Engineering of tissue inhibitor of metalloproteinases TIMP-1 for fine discrimination between closely related stromelysins MMP-3 and MMP-10

Abstract

Matrix metalloproteinases (MMPs) have long been known as key drivers in the development and progression of diseases, including cancer and neurodegenerative, cardiovascular, and many other inflammatory and degenerative diseases, making them attractive potential drug targets. Engineering selective inhibitors based upon tissue inhibitors of metalloproteinases (TIMPs), endogenous human proteins that tightly yet nonspecifically bind to the family of MMPs, represents a promising new avenue for therapeutic development. Here, we used a counter-selective screening strategy for directed evolution of yeast-displayed human TIMP-1 to obtain TIMP-1 variants highly selective for the inhibition of MMP-3 in preference over MMP-10. As MMP-3 and MMP-10 are the most similar MMPs in sequence, structure, and function, our results thus clearly demonstrate the capability for engineering full-length TIMP proteins to be highly selective MMP inhibitors. We show using protein crystal structures and models of MMP-3-selective TIMP-1 variants bound to MMP-3 and counter-target MMP-10 how structural alterations within the N-terminal and C-terminal TIMP-1 domains create new favorable and selective interactions with MMP-3 and disrupt unique interactions with MMP-10. While our MMP-3-selective inhibitors may be of interest for future investigation in diseases where this enzyme drives pathology, our platform and screening strategy can be employed for developing selective inhibitors of additional MMPs implicated as therapeutic targets in disease. Matrix metalloproteinases (MMPs) have long been known as key drivers in the development and progression of diseases, including cancer and neurodegenerative, cardiovascular, and many other inflammatory and degenerative diseases, making them attractive potential drug targets. Engineering selective inhibitors based upon tissue inhibitors of metalloproteinases (TIMPs), endogenous human proteins that tightly yet nonspecifically bind to the family of MMPs, represents a promising new avenue for therapeutic development. Here, we used a counter-selective screening strategy for directed evolution of yeast-displayed human TIMP-1 to obtain TIMP-1 variants highly selective for the inhibition of MMP-3 in preference over MMP-10. As MMP-3 and MMP-10 are the most similar MMPs in sequence, structure, and function, our results thus clearly demonstrate the capability for engineering full-length TIMP proteins to be highly selective MMP inhibitors. We show using protein crystal structures and models of MMP-3-selective TIMP-1 variants bound to MMP-3 and counter-target MMP-10 how structural alterations within the N-terminal and C-terminal TIMP-1 domains create new favorable and selective interactions with MMP-3 and disrupt unique interactions with MMP-10. While our MMP-3-selective inhibitors may be of interest for future investigation in diseases where this enzyme drives pathology, our platform and screening strategy can be employed for developing selective inhibitors of additional MMPs implicated as therapeutic targets in disease. Matrix metalloproteinases (MMPs) are a family of zinc endopeptidases (23 in humans) with important functions in extracellular matrix remodeling and degradation and play central roles in development of many human diseases, including cancer, pulmonary diseases, and neurological disorders (1Radisky E.S. Raeeszadeh-Sarmazdeh M. Radisky D.C. Therapeutic potential of matrix metalloproteinase inhibition in breast cancer.J. Cell. Biochem. 2017; 118: 3531-3548Google Scholar, 2Radisky E.S. Radisky D.C. Matrix metalloproteinases as breast cancer drivers and therapeutic targets.Front. Biosci. (Landmark Ed.). 2015; 20: 1144-1163Google Scholar, 3Hu J. Van den Steen P.E. Sang Q.X. Opdenakker G. Matrix metalloproteinase inhibitors as therapy for inflammatory and vascular diseases.Nat. Rev. Drug Discov. 