Thermodynamic Basis of Selectivity in the Interactions of Tissue Inhibitors of Metalloproteinases N-domains with Matrix Metalloproteinases-1, -3, and -14

Abstract

The four tissue inhibitors of metalloproteinases (TIMPs) are potent inhibitors of the many matrixins (MMPs), except that TIMP1 weakly inhibits some MMPs, including MMP14. The broad-spectrum inhibition of MMPs by TIMPs and their N-domains (NTIMPs) is consistent with the previous isothermal titration calorimetric finding that their interactions are entropy-driven but differ in contributions from solvent and conformational entropy (⌬S solv , ⌬S conf ), estimated using heat capacity changes (⌬C p ). Selective engineered NTIMPs have potential applications for treating MMP-related diseases, including cancer and cardiomyopathy. Here we report isothermal titration calorimetric studies of the effects of selectivitymodifying mutations in NTIMP1 and NTIMP2 on the thermodynamics of their interactions with MMP1, MMP3, and MMP14. The weak inhibition of MMP14 by NTIMP1 reflects a large conformational entropy penalty for binding. The T98L mutation, peripheral to the NTIMP1 reactive site, enhances binding by increasing ⌬S solv but also reduces ⌬S conf . However, the same mutation increases NTIMP1 binding to MMP3 in an interaction that has an unusual positive ⌬C p . This indicates a decrease in solvent entropy compensated by increased conformational entropy, possibly reflecting interactions involving alternative conformers. The NTIMP2 mutant, S2D/S4A is a selective MMP1 inhibitor through electrostatic effects of a unique MMP-1 arginine. Asp-2 increases reactive site polarity, reducing ⌬C p , but increases conformational entropy to maintain strong binding to MMP1. There is a strong negative correlation between ⌬S solv and ⌬S conf for all characterized interactions, but the data for each MMP have characteristic ranges, reflecting intrinsic differences in the structures and dynamics of their free and inhibitor-bound forms.Isothermal titration calorimetry can dissect the sources of the free energy changes for protein interactions with other macromolecules and ligands (1). Measurements of the heat released or absorbed during the titration of a protein with a binding partner can quantify the enthalpy of binding (⌬H), association constant K a , and stoichiometry (N) (1), allowing the calculation of the changes in free energy (⌬G) and entropy (⌬S) of binding. Titrations in buffers with different enthalpies of ionization quantify ionization changes linked to binding, whereas the heat capacity change for binding, ⌬C p , measured as the temperature dependence of ⌬H, can be used to estimate the change in solvation entropy on binding (⌬S solv ). These parameters can be correlated with the character of the interaction interface and differences in dynamics and solvation between the free and bound conformer populations (2-4).Previously, we used isothermal titration calorimetry to elucidate the thermodynamic profiles of interactions between the N-terminal inhibitory domains of tissue inhibitors of metalloproteinases-1 and -2 (NT1 and NT2) 3 and the catalytic domains of matrix metalloproteinases -1 and -3 (MMP1c and MMP3c) (5, 6). TIMP1 and TIMP2 ...

Extracellular Phosphorylation of TIMP-2 by Secreted c-Src Tyrosine Kinase Controls MMP-2 Activity

“…Our findings that TIMP-2:proMMP-2 interaction is coupled to TIMP-2 tyrosine phosphorylation provides an explanation. TIMP-2 Y90 phosphorylation could support conformational changes exposing interaction surfaces that facilitate favorable and stronger complex formation with proMMP-2 in vivo ( Batra et al., 2013 , Sharabi et al., 2014 , Zou et al., 2016 ). Expression of the phosphomimetic TIMP-2 TE in SYF cells was sufficient to restore TIMP-2 interaction with proMMP-2 ( Figure 3 B).…”

Therapeutic Potential of Matrix Metalloproteinase Inhibition in Breast Cancer

“…However, given the large number of residues involved in the TIMP-MMP binding interaction, a challenge comes in identifying the best combination of mutations to optimize a TIMP for selectivity toward an individual MMP. The use of structural knowledge to predict the best mutations is made problematic by the high degree of backbone flexibility evidenced in some regions of the TIMP interface [Wu et al, 2000;Grossman et al, 2010;Batra et al, 2013;Batra and Radisky, 2014], and by the complex and unpredictable nature of entropic contributions to MMP-TIMP binding energies [Arumugam et al, 2003;Wu et al, 2011;Zou et al, 2016]. Fortunately, we are not limited to approaches strictly dependent on the ability to predict advantageous mutations, as directed evolution approaches have now shown substantial promise for development of selective TIMPs.…”

Potential mechanisms of action of celastrol against rheumatoid arthritis: Transcriptomic and proteomic analysis

“…(B) Cartilage oligomeric matrix protein (3FBY) [ 44 ]. (C) Matrix metalloprotease 1 (1SU3) [ 45 ]. (D) Laminin subunit gamma 1 (5XAU) [ 46 ].…”