NEGATIVE CHARGES IN THE C-TERMINAL DOMAIN STABILIZE THE ALPHA-B-CRYSTALLIN COMPLEX

alpha B-Crystallin is one of the six known mammalian small heat-shock proteins (sHsps). These are characterized by the presence of a conserv ed sequence of 80-100 residues, which constitutes the putative C-termi nal domain, Like other sHsps, alpha B-crystallin forms multimeric glob ular complexes, often in combination with related sHsps, Here we show that in a yeast two-hybrid system, alpha B-crystallin can specifically interact with itself as well as with alpha A-crystallin and Hsp27. An alyses of the separate domains show that the conserved C-terminal doma in (C alpha B) is essential for this interaction between subunits. To try and detect residues that are important in subunit interaction, the C alpha B domain was used in a two-hybrid screen as bait to select ra ndomly mutated C alpha B mutants. In this way we obtained nine mutants that were still able to interact with wild-type C alpha B despite the presence of up to 15 replacements, Similarly, we obtained 16 mutants that were unable to bind, because of the presence of just three to nin e replacements, In binding C alpha B mutants, lysine residues were mos t often replaced by glutamic acid residues, and in non-binding C alpha B mutants, acidic residues were often found to be replaced by non-cha rged residues. This indicates that negative charges are important for subunit interaction and we propose a model to explain this role of aci dic residues, Furthermore, we observed that two homologs of alpha B-cr ystallin, alpha A-crystallin and Hsp27, generally interact similarly w ith the binding and non-binding C alpha B mutants as does alpha B-crys tallin, This suggests that interactions involved in the complex format ion of these three sHsps are largely comparable.