2007; 6: 480-498Google Scholar, 4Murphy G. Nagase H. Progress in matrix metalloproteinase research.Mol. Aspects Med. 2008; 29: 290-308Google Scholar, 5Kessenbrock K. Plaks V. Werb Z. Matrix metalloproteinases: Regulators of the tumor microenvironment.Cell. 2010; 141: 52-67Google Scholar, 6Fingleton B. MMPs as therapeutic targets-still a viable option?.Semin. Cell Dev. Biol. 2008; 19: 61-68Google Scholar, 7Raeeszadeh-Sarmazdeh M. Do L.D. Hritz B.G. Metalloproteinases and their inhibitors: Potential for the development of new therapeutics.Cells. 2020; 9: 1313Google Scholar). Several past efforts to develop efficient therapeutics against MMPs failed in the late stages of clinical trials, in part because of nonselective inhibition of many MMPs and related metalloproteinases (6Fingleton B. MMPs as therapeutic targets-still a viable option?.Semin. Cell Dev. Biol. 2008; 19: 61-68Google Scholar, 8Coussens L.M. Fingleton B. Matrisian L.M. Matrix metalloproteinase inhibitors and cancer: Trials and tribulations.Science. 2002; 295: 2387-2392Google Scholar). Lack of selectivity was a critical issue because broad-spectrum metalloproteinase inhibition caused serious dose-limiting musculoskeletal toxicity, and in some cases, it proved to be counterproductive by inhibiting MMPs with protective roles in disease alongside those that drive pathology (9Martin M.D. Matrisian L.M. The other side of MMPs: Protective roles in tumor progression.Cancer Metastasis Rev. 2007; 26: 717-724Google Scholar, 10Decock J. Thirkettle S. Wagstaff L. Edwards D.R. Matrix metalloproteinases: Protective roles in cancer.J. Cell. Mol. Med. 2011; 15: 1254-1265Google Scholar, 11Overall C.M. Kleifeld O. Tumour microenvironment - opinion: Validating matrix metalloproteinases as drug targets and anti-targets for cancer therapy.Nat. Rev. Cancer. 2006; 6: 227-239Google Scholar). Development of highly selective MMP inhibitors has been extremely challenging. However, new strategies for developing selective MMP inhibitors may lead to much-needed molecular probes for better defining individual MMP functions in biology and disease, and ultimately, to more effective therapeutics targeting the specific MMPs that are underlying causes of disease. Protein engineering using directed evolution offers a robust method to define the sequence–structure–function relationships of proteins, and directed evolution was previously used to develop new proteins with novel based the of proteins M. H. and Rev. Scholar, H. evolution of and using Cell Scholar). MMPs are in by tissue inhibitors of metalloproteinases J. Radisky E.S. inhibitors of metalloproteinases of of and and proteins for inhibition of many MMPs, we and have that their can be protein engineering using and directed has engineering the N-terminal of and that MMP capability K. Nagase H. S. Van K. and of the of human tissue of in Scholar, G. M. The N-terminal of tissue of metalloproteinases metalloproteinase Scholar). of the N-terminal of was used as a directed evolution platform to develop inhibitors of and with and selectivity V. G. J. M. Radisky E.S. Development of and inhibitors of matrix metalloproteinase and directed Biol. 2017; Scholar, J. V. Radisky E.S. a matrix metalloproteinase family a specific of and Scholar, G. V. the with for cancer Biol. Scholar, V. Radisky E.S. engineering of variants that and in the 9: Scholar). we that full-length as for engineering inhibitors with evolution of full-length domains that the that in N-terminal and C-terminal domains to the MMP-3 M. B. Radisky D.C. Radisky E.S. evolution of the metalloproteinase TIMP-1 that and C-terminal domains in matrix metalloproteinase Biol. Scholar). was to MMP-3 to with TIMP-1 and MMP-3 with inhibition in the M. B. Radisky D.C. Radisky E.S. evolution of the metalloproteinase TIMP-1 that and C-terminal domains in matrix metalloproteinase Biol. Scholar). the we developing and the potential of our platform for selective TIMP-1 variants of related MMP the MMP-3 and MMP-10 are the most similar in structure, and and thus a of our strategy to develop highly selective J. J. Radisky D.C. Radisky E.S. Matrix with tissue inhibitors of metalloproteinases