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

[摘要] Purpose: α-Crystallin, a hetero-oligomer of αA- andαB-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 isalmost exclusively restricted to the central, conserved "αA-crystallin domain". The flanking N-terminal domain and C-terminalextension are highly variable both in sequence and length. Mutations andage-related post-translational modifications of these proteins areassociated with congenital and age-onset cataracts. Interestingly, mostmutations or truncations in the C-terminal extensions of theα-crystallins and other α-sHsps like Hsp27 lead to pathology.It is therefore important to understand the structure/functionrelationship of this region. Sequence alignment of the C-terminalextensions of αA- and αB-crystallin with other homologuesshows a conserved IXI/V motif. The purpose of this study was toinvestigate the role of this conserved motif, IPV in αA-crystallinand IPI in αB-crystallin (corresponding to residues 159-161 inboth 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 toglycine and studied the secondary and tertiary structure of the mutantproteins using circular dichroism and fluorescence spectroscopy, and thequaternary structure using glycerol density gradient centrifugation anddynamic light scattering. Chaperone activity was studied at 37 °Cand 25 °C using DTT induced aggregation of insulin as a modelsystem. We have performed fluorescence resonance energy transfer (FRET)experiments to investigate the interactions of this motif in homo- andhetero-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 doesnot result in oligomer dissociation or loss of chaperone activity. Onthe contrary, the mutant proteins retain their capacity to oligomerizeand show enhanced chaperone activity at 37 °C. The mutants alsoexhibit significantly higher chaperone-like activity at 25 °C. FRETexperiments show that the region spanning the IPI/V motif comes inproximity either to the β-strands of the "α-crystallin" domainor the corresponding IPI/V region of another subunit.Conclusions: Our mutational studies show that the IPI/V motif has apropensity to participate in inter-subunit interactions, either withregions in the α-crystallin domain or with the corresponding IPI/Vregion on another monomer. These interactions are important in thestructure and function of α-crystallins. This motif also appears tobe important in the temperature dependent chaperone-like activity of theα-crystallins. The propensity of the IPI/V motif to form multipleinter-subunit interactions may contribute to the diversity in structureand function seen in the α-crystallin/sHsp family.

[学科分类] 生物化学/生物物理