Expression of the protease inhibitor ecotin and its co-crystallization with trypsin

McGrath, Mary E.; Erpel, Thorsten; Browner, Michelle F.; Fletterick, Robert J.

doi:10.1016/0022-2836(91)90199-g

Cited by 29 publications

(17 citation statements)

References 27 publications

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“…Different methods have been used to better understand ecotin binding to serine-proteases including site-directed mutagenesis, enzyme kinetics, and X-ray crystallography [18][19][20][21]. These techniques have been used to study ecotin structure-activity relationships, as well as the binding mechanism of ecotin to its target enzyme.…”

Section: Resultsmentioning

confidence: 99%

Engineering Ecotin for Identifying Proteins with a Trypsin Fold

Sathler

Craik

Takeuchi

et al. 2009

Appl Biochem Biotechnol

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Ecotin is a bidentate, fold-specific inhibitor of mammalian serine-proteases produced by Escherichia coli. This molecule may be engineered to increase and/or change its affinity and specificity providing significant biotechnological potential. Since ecotin binds tightly to serine proteases of the trypsin fold, it may help to identify the role of these enzymes in different biological processes. In this work, we tested ecotin variants as an affinity purification reagent for identifying enzymes in samples of tumor progression and mammary gland involution. Initially, we used a commercial source of urokinase-type plasminogen activator (u-PA) that remained fully active after elution from an affinity column of the ecotin variant (M84R, M85R). We then successfully identified u-PA from more complex mixtures including lysates from a prostate cancer cell line and involuting mouse mammary glands. Interestingly, a membrane-type serine protease 1 was isolated from the Triton X-100-solubilized PC-3 cell lysates, and surprisingly, haptoglobin, a serine-protease homolog protein, was also identified in mammary gland lysates and in blood. Haptoglobin does not prevent ecotin inhibition of u-PA, but it may act as a carrier within blood when ecotin is used in vivo. Finally, this affinity purification matrix was also able to identify a thrombin-like enzyme from snake venom using an ecotin variant directed against thrombin. Overall, the ecotin variants acted as robust tools for the isolation and characterization of proteins with a trypsin fold. Thus, they may assist in the understanding of the role of these serine proteases and homologous proteins in different biological processes.

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Section: Resultsmentioning

confidence: 99%

Engineering Ecotin for Identifying Proteins with a Trypsin Fold

Sathler

Craik

Takeuchi

et al. 2009

Appl Biochem Biotechnol

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“…EcotinP was generated using ecotinD as a template. The methods for the expression and purification of ecotin mutants were similar to those described previously (24). Minor modifications included transformation of the ecotin plasmids into the E. coli strain BL21 and further purification of the ecotin mutants using a POROS HQ (Applied Biosystems) column (4.6 ϫ 50 mm) mounted on a BioCAD 700E in buffer containing 5 mM MgCl 2 and 10 mM Tris-HCl, pH 7.8.…”

Section: Methodsmentioning

confidence: 99%

Crystal Structures of the FXIa Catalytic Domain in Complex with Ecotin Mutants Reveal Substrate-like Interactions

Jin¹,

Pandey²,

Babine³

et al. 2005

Journal of Biological Chemistry

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Thrombosis can lead to life-threatening conditions such as acute myocardial infarction, pulmonary embolism, and stroke. Although commonly used anti-coagulant drugs, such as low molecular weight heparin and warfarin, are effective, they carry a significant risk of inducing severe bleeding complications, and there is a need for safer drugs. Activated Factor XI (FXIa) is a key enzyme in the amplification phase of the coagulation cascade. Anti-human FXI antibody significantly reduces thrombus growth in a baboon thrombosis model without bleeding problems (Gruber, A., and Hanson, S. R. (2003) Blood 102, 953-955). Therefore, FXIa is a potential target for anti-thrombosis therapy. To determine the structure of FXIa, we derived a recombinant catalytic domain of FXI, consisting of residues 370 -607 (rhFXI 370 -607 ). Here we report the first crystal structure of rhFXI 370 -607 in complex with a substitution mutant of ecotin, a panserine protease protein inhibitor secreted by Escherichia coli, to 2.2 Å resolution. The presence of ecotin not only assisted in the crystallization of the enzyme but also revealed unique structural features in the active site of FXIa. Subsequently, the sequence from P5 to P2 in ecotin was mutated to the FXIa substrate sequence, and the structures of the rhFXI 370 -607 -ecotin mutant complexes were determined. These structures provide us with an understanding of substrate binding interactions of FXIa, the structural information essential for the structure-based design of FXIa-selective inhibitors.

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“…Ecotin was cocrystallized with a well-characterized mutant of rat anionic trypsin (McGrath et al, 1991a), as well as by itself (Shin et al, 1993). Because no suitable structural homolog of ecotin existed to allow molecular replacement, the strategy for structure determination was to use trypsin as the search model.…”

Section: ~~mentioning

confidence: 99%

Ecotin: Lessons on survival in a protease‐filled world

1995

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Ecotin, an Escherichia coli periplasmic protein of 142 amino acids, has been shown to be a potent inhibitor of a group of homologous serine proteases with widely differing substrate recognition. It is highly effective against a number of enzymes, including both pancreatic and neutrophil-derived elastases, chymotrypsin, trypsin, factor Xa, and kallikrein. Recent structural and functional studies on ecotin and its interactions with different serine proteases have clarified these initial observations and revealed the remarkable features of this protein in inhibiting a strikingly large subset of the chymotrypsin family of serine proteases. The structures of the ecotinserine protease complexes provide the first examples of protein-protein recognition where the concept of specificity of interactions needs to be reexamined. The binding sites show a fluidity of protein contacts derived from ecotin's innate flexibility in fitting itself to proteases while strongly interfering with their function.

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Expression of the protease inhibitor ecotin and its co-crystallization with trypsin

Cited by 29 publications

References 27 publications

Engineering Ecotin for Identifying Proteins with a Trypsin Fold

Engineering Ecotin for Identifying Proteins with a Trypsin Fold

Crystal Structures of the FXIa Catalytic Domain in Complex with Ecotin Mutants Reveal Substrate-like Interactions

Ecotin: Lessons on survival in a protease‐filled world

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