TIMP-1 and and crystal Biol. Scholar, J. Radisky E.S. Matrix and define interactions and interactions in Scholar). MMP-3 a therapeutic MMP-3 has a in and tumor progression of and in it a of to M.D. Werb Z. The matrix metalloproteinase as a tumor 19: Scholar, M.D. B. Werb Z. The Scholar, D.C. H. C.M. Werb Z. and and Scholar, J. Radisky E.S. Radisky D.C. Matrix metalloproteinase of a key of cancer Med. Scholar, L. J. Radisky E.S. and tissue alterations that Scholar). a therapeutic in other inflammatory and degenerative diseases, including where MMP-3 in with M. J. J. MMP-3 and and progression of in where MMP-3 in with pulmonary and drives C.M. S. The of matrix pulmonary in a of J. Scholar, C.M. Radisky D.C. The of matrix in Rev. Med. Scholar, C.M. L. J. J. Radisky D.C. G. Matrix metalloproteinase a of pulmonary J. 2011; and in the where of MMP-3 with L. J. of and in J. Cell Mol. Biol. Scholar, J. Matrix in inflammatory tissue Mol. 2007; Scholar, J. Matrix metalloproteinases and matrix metalloproteinase inhibitors in 2006; Scholar). Here, we to TIMP variants of MMP-3 MMP-10 to of selective MMP and inhibition and as a of the of our TIMP engineering counter-selective we a TIMP-1 using and variants that bind to MMP-3 in the of of the MMP-10. TIMP-1 of screening to in selectivity MMP-3 MMP-10 to of TIMP-1 variants with MMP-3 selectivity key that are for in Protein crystal structures of the TIMP-1 variants in MMP-3 selectivity in with the and of structures with models of with the counter-target MMP-10 the structural alterations for the We previously MMP-3 a of human TIMP-1 the M. B. Radisky D.C. Radisky E.S. evolution of the metalloproteinase TIMP-1 that and C-terminal domains in matrix metalloproteinase Biol. Scholar). the for MMP-3 TIMP-1 variants in MMP-10 as to the TIMP-1 selectivity MMP-3 we a counter-selective strategy to our of TIMP-1 to selective for MMP-3 over MMP-10. of TIMP-1 variants within the N-terminal and within the C-terminal that are the with of to and our counter-selective screening the yeast-displayed of TIMP-1 was for MMP-3 in the of MMP-10 was with and protein using by The TIMP-1 of of screening the of to for variants with selectivity TIMP-1 variants with have bound to the of in preference to and thus have and been the screening of counter-selective the of TIMP-1 selectivity to with TIMP-1 similar to and The TIMP-1 variants of counter-selective screening to with human and of the the most TIMP-1 variants in the to within the of as the TIMP-1 by of their in the and and in the the most The been the MMP-3 variants that previously M. B. Radisky D.C. Radisky E.S. evolution of the metalloproteinase TIMP-1 that and C-terminal domains in matrix metalloproteinase Biol. that the has a key in of TIMP-1 of the and to and TIMP-1 using of the TIMP-1 in and the selectivity and and thus for The for the most selective variants and and to The and proteins for and inhibition using a in the of of the inhibitors to to and of TIMP-1 proteins for inhibition of and using and variants inhibition of that be in the and variants inhibition of that be in the for TIMP-1 and variants inhibition of that be in the in a new for We to of for selectivity for and The using TIMP-1 proteins are with the results and that the TIMP-1 variants the selectivity by the TIMP-1 a of J. Van den Steen P.E. Opdenakker G. and molecular biology of matrix The Rev. Biochem. Mol. Biol. we inhibition of the MMP-3-selective TIMP-1 the TIMP-1 with was using the because of the inhibition of the selective variants demonstrate that MMP-3-selective TIMP-1 variants and with with the potential for counter-selective screening with MMP to to other MMPs as in the better the of TIMP-1 selective to we of the most selective counter-selective and in with We the and structures by molecular and against to of and and are in The structures have been in the Protein with and the protein of TIMP-1 and as previously for MMP-3 with TIMP-1 K. M. K. Nagase H. K. H. of inhibition of the human matrix metalloproteinase by and other TIMP-1 variants with M. B. Radisky D.C. Radisky