Site-directed mutagenesis induces domain swapping and fibril formation in human pancreatic ribonuclease

The aggregation of intracellular, normally soluble, proteins into insoluble fibers has been now seen as the common cause of many diseases. Neverthless, it is known that some proteins form fibers that are non-toxic and even functionally relevant, whereas others form toxic aggregates without forming fibers. The structures of all these protein aggregates is difficult to obtain by conventional structural methods such as X-ray crystallography, solution NMR and electron microscopy. Recently, to gain insight into the structural chainges that take place when proteins form fibrils, D. Eisenberg and his coworkers designed a bovine pancreatic ribonuclease (RNase A) variant that is able to form amyloid-like fibrils that contains 3D domain swapped molecules capable of enzymatic activity. Along this line, by deleting five residues in the hinge loop linking the N-terminal a-helix of human pancreatic RNase (HP-RNase) to the rest of the protein, we found that HP-RNase is able to form domain swapped dimers. X-ray structure analysis has revealed a tetrameric association of two swapped HP-RNase dimers, which have a different quaternary structure. The analysis of packing interactions in this structure suggests a pathway of large-scale oligomerization of the protein. On the basis of the observed supramolecular assembly, fibril formation was predicted. This expectation was confirmed by the observation of the protein aggregates by electron microscopy. The biophysical and biochemical characterization of those fibrils is now in progress. These data provide an interesting example in which a single deletion of a few residues can be the primary event irreversibly leading to a monomer to dimer transition by domain swapping and to protein aggregation by fibril formation.