TY - JOUR
T1 - Utilizing genetic code expansion to modify N-TIMP2 specificity towards MMP-2, MMP-9, and MMP-14
AU - Hayun, Hezi
AU - Coban, Matt
AU - Bhagat, Ashok Kumar
AU - Ozer, Eden
AU - Alfonta, Lital
AU - Caulfield, Thomas R.
AU - Radisky, Evette S.
AU - Papo, Niv
N1 - Funding Information:
The authors thank Yuval Pinkert for her assistance with the SDS gel preparation, Dr. Mark Karpasas for his professional assistance with the MS experiments that were performed at the Ilse Katz Institute for Nanoscale Science and Technology, BGU. They also thank Ms. Inez Mureinik for careful reading of the manuscript. This work was supported by the Israel Cancer Research Fund (ICRF) (contract grant number: 846497) to N.P., the US-Israel Binational Science Foundation (BSF) grant (contract grant number: 2019303) to N.P. and E.S.R., and by U.S. National Institutes of Health grants R01 GM132100 and R01 CA258274 to E.S.R. and R01 GM144393 to T.R.C, E.S.R. and N.P.
Funding Information:
The authors thank Yuval Pinkert for her assistance with the SDS gel preparation, Dr. Mark Karpasas for his professional assistance with the MS experiments that were performed at the Ilse Katz Institute for Nanoscale Science and Technology, BGU. They also thank Ms. Inez Mureinik for careful reading of the manuscript. This work was supported by the Israel Cancer Research Fund (ICRF) (contract grant number: 846497) to N.P., the US-Israel Binational Science Foundation (BSF) grant (contract grant number: 2019303) to N.P. and E.S.R., and by U.S. National Institutes of Health grants R01 GM132100 and R01 CA258274 to E.S.R. and R01 GM144393 to T.R.C, E.S.R. and N.P.
Publisher Copyright:
© 2023, The Author(s).
PY - 2023/12/1
Y1 - 2023/12/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85151315788&partnerID=8YFLogxK
U2 - 10.1038/s41598-023-32019-3
DO - 10.1038/s41598-023-32019-3
M3 - Article
C2 - 36997589
AN - SCOPUS:85151315788
SN - 2045-2322
VL - 13
JO - Scientific Reports
JF - Scientific Reports
IS - 1
M1 - 5186
ER -