E.S. evolution of the metalloproteinase TIMP-1 that and C-terminal domains in matrix metalloproteinase Biol. Scholar). structural of the of the variants for inhibition of MMP-3 in preference to we the of TIMP-1 variants with using in and molecular based upon our previously crystal of the of with TIMP-1 J. J. Radisky D.C. Radisky E.S. Matrix with tissue inhibitors of metalloproteinases TIMP-1 and and crystal Biol. and the and models of the as and used in of Protein are in in a new are in The most the and variants was a previously TIMP-1 variants with MMP-3 and for structural have been previously M. B. Radisky D.C. Radisky E.S. evolution of the metalloproteinase TIMP-1 that and C-terminal domains in matrix metalloproteinase Biol. Scholar). the new crystal structures of with and we that this in the MMP-3 and the TIMP-1 While TIMP-1 and MMP-3 are because of the this more by of a in TIMP-1 and side the to with the protein The favorable and interactions with the with the of this for have that the TIMP-1 and inhibition M. B. Radisky D.C. Radisky E.S. evolution of the metalloproteinase TIMP-1 that and C-terminal domains in matrix metalloproteinase Biol. Scholar). The with MMP-10. of in in the TIMP-1 in to to the it in the crystal of TIMP-1 and to in MMP-10 with and variants As in the with the TIMP-1 results in of favorable interactions with of MMP-10 as in this new interactions the it that by the the of TIMP-1 variants with MMP-3 to selectivity in to The of this in selectivity by the of selectivity of the in the of the and TIMP-1 variants in the in with of the TIMP-1 interactions with MMP-3 with MMP-10 that are in the MMP-3 with the and MMP-3 and the in MMP-10 are and with the TIMP-1 side of and The in the the the with the with the side the favorable of with and in the with MMP-10 and of with in the favorable interactions with the with MMP-3 in this our crystal of bound to for the side was the of of interactions to this side in a of potential of in MMP-3 and MMP-10 the of potential interactions with this The of the may be for MMP-3 and MMP-10 with of interactions with the of TIMP-1 with a with the of MMP-3 the with this with the and with TIMP-1 of to the in MMP the of with the in this because of of the of by with the of The may have a MMP-10 in this the results in of the and the in this to be by interactions Protein crystal structures and structural models of with and how in the TIMP-1 C-terminal selectivity of The the within the and the the of MMP-3 and MMP-10 in this part of the and interactions in the of with of a with and this by the the in interactions and interactions of with MMP-3 are to a the of TIMP-1 with in to the MMP and the N-terminal of the with MMP-10 and and with TIMP-1 and and favorable interactions are to be in the with the the of the to the MMP in this by of with and are in because of the and the of with MMP-10 with of the and are to be for MMP-3 for MMP-10 the of in and in structures of the TIMP-1 with in the as a of the of the it the as this to with However, the in the may selectivity for MMP-3 over MMP-10. The TIMP-1 with a in this the TIMP-1 and MMP-3 side The of the TIMP-1 in the results in of this with new interactions of the side with MMP-3 and and TIMP N-terminal the MMP-10 by the in the are to interactions with the where the TIMP-1 and MMP-10 the in the of may in of MMP-3 inhibition Protein engineering using a for and selectivity of protein and has been used to as Raeeszadeh-Sarmazdeh M. Engineering by Biochem. Scholar, for screening 15: Scholar). Protein engineering directed evolution can be used to protein sequence–structure–function relationships and the of and interactions B. in protein and Rev. Mol. Cell Biol. 20: Scholar). Here, we used a of protein directed and structural to variants of TIMP-1 of related MMPs and to the and structural for the We used a counter-selective screening strategy to TIMP-1 variants with selectivity for MMP-3 with the most related MMP in of protein structure, and our of for the capability of full-length to as for development of highly selective MMP inhibitors and the of our