Autor: |
Hezi Hayun, Matt Coban, Ashok Kumar Bhagat, Eden Ozer, Lital Alfonta, Thomas R. Caulfield, Evette S. Radisky, Niv Papo |
Jazyk: |
angličtina |
Rok vydání: |
2023 |
Předmět: |
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Zdroj: |
Scientific Reports, Vol 13, Iss 1, Pp 1-14 (2023) |
Druh dokumentu: |
article |
ISSN: |
2045-2322 |
DOI: |
10.1038/s41598-023-32019-3 |
Popis: |
Abstract 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. |
Databáze: |
Directory of Open Access Journals |
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