The role of the conserved COOH-terminal triad in alphaA-crystallin aggregation and functionality

The sequentially variable COOH-terminal region of small heat shock protein superfamily members usually contains a conserved IXI/V feature where X is typically a proline. When present in solved sHsp crystal structures (e.g. MjHsp16.5 and wheat Hsp16.9), this short sequence forms an isolated beta strand apparently involved in the alignment of dimers into larger oligomers. Because it is a common feature of many sHsp family members, it is possible that this triad has a similar role in alphaA-crystallin. This study was undertaken to determine the contribution of this conserved triad to the quaternary structure and function of alphaA-crystallin.

A series of site-directed mutants was generated in both wild type alphaA and in an alphaA deletion mutant lacking the NH2-terminal residues 1-50. After overexpression and purification, each protein's oligomer size was characterized by size-exclusion fast protein liquid chromatography (FPLC), thermal transition temperature by non-denaturing composite gel electrophoresis, and chaperone activity by the inhibition of DL-dithiothreitol (DTT)-induced insulin aggregation.

Using the alphaA-crystallin NH2-deletion mutant, the hydrophobic triad was changed from IPV to TPT, GPG, IGV, ITV, or GGG. All six D51 mutants associated into tetramers with small amounts of dimer and monomer also present. Chaperone-like activity was reduced but not eliminated in some of these triad mutants with GGG and ITV the most strongly affected. Similar modifications to wild type alphaA-crystallin (IPV to ITV, IGV, or GGG) restored oligomer sizes similar, but not identical to, native alphaA-crystallin, with additional small amounts of tetramer and dimer. Interestingly, equivalent mutants of wild type alphaA-crystallin did not have reduced chaperone-like activity but differed considerably in their thermal transition temperatures.

The conserved COOH-terminal triad does not appear to have a strong effect on the steady-state aggregation of wild type alphaA-crystallin or its 50-residue deletion mutant at 25 degrees C. However, it can exert a considerable effect on chaperone-like activity in the absence of the NH2-terminal 50-residue sequence extension and can influence the thermal transition temperature in its presence. These results suggest that the conserved triad in alphaA-crystallin contributes to the stability of higher order oligomers but is not essential for the formation of tetramers.