platform and counter-selective screening strategy to MMPs play roles in many diseases, and of specific MMPs roles in disease are many in other MMPs can have in the disease (9Martin M.D. Matrisian L.M. The other side of MMPs: Protective roles in tumor progression.Cancer Metastasis Rev. 2007; 26: 717-724Google Scholar, 10Decock J. Thirkettle S. Wagstaff L. Edwards D.R. Matrix metalloproteinases: Protective roles in cancer.J. Cell. Mol. Med. 2011; 15: 1254-1265Google Scholar, B. Matrix metalloproteinases as of inflammatory Mol. Cell 2017; Scholar). the of broad-spectrum MMP inhibitors in clinical for cancer and MMPs alongside those drivers of pathology, and because of side (1Radisky E.S. Raeeszadeh-Sarmazdeh M. Radisky D.C. Therapeutic potential of matrix metalloproteinase inhibition in breast cancer.J. Cell. Biochem. 2017; 118: 3531-3548Google Scholar, 6Fingleton B. MMPs as therapeutic targets-still a viable option?.Semin. Cell Dev. Biol. 2008; 19: 61-68Google Scholar, 7Raeeszadeh-Sarmazdeh M. Do L.D. Hritz B.G. Metalloproteinases and their inhibitors: Potential for the development of new therapeutics.Cells. 2020; 9: 1313Google Scholar, 8Coussens L.M. Fingleton B. Matrisian L.M. Matrix metalloproteinase inhibitors and cancer: Trials and tribulations.Science. 2002; 295: 2387-2392Google Scholar, targets in Metalloproteinases and their Drug 2007; Scholar). more selective inhibitors are selective inhibitors have been to develop for the MMPs because of structural in the of the the many of this enzyme with inhibitors can to because with proteins Here, we have of the protein of full-length has to metalloproteinase domains a of the the MMP the to the potential to J. Radisky E.S. Matrix and define interactions and interactions in Scholar). Several other strategies have to selective MMP inhibitors by screening of TIMP of by of with human protein and for MMP-10 the MMP-10 in this that MMP-10 of to be selective for MMP-10 over MMP-3 inhibition of matrix metalloproteinase with a Biol. 2020; 295: Scholar). and of domains have been to selective inhibitors of as we have previously (1Radisky E.S. Raeeszadeh-Sarmazdeh M. Radisky D.C. Therapeutic potential of matrix metalloproteinase inhibition in breast cancer.J. Cell. Biochem. 2017; 118: 3531-3548Google Scholar). human with that of was using a and to MMP-10 in preference to inhibitors S. Scholar). of the of developing are that many against the MMP bind in and MMP and it may be a of the that using a to selective MMP the based a that to bind to the of MMPs in a Here, the selectivity MMPs, most employed the N-terminal of as a for engineering selective inhibitors of and V. G. J. M. Radisky E.S. Development of and inhibitors of matrix metalloproteinase and directed Biol. 2017; Scholar, J. V. Radisky E.S. a matrix metalloproteinase family a specific of and Scholar). to the that in the enzyme this the TIMP the MMP a for evolution of the by full-length TIMP-1 as a we of the TIMP C-terminal the and the that additional for selectivity by additional the MMP we employed a screening for our for the most similar of the MMP family and structural that in of N-terminal and C-terminal domains to in in of structural and in alterations to TIMP-1 are in the with our results are to have been by of other our counter-selective screening directed the evolution of TIMP-1 selectivity in have been by the selective variants screening MMP-3 in our M. B. Radisky D.C. Radisky E.S. evolution of the metalloproteinase TIMP-1 that and C-terminal domains in matrix metalloproteinase Biol. Scholar). the screening employed selective inhibitors of the most related MMPs, that it be of inhibitors selective for other MMP-3 has been as a potential therapeutic for inflammatory and degenerative we have that MMP-3 in with where it of to to C.M. Radisky D.C. The of matrix in Rev. Med. Scholar, C.M. L. J. J. Radisky D.C. G. Matrix metalloproteinase a of pulmonary J. 2011; Scholar). are against pulmonary that selective MMP-3 inhibition effective therapeutic strategy to MMP-3 in inflammatory of and where it a and therapeutic the that