Structural study of protein-protein and nucleic acid-protein complexes: stability and recognition specificity

This thesis is divided in two parts. In the first part, modulation of human ?-thrombin activity with nucleic acid aptamers is investigated from a structural point of view. The molecular determinants in the recognition between thrombin and the best known 15-mer aptamer HD1 are deeply reviewed and discussed on the basis of the structure of two complexes between the protein and two deletion mutants of HD1. Moreover, the structure of the more powerful aptamer HD22, targeting a different thrombin exosite, is finally unraveled and discussed. On the basis of this structure, a bunch of literature data, till now been considered unclear and ambiguous, could be explained and rationalized. The second part of the thesis is focused on protein self-recognition and oligomerization through 3D domain swapping. It has been shown how this mechanism can lead to the formation of dimers, oligomers, and, as an extreme case, fibrils. Mammalian pancreatic-type ribonuclease family has been used as a model system. Indeed, 3D domain swapping has been found to endow some of this proteins with special functions, besides their normal enzymatic activity, such as cytotoxic activity. In particular, the oligomerization has been used to convert the non-cytotoxic bovine seminal ribonuclease (RNase A) in multimeric protein with medical relevance as antitumor drugs. The final aim is the production of new ribonuclease oligomers with improved antitumor activity for the treatment of human cancer and able to be well-tolerated by patients. The main idea developed in this thesis has been the use of protein engineering to force monomeric proteins to form high stable domain-swapped dimers or oligomers that could be used as a new generation of antitumor agents. Finally, a new very interesting evidence that domain swapping is involved in the assembly of fibrillar aggregates has been obtained. These results support the hypothesis that the subunits of the fibril retain much of the native structure of the protein.