The IXI/V motif in the C-terminal extension of α-crystallins: alternative interactions and oligomeric assemblies

Abstract

Purpose: α-Crystallin, a hetero-oligomer of αA- and αB-crystallin, is involved in maintaining eye lens transparency, primarily by its structural packing and chaperone activity. αA- and αB-crystallin share significant sequence homology, which is almost exclusively restricted to the central, conserved "αA-crystallin domain". The flanking N-terminal domain and C-terminal extension are highly variable both in sequence and length. Mutations and age-related post-translational modifications of these proteins are associated with congenital and age-onset cataracts. Interestingly, most mutations or truncations in the C-terminal extensions of the α-crystallins and other α-sHsps like Hsp27 lead to pathology. It is therefore important to understand the structure/function relationship of this region. Sequence alignment of the C-terminal extensions of αA- and αB-crystallin with other homologues shows a conserved IXI/V motif. The purpose of this study was to investigate the role of this conserved motif, IPV in αA-crystallin and IPI in αB-crystallin (corresponding to residues 159-161 in both crystallins), in the structure and chaperone activity. Methods: The isoleucine/valine residues in the IPV motif of αA-crystallin and the IPI motif of αB-crystallin were mutated to glycine and studied the secondary and tertiary structure of the mutant proteins using circular dichroism and fluorescence spectroscopy, and the quaternary structure using glycerol density gradient centrifugation and dynamic light scattering. Chaperone activity was studied at 37 °C and 25 °C using DTT induced aggregation of insulin as a model system. We have performed fluorescence resonance energy transfer (FRET) experiments to investigate the interactions of this motif in homo- and hetero-oligomers. Since αB-crystallin is devoid of Cys residues, we have introduced a Cys residue flanking the IPI motif (T162CαB-crystallin) to facilitate fluorescence labeling studies. Results: Unlike in other homologues from plants or prokaryotes, mutation of the isoleucine/valine residues in α-crystallins does not result in oligomer dissociation or loss of chaperone activity. On the contrary, the mutant proteins retain their capacity to oligomerize and show enhanced chaperone activity at 37 °C. The mutants also exhibit significantly higher chaperone-like activity at 25 °C. FRET experiments show that the region spanning the IPI/V motif comes in proximity either to the β-strands of the "α-crystallin" domain or the corresponding IPI/V region of another subunit. Conclusions: Our mutational studies show that the IPI/V motif has a propensity to participate in inter-subunit interactions, either with regions in the α-crystallin domain or with the corresponding IPI/V region on another monomer. These interactions are important in the structure and function of α-crystallins. This motif also appears to be important in the temperature dependent chaperone-like activity of the α-crystallins. The propensity of the IPI/V motif to form multiple inter-subunit interactions may contribute to the diversity in structure and function seen in the α-crystallin/sHsp family.