diseases L. J. of and in J. Cell Mol. Biol. Scholar, J. Matrix in inflammatory tissue Mol. 2007; Scholar, J. Matrix metalloproteinases and matrix metalloproteinase inhibitors in 2006; Scholar, S. with in the 2020; Scholar, S. H. S. by the in the of 141: Scholar, V. C.M. Lack of matrix metalloproteinase in models of the pulmonary inflammatory in in Scholar). in MMP-3 have been to be in models L. J. of and in J. Cell Mol. Biol. Scholar, J. Matrix in inflammatory tissue Mol. 2007; the therapeutic potential of selective MMP-3 inhibition for of Several other MMPs are in diseases are in the including some that to have protective disease and thus may in the development of MMP-3 the inflammatory by MMP-10 by and against tissue B. Matrix metalloproteinases as of inflammatory Mol. Cell 2017; Scholar, by Scholar, matrix metalloproteinases in with 2007; Scholar). protective are in caused by of long S. and and to long J. 2017; Scholar). of in disease have and in models of L. Matrix metalloproteinases as therapeutic targets for pulmonary J. Cell Mol. Biol. 2015; Scholar). models of by by was to have protective and more in models Lack of matrix J. Cell Mol. 2008; Scholar, Protective of matrix in 2007; Scholar). MMPs that protective roles in disease against the development of G. V. S. Matrix a key of in and J. Med. in of matrix 2010; and of B. G. J. of matrix metalloproteinase in extracellular matrix in pulmonary Scholar, S. M. J. M. M. of pulmonary in of J. Cell Mol. and and of B. Matrix metalloproteinases as of inflammatory Mol. Cell 2017; Scholar, M. Van J. J. in Scholar). this we TIMP-1 variants that capability MMP-3 inhibition of MMP-10 and inhibition of thus the selectivity for therapeutic for we have that the full-length TIMP-1 protein can be for the most similar of We a promising protein engineering strategy that for of MMP-3-selective inhibitors as as selective inhibitors of other MMPs of clinical interest as therapeutic targets. structural the alterations in interactions the that are for in our results the of with directed evolution using and counter-selective protein engineering can be to protein therapeutic development based other enzyme selectivity to for TIMP-1 variants the of protein the the J. B. H. of with a Mol. Biol. as previously M. B. Radisky D.C. Radisky E.S. evolution of the metalloproteinase TIMP-1 that and C-terminal domains in matrix metalloproteinase Biol. Scholar). the of full-length human TIMP-1 and TIMP-1 the was MMP proteins the C-terminal in using a was a of Nagase K. K. Nagase H. of human metalloproteinase that the N-terminal in and with tissue of metalloproteinase Scholar). was and and as previously M. B. Radisky D.C. Radisky E.S. evolution of the metalloproteinase TIMP-1 that and C-terminal domains in matrix metalloproteinase Biol. Scholar, K. K. Nagase H. of human metalloproteinase that the N-terminal in and with tissue of metalloproteinase Scholar). The protein was and by by in the of the and of a Scholar, K. G. Nagase H. of of tissue by matrix metalloproteinase 29: Scholar, H. K. G. of the of matrix metalloproteinase by and 29: to our M. B. Radisky D.C. Radisky E.S. evolution of the metalloproteinase TIMP-1 that and C-terminal domains in matrix metalloproteinase Biol. Scholar). was and using similar to those for as we have previously J. J. Radisky D.C. Radisky E.S. Matrix with tissue inhibitors of metalloproteinases TIMP-1 and and crystal Biol. Scholar). MMP-10 was for and the was and degradation by J. J. Radisky D.C. Radisky E.S. Matrix with tissue inhibitors of metalloproteinases TIMP-1 and and crystal Biol. Scholar). for of human was by of the a human to using and The was with and and with and The protein was by and as previously M.D. of the human a matrix Biol. 2002; Scholar). was by using a of in and for and using the to the with of in to and for The MMPs using and for of using the to the of full-length human TIMP-1 and TIMP-1 proteins, the protein was used L. B. B. B. of of the human tissue of metalloproteinases Scholar). The TIMP-1 the using and the using and TIMP-1 and TIMP-1 variants in human