Affinity- and specificity-enhancing mutations are frequent in multispecific interactions between TIMP2 and MMPs

Abstract

Multispecific proteins play a major role in controlling various functions such as signaling, regulation of transcription/translation, and immune response. Hence, a thorough understanding of the atomic-level principles governing multispecific interactions is important not only for the advancement of basic science but also for applied research such as drug design. Here, we study evolution of an exemplary multispecific protein, a Tissue Inhibitor of Matrix Metalloproteinases 2 (TIMP2) that binds with comparable affinities to more than twenty-six members of the Matrix Metalloproteinase (MMP) and the related ADAMs families. We postulate that due to its multispecific nature, TIMP2 is not optimized to bind to any individual MMP type, but rather embodies a compromise required for interactions with all MMPs. To explore this hypothesis, we perform computational saturation mutagenesis of the TIMP2 binding interface and predict changes in free energy of binding to eight MMP targets. Computational results reveal the non-optimality of the TIMP2 binding interface for all studied proteins, identifying many affinity-enhancing mutations at multiple positions. Several TIMP2 point mutants predicted to enhance binding affinity and/or binding specificity towards MMP14 were selected for experimental verification. Experimental results show high abundance of affinity-enhancing mutations in TIMP2, with some point mutations producing more than ten-fold improvement in affinity to MMP14. Our computational and experimental results collaboratively demonstrate that the TIMP2 sequence lies far from the fitness maximum when interacting with its target enzymes. This non-optimality of the binding interface and high potential for improvement might characterize all proteins evolved for binding to multiple targets.

Figure 1. Structural Analysis of MMP/N-TIMP interactions.

(A) MMP-14 interacting with N-TIMP2 (PDB ID 1BUV). MMP14 is shown in red, N-TIMP2 – in cyan. The catalytic Zn²⁺ ion is shown as a blue sphere. The interacting regions on N-TIMP2 are colored in blue and their boundaries are numbered. (B) N-TIMP2 binding interface on MMPs. Superposition of backbones for MMP14 (red) and MMP9 (green). The regions that contact N-TIMP2 are shown in purple for MMP14 and in blue for MMP9. MMP14 and MMP9 exhibit 59% sequence identity and 70% sequence similarity in the binding interface region and exhibit Cα RMSD of 0.66 Å.

Figure 2. Computational binding landscapes for the N-TIMP2/MMP14 (A) and the N-TIMP2/MMP9 (B) interactions.

N-TIMP2 binding interface positions with their WT identity are displayed on the left, the mutated amino acid identity is on the top. Calculated ΔΔG_bind value for each mutation is color coded: ΔΔG_bind ≥1.5 kcal/mol: red, 0.5 kcal/mol ≤ ΔΔG_bind <1.5 kcal/mol: yellow, 0.5 kcal/mol ≤ ΔΔG_bind < −0.5 kcal/mol: green and ΔΔG_bind ≤ −0.5 kcal/mol: blue. Mutations where negative ΔΔG_bind is coupled to significant destabilization of a single chain (>2 kcal/mol) are shown in gray. For these mutations we cannot reliably predict ΔΔG_bind. Positions are divided into tolerant, semi-tolerant and non-tolerant denoted by T, S, and N on the right of the figure.

Figure 3. Binding affinity measurements between N-TIMP2 mutants and MMP14/MMP9.

(A) Enzyme activity assay is performed in the presence and the absence of N-TIMP2 and the fraction of enzyme activity is plotted vs. log of N-TIMP2 concentration. The curves were fitted to equation 1 to determine K_d of the interaction. (B) ΔΔG_bind calculated from the K_d measured in (A) for each studied N-TIMP2 mutation when interacting with MMP14 (black bars) and with MMP9 (gray bars).

Figure 4. Per-position ΔΔG_bind predictions for N-TIMP2 interacting with eight studied MMPs.

Color coding is the same as in Figure 2. Mutations with the standard deviation greater than one are marked by stars (see Methods for calculation of the standard deviation).

Figure 5. Structure-based sequence alignment of the N-TIMP2 contacting residues for eight MMPs under study.

Negatively charged amino acids are colored blue while positively charged residues are colored red. Shannon entropy that represents sequence variability at a particular position is shown below. N-TIMP2 positions with the entropy greater or equal to 1.6 are underlined. Positions on N-TIMP2 that contact these high-entropy positions (among those explored in this work) are shown on top of the table above the corresponding MMP position.

Figure 6. Structural analysis of the affinity-enhancing mutations.

H97R (A); I35K (B); S68Y (C). The left panel shows WT interaction and the right panel shows interaction after mutation. N-TIMP2 is shown in blue and MMP14 is shown in green. Mutated residues and surrounding residues are shown as sticks and hydrogen bonds and salt bridges are shown as yellow dots.