Birgt die Tertiärstruktur des Corticoide-bindenden Globulins (CBG) einen einzigartigen Mechanismus für die Steroidfreisetzung im Zielgewebe?
Zusammenfassung der Projektergebnisse
The 1.9 Å crystal structure of native rat corticoid-binding globulin (CBG) showed for the first time in molecular detail how steroids are recognized and transported in the blood by CBG. We were able to identify and delineate the steroid-binding pocket on ?-sheet B of the SERPIN fold together with the major binding determinants. Our results also suggest an allosteric mechanism that couples the protease-triggered insertion of the reactive centre loop (RCL) into ?-sheet A of CBG to the ejection of the steroid from its binding pocket. In this mechanism helix D plays a central role, as a link between the conformation of the RCL and the steroid-binding pocket. This hypothesis was further supported by the publication of the structure of an engineered cleaved human CBG by a competing group. Overall, the allosteric coupling mechanism resembles the regulatory mechanism for antithrombin activation by heparin. Starting out with our structure of rat CBG and the published structures of the human CBG-antitrypsin chimera we sought to validate the postulated protease-triggered insertion of the RCL for wild-type human CBG. Together with our collaborators, we examined single residues in human CBG which influence steroid-binding affinity before and after elastase cleavage. The introduction of bulkier or sterically more hindered residues in parts of the RCL important for insertion yielded CBG variants that unlike wild-type CBG did not lose their steroid-binding affinity after elastase cleavage clearly revealing a direct link between the SERPIN-typical mechanism of RCL insertion and the modulation of the steroid-binding affinity in human CBG. While a ligand-bound structure of rat CBG, two cleaved structures of human CBG with different ligands and a cleaved ligand-free human CBG structure are now available, no crystal structure has so far been determined of an uncleaved ligand-free CBG. Such a structure could bear interesting findings. From a biological point of view it would make sense that in the absence of steroids, the RCL is partially inserted and that this prevents its cleavage by proteases. Steroid-binding would then induce full exposure of the RCL. At sites of inflammations, the exposed RCL gets cleaved by neutrophil elastase and triggers a SERPIN- typical S to R transition in CBG that is characterized by the insertion of the RCL into ?-sheet A. At the same time anti-inflammatory corticoids are released from human CBG through the allosteric coupling mechanism that we succeeded in identifying.
Projektbezogene Publikationen (Auswahl)
- (2007). Corticosteroidbinding globulin: Structural basis for steroid transport and proteinase-triggered release. J. Biol. Chem. 282, 29594-29603
Klieber, M. A., Underhill, C., Hammond, G. L. & Muller, Y. A.
- (2009). Residues in the human corticosteroid-binding globulin reactive center loop that influence steroid binding before and after elastase cleavage. J. Biol. Chem. 284, 884-896
Lin, H.-Y., Underhill, C., Gardill, B.R., Muller, Y.A. & Hammond, G. L.
