RNase A dimer with implications for amyloid formation

Bovine pancreatic ribonuclease (RNase A) forms two types of dimers (a major and a minor component) upon concentration in mild acid. These two dimers exhibit different biophysical and biochemical properties. Earlier we reported that the minor dimer forms by swapping its N-terminal α-helix with that of an identical molecule. Here we find that the major dimer forms by swapping its C-terminal β-strand, thus revealing the first example of three-dimensional (3D) domain swapping taking place in different parts of the same protein. This feature permits RNase A to form tightly bonded higher oligomers. The hinge loop of the major dimer, connecting the swapped β-strand to the protein core, resembles a short segment of the polar zipper proposed by Perutz and suggests a model for aggregate formation by 3D domain swapping with a polar zipper. The term 3D domain swapping was introduced to describe a dimer of diphtheria toxin1. In a 3D domain-swapped protein, two or more protein chains exchange identical ‘domains’, forming a strongly bound oligomer1. To date, some 20 crystal structures of 3D domain-swapped dimers and trimers have been reported but the physiological significance of these tightly bound oligomers is not fully understood. The structure described here of a second 3D domain-swapped form of an RNase A dimer vastly expands the repertoire of types of oligomeric proteins that may form by swapping domains. In 1962, Crestfield et al.2 reported that RNase A forms dimers and higher order oligomers after lyophilization in 50% acetic acid. From elegant studies of chemical modification of active site residues, they proposed that RNase A forms its dimer by exchanging its N-terminal segment, a mechanism essentially identical to what we now term 3D domain swapping. From the crystal structure of an RNase A dimer3 we confirmed that the structure is indeed 3D domainswapped by exchange of the N-terminal helix (residues 1–15). A follow-up experiment in 1965 showed that RNase A forms two types of dimers, which can be separated by cation exchange chromatography4. Study of these two dimers was not pursued until 1996 when Libonati et al.5,6 found that one component predominates (the ‘major dimer’). The two dimers show different biophysical properties on gel filtration chromatography, ion exchange chromatography and native gel electrophoresis, suggesting that they have different quaternary structures. Both dimers possess higher enzyme activity on double-stranded (ds) RNA than does the monomer5,6. Because our previous RNase A crystals were obtained from a mixture of the two dimers, we seeded solutions of the separated dimers each with the previous crystals. After seeding, crystals grew from the minor dimer solution, but not the major dimer solution. This showed that the dimeric structure determined previously3 is the minor dimer. A crystal screen then yielded crystals of the major dimer.