On the molecular mechanism of the chaperone function of mini-alpha crystallin (MAC), a 19-residue peptide of human alpha-crystallin.

Alpha-crystallin is the archetypical chaperone of the small heat shock protein family, all members of which contain the so called "alpha-crystallin domain (ACD)". This domain along with the N- and C-terminal extensions, are considered the main functional units in its chaperone function. Previous studies have shown that a 19-residue fragment of the ACD of human alphaA-crystallin called mini-alpha-crystallin (MAC) shows similar chaperone properties as the parent protein. Subsequent studies have confirmed the function of this peptide, but there are no studies to date that address the mechanistic basis for the chaperone function of MAC. Using human gammaD-crystallin (HGD), a key substrate protein for parent alpha-crystallin in the ocular lens, we show here that MAC not only protects HGD from aggregation during thermal and chemical unfolding, but also binds weakly to HGD (Kd ≈ 200-700 micromolar) even when HGD is in the native state. This type of binding is reversible. However, at temperatures favoring the unfolding of HGD, MAC forms a stable complex with HGD similar to parent alpha-crystallin. Using NMR spectroscopy we identify the residues in HGD that are involved in these two modes of binding and show that MAC protects HGD from aggregation by binding to Phe 56 and Val 132 at the domain-interface of the target protein, and residues Val 164 to Leu 167 in the core of the C-terminal domain. Furthermore, we suggest that the low-affinity, reversible binding of MAC on the surface of HGD in the native state, is involved in facilitating its binding to both the domain interface and core regions at the early stages of the unfolding of HGD. This work highlights some structural features of MAC and MAC-like peptides which affect their chaperone activity, and which can potentially be manipulated for translational studies.