complex, albeit with some differences in their geometry. Molecular dynamics simulations of the 10 binders in complex with MMP-14 suggested that these scaffolds could allow reproducing in part the native intermolecular interactions, but some differences in geometry and stability could contribute to the lower affinity of the artificial protein binders as compared to the natural one. Nevertheless, these results show that the in silico design method used can provide sets of starting protein binders targeting a specific binding site with a good rate of success. This approach could constitute the first step of an efficient hybrid computational-experimental protein binder design approach.
