Computational design of matrix metalloprotenaise-9 (MMP-9) resistant to auto-cleavage

Abstract

Matrix metalloproteinase-9 (MMP-9) is an endopeptidase that remodels the extracellular matrix. MMP-9 has been implicated in several diseases including neurodegeneration, arthritis, cardiovascular diseases, fibrosis and several types of cancer, resulting in a high demand for MMP-9 inhibitors for therapeutic purposes. For such drug design efforts, large amounts of MMP-9 are required. Yet, the catalytic domain of MMP-9 (MMP-9Cat) is an intrinsically unstable enzyme that tends to auto-cleave within minutes, making it difficult to use in drug design experiments and other biophysical studies. We set our goal to design MMP-9Cat variant that is active but stable to auto-cleavage. For this purpose, we first identified potential auto-cleavage sites on MMP-9Cat using mass spectroscopy and then eliminated the auto-cleavage site by predicting mutations that minimize auto-cleavage potential without reducing enzyme stability. Four computationally designed MMP-9Cat variants were experimentally constructed and evaluated for auto-cleavage and enzyme activity. Our best variant, Des2, with 2 mutations, was as active as the wild-type enzyme but did not exhibit auto-cleavage after 7 days of incubation at 37°C. This MMP-9Cat variant, with an identical with MMP-9Cat WT active site, is an ideal candidate for drug design experiments targeting MMP-9 and enzyme crystallization experiments. The developed strategy for MMP-9CAT stabilization could be applied to redesign other proteases to improve their stability for various biotechnological applications.

Keywords: MMP-9; auto-cleavage; auto-degradation; enzyme engineering; matrix metalloproteases; protein engineering.

Figure 1.. MMP-9 auto-degradation site is a partially exposed loop.

(A) SDS–PAGE gel analysis of MMP-9_Cat WT at 25°C showing the full-length (blue arrow) and degradation products (red arrows). (B) The relative auto-cleavage preference of MMP-9_Cat WT. Cleavage preference was calculated from the LS/MS count of each peptide product contributing to a given cleavage site at a specific sequence position. The X-axis represents the amino acid residue number of the full MMP-9 sequence. The C-terminal cleavage positions with the highest peptide spectral match (PSM) were after positions 405 and 417. (C) Structure of MMP-9_Cat WT (orange) in cartoon representation, showing the catalytic Zn²⁺ and the predicted auto-cleavage site in the partially exposed loop (colored in blue). Amino acids at the interface with the MMP protein inhibitor TIMP-2 are colored in green. Zn²⁺ ligating residues are colored in cyan.

Figure 2.. Design of non-degrading MMP-9_Cat variants.

(A) Saturation mutagenesis scan of the cleavage loop displaying the auto-cleavage score according to the Procleave webserver [30]. (B) Saturation mutagenesis scan of the cleavage loop displaying the Rosetta energy. (C) Alignment of eight representative MMPs in the region of the predicted auto-cleavage site. Alignment of all 23 human MMPs is shown in Supplementary Figure S1. D414, N414, T415, and I418 are frequently present in MMP-9 homologs and confer lower predicted auto-cleavage scores compared with that of the MMP-9_Cat WT sequence and exhibit favorable energy according to Rosetta. (D) A model of the Des2 loop according to AlphaFold (light gray) superimposed onto the structures of MMP-3 (blue, PDB: 4DP3) and MMP-9 (orange, PDB: 5TH9). The mutated N414 and T415 in Des2 have the same rotamers as in the homologous MMP-3 structure as well as the same neighboring amino acids and most of the same rotamers among neighboring positions, increasing the confidence in Des2 foldability.

Figure 3.. Evaluation of MMP-9_Cat WT and Des2 stability and enzyme activity at different time points.

(A) Silver-stained SDS–PAGE analysis showing full-length and cleavage products of MMP-9_Cat WT (top) and Des2 (bottom) after 37°C at several time intervals, T = 0, T = 1 h (T_1hr), T = 1 day (T_1d), T = 3 days (T_3d), and T = 7 days (T_7d). (B) Enzyme activity of MMP-9_Cat WT (top) and Des2 (bottom) at T₀, T_3d, and T_7d using a fluorogenic peptide substrate. Substrate digestion was monitored by the appearance of a fluorescence signal at 395 nm. (C) Inhibition of MMP-9_Cat WT (top) and Des2 (bottom) at T₀, T_3d, and T_7d using prinomastat as an inhibitor, using the same fluorogenic substrate as in (B). K_i^app was determined by fitting the data to Morrison's equation (see equation (1) in Methods) [26].