Species-Specific Structural and Functional Divergence of α-Crystallins: Zebrafish αBa- and Rodent αAins-Crystallin Encode Activated Chaperones

In addition to contributing to lens optical properties, the alpha-crystallins are small heat shock proteins that possess chaperone activity and are predicted to bind and sequester destabilized proteins to delay cataract formation. The current model of a-crystallin chaperone mechanism envisions a transition from the native oligomer to an activated form that has higher affinity to non-native states of the substrate. Previous studies have suggested that this oligomeric plasticity is encoded in the primary sequence and controls access to high affinity binding sites within the N-terminal domain. Here, we further examined the role of sequence variation in the context of species-specific a-crystallins from rat and zebrafish. Alternative splicing of the alpha A gene in rodents produces alpha A(ins), which is distinguished by a longer N-terminal domain. The zebrafish genome includes duplicate alpha B-crystallin genes, alpha Ba and alpha Bb, which display divergent primary sequence and tissue expression patterns. Equilibrium binding experiments were employed to quantitatively define chaperone interactions with a destabilized model substrate, T4 lysozyme. In combination with multiangle light scattering, we show that rat ales and zebrafish alpha-crystallins display distinct global structural properties and chaperone activities. Notably, we find that alpha A(ins) and alpha Ba demonstrate substantially enhanced chaperone function relative to other alpha-crystallins, binding the same substrate more than 2 orders of magnitude higher affinity and mimicking the activity of fully activated mammalian small heat shock proteins. These results emphasize the role of sequence divergence as an evolutionary strategy to tune chaperone function to the requirements of the tissues and organisms in which they are expressed.