Structure and function of the plasma protein fetuin-B

Fetuin-B is a hepatic plasma protein and essential for the fertilization of the mammalian oocyte. In vitro studies confirmed fetuin-B as a potent inhibitor of the metalloproteinase ovastacin. In female fetuin-B deficient (Fetub-/-) mice, ovastacin is not inhibited prior to fertilization, leading to premature remodeling of the zona pellucida and thus to female infertility. Within this study the fertility of female Fetub-/- mice could be restored by the introduction of an additional ovastacin deficiency. In the absence of the target proteinase ovastacin, the absence of its specific inhibitor fetuin-B had no further effect. Thus, the proteinase inhibitor pair could also be confirmed in vivo. The main objective of this work was to investigate the structure-function relationship between fetuin-B and ovastacin in more detail. Two main approaches were applied: On the one hand, the inhibitory active region of fetuin-B had to be defined more closely by fetuin chimeras and single domain proteins. In a second approach, suitable material for co-crystallization experiments of fetuin-B and ovastacin should be produced. Finally, crystals of murine fetuin-B were obtained from insect cells in complex with crayfish astacin, an ovastacin homolog, in the laboratory of our cooperation partner. The structure could be solved with a resolution of 3.1 Å. Fetuin-B consists of three domains, two N-terminal cystatin-like domains (CY1, CY2), followed by a proline-rich C-terminal region (CTR). CY1 and CY2 adopt the typical cystatin folding consisting of a five-stranded antiparallel β-sheet wrapped around a central α-helix. The following CTR has only a few secondary structural elements and is therefore very flexible and partially intrinsically disordered. CY1 and CY2 are connected via a linker segment, which has a CPDCP motif essential for astacin inhibition. Together with the hairpin loop I of CY2, this motif forms a bipartite wedge which protrudes into the active site cleft of astacin and blocks it. CY1 and the hairpin loop II of CY2 only have a scaffolding role, while the CTR is not involved in inhibition at all. This inhibition mechanism was designated ’raised elephant-trunk model’ according to papain inhibition by single domain cystatins. The structure-based mechanism of inhibition was confirmed by fetuin chimeras and point mutants produced within this dissertation. Enzyme kinetics of selected mutants also confirmed that the inhibition mechanism described for astacin is also valid for ovastacin in mammals. The two essential inhibitory motifs, the CPDCP motif and the hairpin loop I in CY2, are also highly conserved in other vertebrate classes such as reptiles and birds. Since astacin-like proteinases are also widespread in the animal kingdom, it is likely that the proposed ’raised elephant-trunk model’ is effective across vertebrate classes