Denaturation and unfolding of human anaphylatoxin C3a: an unusually low covalent stability of its native disulfide bonds.

The complement C3a anaphylatoxin is a major molecular mediator of innate immunity. It is a potent activator of mast cells, basophils and eosinophils and causes smooth muscle contraction. Structurally, C3a is a relatively small protein (77 amino acids) comprising a N-terminal domain connected by 3 native disulfide bonds and a helical C-terminal segment. The structural stability of C3a has been investigated here using three different methods: Disulfide scrambling; Differential CD spectroscopy; and Reductive unfolding. Two uncommon features regarding the stability of C3a and the structure of denatured C3a have been observed in this study. (a) There is an unusual disconnection between the conformational stability of C3a and the covalent stability of its three native disulfide bonds that is not seen with other disulfide proteins. As measured by both methods of disulfide scrambling and differential CD spectroscopy, the native C3a exhibits a global conformational stability that is comparable to numerous proteins with similar size and disulfide content, all with mid-point denaturation of [GdmCl](1/2) at 3.4-5M. These proteins include hirudin, tick anticoagulant protein and leech carboxypeptidase inhibitor. However, the native disulfide bonds of C3a is 150-1000 fold less stable than those proteins as evaluated by the method of reductive unfolding. The 3 native disulfide bonds of C3a can be collectively and quantitatively reduced with as low as 1mM of dithiothreitol within 5 min. The fragility of the native disulfide bonds of C3a has not yet been observed with other native disulfide proteins. (b) Using the method of disulfide scrambling, denatured C3a was shown to consist of diverse isomers adopting varied extent of unfolding. Among them, the most extensively unfolded isomer of denatured C3a is found to assume beads-form disulfide pattern, comprising Cys(36)-Cys(49) and two disulfide bonds formed by two pair of consecutive cysteines, Cys(22)-Cys(23) and Cys(56)-Cys(57), a unique disulfide structure of polypeptide that has not been documented previously.

Molecular interactions of the central conserved domain of p53

p53 mutations are the most commonly observed genetic alterations in human cancers to date. A majority of these point mutations cluster in four evolutionarily conserved domains spanning amino acids 100-300. This region of p53 has been called its central conserved, or conformational domain. This domain of p53 is also targeted by the SV40 T antigen. Mutation, as well as interaction with SV40 T antigen results in inactivation of p53. We hypothesized that mutations and SV40 T antigen disrupt p53 function by interfering with the molecular interactions of the central conserved domain. Using a chimeric protein consisting of the central conserved domain of wild-type p53 (amino acids 115-295) and a protein A affinity tail, we isolated several cellular proteins that interact specifically with this domain of p53. These proteins range in size from 30K to 90K M$\rm\sb{r}.$ We also employed the p53 fusion protein to demonstrate that the central conserved domain of p53 possesses sequence-specific DNA-binding activity. Interestingly, the cellular proteins binding to the central conserved domain of p53 enhance the sequence-specific DNA-binding activity of full length p53. Partial purification of the individual proteins binding to the conformational domain of p53 by utilizing a sodium chloride step-gradient enabled further characterization of two proteins: (1) a 42K M$\rm\sb{r}$ protein that eluted at 0.5M NaCl, and bound DNA nonspecifically, and (2) a 35K M$\rm\sb{r}$ protein eluting into the 1.0M NaCl fraction, capable of enhancing the sequence-specific DNA-binding activity of p53. In order to determine the physiologic relevance of the molecular interactions of the conformational domain of p53, we examined the biochemical processes underlying the TNF-$\alpha$ mediated growth suppression of the NSCLC cell line H460. While growth suppression was accompanied by enhanced sequence-specific p53-DNA binding activity in TNF-$\alpha$ treated H460 nuclei, there was no increase in p53 protein levels. Furthermore, p35 was upregulated in TNF-$\alpha$ treated H460 cells, suggesting that the enhanced p53-DNA binding seen in these cells may be mediated by p35. Our studies define two novel interactions involving the central conserved domain of p53 that appear to be functionally relevant: (1) sequence-specific DNA-binding, and (2) interaction with other cellular proteins. ^