RESONANCE ASSIGNMENT AND SECONDARY STRUCTURE DETERMINATION AND STABILITY OF THE RECOMBINANT HUMAN UTEROGLOBIN WITH HETERONUCLEAR MULTIDIMENSIONAL NMR

Human uteroglobin (h-UG) or Clara cell 10kDa (cc10kDa) is a steroid-de pendent, 17 kDa homodimeric, secretory protein with potent anti-inflam matory/immunomodulatory properties. However, the exact physiological r ole still remains to be determined. It has been hypothesised that its activity is exerted through the binding of a specific target represent ed by a small molecule (still unknown), and that the binding is regula ted by the formation/disruption of two cysteine bonds. The binding pro perties of the reduced UG have been proved in vitro for several differ ent molecules, but no in vivo data are available to date. However, bin ding has been observed between reduced rabbit UG and a protein of an a pparent molecular mass of 90 kDa and, more recently, we found an h-UG- binding protein (putative receptor), of an apparent molecular mass of 190 kDa, on the surface of several cell types. The recognition involve s oxidised h-UG. These findings pose the problem of the relevance of t he oxidation state in the recognition process. To determine the soluti on structure of the oxidised h-UG, we produced wild-type as well as un iformly N-15- and N-15/C-13-labelled samples of the recombinant protei n. The assignments of the H-1, N-15 and C-13 resonances are presented, based on a series of homonuclear 2D and 3D and heteronuclear 2D and 3 D double and triple resonance NMR experiments. Our results indicate th at h-UG is an extremely stable protein under a wide range of temperatu res and pH conditions. The secondary structure in solution is in gener al agreement with previously reported crystal structures of rabbit UG, suggesting that cc10kDa and h-UG are indeed the same protein. Small l ocal differences found in the N- and C-terminal helices seem to suppor t the hypothesis that flexibility involves these residues; moreover, i t possibly accounts for the residual binding properties observed when the protein is in the oxidised state.