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

PURPOSE: Alpha-crystallin, a hetero-oligomer of alphaA- and alphaB-crystallin, is involved in maintaining eye lens transparency, primarily by its structural packing and chaperone activity. alphaA- and alphaB-crystallin share significant sequence homology, which is almost exclusively restricted to the central, conserved "alphaA-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 alpha-crystallins and other alpha-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 alphaA- and alphaB-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 alphaA-crystallin and IPI in alphaB-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 alphaA-crystallin and the IPI motif of alphaB-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 degrees C and 25 degrees 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 alphaB-crystallin is devoid of Cys residues, we have introduced a Cys residue flanking the IPI motif (T162CalphaB-crystallin) to facilitate fluorescence labeling studies. RESULTS: Unlike in other homologues from plants or prokaryotes, mutation of the isoleucine/valine residues in alpha-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 degrees C. The mutants also exhibit significantly higher chaperone-like activity at 25 degrees C. FRET experiments show that the region spanning the IPI/V motif comes in proximity either to the beta-strands of the "alpha-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 alpha-crystallin domain or with the corresponding IPI/V region on another monomer. These interactions are important in the structure and function of alpha-crystallins. This motif also appears to be important in the temperature dependent chaperone-like activity of the alpha-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 alpha-crystallin/sHsp family.

Role of the conserved SRLFDQFFG region of alpha-crystallin, a small heat shock protein. Effect on oligomeric size, subunit exchange, and chaperone-like activity.

Small heat shock proteins (sHsps) are necessary for several cellular functions and in stress tolerance. Most sHsps are oligomers; intersubunit interactions leading to changes in oligomeric structure and exposure of specific regions may modulate their functioning. Many sHsps, including alpha A- and alpha B-crystallin, contain a well conserved SRLFDQFFG sequence motif in the N-terminal region. Sequence-based prediction shows that it exhibits helical propensity with amphipathic character, suggesting that it plays a critical role in the structure and function of alpha-crystallins. In order to investigate the role of this motif in the structure and function of sHsps, we have made constructs deleting this sequence from alpha A- and alpha B-crystallin, overexpressed, purified, and studied these engineered proteins. Circular dichroism spectroscopic studies show changes in tertiary and secondary structure on deletion of the sequence. Glycerol density gradient centrifugation and dynamic light scattering studies show that the multimeric size of the mutant proteins is significantly reduced, indicating a role for this motif in higher order organization of the subunits. Both deletion mutants exhibit similar oligomeric size and increased chaperone-like activity. Urea-induced denaturation study shows that the SRLFDQFFG sequence contributes significantly to the structural stability. Fluorescence resonance energy transfer studies show that the rate of exchange of the subunits in the alpha Adel-crystallin oligomer is higher compared with that in the alpha A-crystallin oligomer, suggesting that this region contributes to the oligomer dynamics in addition to the higher order assembly and structural stability. Thus, our study shows that the SRLFDQFFG sequence is one of the critical motifs in structure-function regulation of alpha A- and alpha B-crystallin.

Role of the C-terminal extensions of alpha-crystallins. Swapping the C-terminal extension of alpha-crystallin to alphaB-crystallin results in enhanced chaperone activity.

Several small heat shock proteins contain a well conserved alpha-crystallin domain, flanked by an N-terminal domain and a C-terminal extension, both of which vary in length and sequence. The structural and functional role of the C-terminal extension of small heat shock proteins, particularly of alphaA- and alphaB-crystallins, is not well understood. We have swapped the C-terminal extensions between alphaA- and alphaB-crystallins and generated two novel chimeric proteins, alphaABc and alphaBAc. We have investigated the domain-swapped chimeras for structural and functional alterations. We have used thermal and non-thermal models of protein aggregation and found that the chimeric alphaB with the C-terminal extension of alphaA-crystallin, alphaBAc, exhibits dramatically enhanced chaperone-like activity. Interestingly, however, the chimeric alphaA with the C-terminal extension of alphaB-crystallin, alphaABc, has almost lost its activity. Pyrene solubilization and bis-1-anilino-8-naphthalenesulfonate binding studies show that alphaBAc exhibits more solvent-exposed hydrophobic pockets than alphaA, alphaB, or alphaABc. Significant tertiary structural changes are revealed by tryptophan fluorescence and near-UV CD studies upon swapping the C-terminal extensions. The far-UV CD spectrum of alphaBAc differs from that of alphaB-crystallin whereas that of alphaABc overlaps with that of alphaA-crystallin. Gel filtration chromatography shows alteration in the size of the proteins upon swapping the C-terminal extensions. Our study demonstrates that the unstructured C-terminal extensions play a crucial role in the structure and chaperone activity, in addition to generally believed electrostatic "solubilizer" function.