title: Structural characterization of thyroglobulin type-1 domains of equistatin.
abstract: Equistatin is a protein composed of three thyroglobulin type-1 domains. It inhibits papain-like cysteine proteinases and the aspartic proteinase, cathepsin D. To determine the structural basis for this inhibition we cloned and expressed the separated domains (eq d-1, eq d-2, eq d-3) in Pichia pastoris. Kinetic constants for the interaction of eq d-1 with papain and that of eq d-2 with cathepsin D are of similar order (subnanomolar) and are comparable to the constants obtained for full-length equistatin. The target proteinase for the third domain remains unknown. Thus, we demonstrate here that thyroglobulin type-1 motifs per se are able to support specific structural features that enable them to inhibit proteases from different classes. The overall conformation of three domains in equistatin is such that the interaction of domains 1 or 2 with their respective target enzymes is not hindered sterically by either domain. In addition, we show that the interaction of eq d-2 with cathepsin D results in conformational changes, which is not the case for the eq d-1/papain interaction.
authors.affiliation: Department of Biochemistry and Molecular Biology, J. Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia.
authors.forename: Katja
authors.initials: K
authors.lastname: Galesa
authors.forename: Roger
authors.initials: R
authors.lastname: Pain
authors.forename: Maarten A
authors.initials: MA
authors.lastname: Jongsma
authors.forename: Vito
authors.initials: V
authors.lastname: Turk
authors.forename: Brigita
authors.initials: B
authors.lastname: Lenarcic
mesh_terms: D002402:Cathepsin D / Q000037:antagonists & inhibitors; D002942:Circular Dichroism; D003001:Cloning, Molecular; D007700:Kinetics; D010206:Papain / Q000378:metabolism; D010843:Pichia; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013954:Thyroglobulin / Q000737:chemistry
chemical_list: D011506:Proteins; D011994:Recombinant Proteins; C106356:equistatin; D013954:Thyroglobulin; D010206:Papain; D002402:Cathepsin D