and by and as previously M. B. Radisky D.C. Radisky E.S. evolution of the metalloproteinase TIMP-1 that and C-terminal domains in matrix metalloproteinase Biol. Scholar). TIMP-1 protein was using against protein of known as previously M. B. Radisky D.C. Radisky E.S. evolution of the metalloproteinase TIMP-1 that and C-terminal domains in matrix metalloproteinase Biol. Scholar). TIMP-1 proteins with to the to proteins for TIMP-1 and proteins by using a using and The highly TIMP-1 and using with molecular of of the of TIMP-1 variants with within as in and our and have been previously in M. B. Radisky D.C. Radisky E.S. evolution of the metalloproteinase TIMP-1 that and C-terminal domains in matrix metalloproteinase Biol. Scholar). to of and of and with a of and in with and for and in and for and with and with for and in a for in with and and protein was by in and The was to of screening and using a of in the of was additional for a of to of was with and was the individual using the using with and of the and was for and using the and for using by and by using similar to those previously M. B. Radisky D.C. Radisky E.S. evolution of the metalloproteinase TIMP-1 that and C-terminal domains in matrix metalloproteinase Biol. Scholar). The TIMP-1 variants with to in for by and in for and for and with and in of and and using of TIMP-1 and TIMP-1 variants and using a method for as we have previously V. G. J. M. Radisky E.S. Development of and inhibitors of matrix metalloproteinase and directed Biol. 2017; Scholar, M. B. Radisky D.C. Radisky E.S. evolution of the metalloproteinase TIMP-1 that and C-terminal domains in matrix metalloproteinase Biol. Scholar). was with to in and for the was to using the and the was to the a of and was with and using a for and the of the of the as TIMP and by to inhibition of the inhibition of by where enzyme in the of enzyme in the of enzyme the and inhibition by using inhibition are with and the using a of for for and for as in in our TIMP-1 variants and protein a of and protein to to to the by the protein with a B. of for Biol. Scholar). protein in of the protein in of the TIMP-1 protein in a and over a in using TIMP-1 structures that to and The with L. O. new for with Biol. 2011; for and with and of Biol. 2006; and are and the Biol. for and The and efforts to their the and within the efforts and to a of and this was stages of and crystal structures of the protein of variants by molecular using the with Biol. 2010; structures of protein by molecular with Biol. 2007; Scholar, M.D. 2007; The previously of human TIMP-1 the and was used as a molecular was used for The with alterations in K. for molecular Biol. Scholar). stages of a within a of the method J. of a protein in of Biol. 2006; Scholar). a of in MMP-3 and for for a of for MMP-3 and for As within to and was highly in and N-terminal to and C-terminal to of and to and C-terminal to of in and C-terminal to of and to and C-terminal to of in using of TIMP-1 for J. J. Radisky D.C. Radisky E.S. Matrix with tissue inhibitors of metalloproteinases TIMP-1 and and crystal Biol. using that we have in previously O. Radisky E.S. a for in Biol. Scholar, M. B. S. Radisky E.S. engineering of human inhibitors and Biol. Scholar, J. S. Radisky E.S. C.M. H. progression using therapy for Scholar). as of side with and of TIMP-1 and using the TIMP-1 the crystal K. M. K. Nagase H. K. H. of inhibition of the human matrix metalloproteinase by as a in with the most models G. G. the of models with 2002; where and with was with protein the potential models a of and The was to to and based was to the to the was to for The crystal structures of have been in the and The that have of interest with the of this We in the for with of TIMP-1 The are in part by of The a of of M. and S. M. and S. M. M. and S. M. M. B. and S. M. M. S. and B. S. B. and S. M. M. and S. M. M. and S. M. M. B. G. and S. M. and S. M. G. and S. M. and S. G. and S. was by of and S. G. and S. and of G. and M. in part by of The the of the and the of the of

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