Immobilization of the C-terminal Extension of Bovine αA-Crystallin Reduces Chaperone-like Activity

Abstract

␣-Crystallins occur as multimeric complexes, which are able to suppress precipitation of unfolding proteins. Although the mechanism of this chaperone-like activity is unknown, the affinity of ␣-crystallin for aggregationprone proteins is probably based on hydrophobic interactions. ␣-Crystallins expose a considerable hydrophobic surface to solution, but nevertheless they are very stable and highly soluble. An explanation for this paradox may be that ␣-crystallin subunits have a polar and unstructured C-terminal extension that functions as a sort of solubilizer. In this paper we have described five ␣A-crystallins in which charged and hydrophobic residues were inserted in the C-terminal extension. Introduction of lysine, arginine, and aspartate does not substantially influence chaperone-like activity. In contrast, introduction of a hydrophobic tryptophan greatly diminishes functional activity. CD experiments indicate that this mutant has a normal secondary structure and fluorescence measurements show that the inserted tryptophan is located in a polar environment. However, NMR spectroscopy clearly demonstrates that the presence of the tryptophan residue dramatically reduces the flexibility of the C-terminal extension. Furthermore, the introduction of this tryptophan results in a considerably decreased thermostability of the protein. We conclude that changing the polarity of the C-terminal extension of ␣A-crystallin by insertion of a highly hydrophobic residue can seriously disturb structural and functional integrity.The composition of the vertebrate eye lens is dominated by a group of structural proteins known as crystallins. The largest of these, ␣-crystallin, is a dynamic multimeric complex composed of two types of homologous subunits, ␣A-and ␣B-crystallin (1). A few years ago, it was discovered that these subunits are also constitutively expressed in various non-lenticular tissues, suggesting that their function is more than merely structural (2-5). In addition, increased expression of ␣B-crystallin has been observed in a variety of neurodegenerative disorders such as multiple sclerosis (6), Alexander's disease (7,8), and Alzheimer's disease (9, 10). Although the physiological significance of this extralenticular expression is unknown, it certainly relates to the fact that ␣-crystallins belong to the family of small heat shock proteins (hsp) 1 (11, 12). Among the shared features of ␣-crystallins and other small hsp are the conferring of thermotolerance (13,14), interaction with actin (15, 16), phosphorylation (17, 18), and intracellular relocalization upon stress (19,20). Furthermore, in vitro experiments have shown that ␣A-and ␣B-crystallin as well as hsp25 can act as molecular chaperones by suppressing aggregation of denaturing proteins (21-23). Unfortunately, the mechanism of this functional activity is unclear because the three-dimensional structure of ␣-crystallin is unknown.Native ␣-crystallins occur as polydisperse particles with an average molecular mass of about 800 kDa. Understanding the multimeric arra...

Synuclein γ Compromises Spindle Assembly Checkpoint and Renders Resistance to Antimicrotubule Drugs

“…The polar and flexible C-terminal tail is thought to promote the interaction of the chaperone with the hydrophobic region in the denatured protein and thus play a critical role in substrate-chaperone association. Indeed, previous reports indicated that the C-terminus of SNCG is particularly important in performing the protein binding and chaperone function (34,44). Consistent with the molecular modeling analysis, our studies using SNCG deletion mutants indicate that the C-terminal AA 106-127 is responsible for its specific binding to BubR1 and such binding is able to reduce the BubR1 interaction with Cdc20.…”

“…A unique feature of chaperone proteins is a flexible hydrophilic tail region in the C-terminal. Nuclear magnetic resonance spectroscopy has revealed that proteins with chaperone-like activities have unstructured flexible solvent-exposed C-terminal extensions (44). The polar and flexible C-terminal tail is thought to promote the interaction of the chaperone with the hydrophobic region in the denatured protein and thus play a critical role in substrate-chaperone association.…”

The mammalian small heat‐shock protein Hsp20 forms dimers and is a poor chaperone

“…5). There are good reasons to assume that charged and otherwise polar residues in this extension are important to pro- vide the solubilizing power to keep the complex of sHsp and unfolding substrates in solution [23,30]. Although Hsp20 has a flexible C-terminal extension similar in length to that of ABcrystallin, this is apparently not sufficient in itself for an optimal chaperone capacity.…”

“…Samples containing reconstituted recombinant Hsp20 and AB-crystallin were diluted with phosphate buffer (0.1 M Na 2 SO 4 , 20 mM NaP i , pH 6.9) to concentrations ranging from 100Ϫ300 µg/ml and scattering was measured as a function of the temperature, essentially as described previously [23].…”

“…1 H-NMR spectroscopy has shown that the last 8, 10 and 18 residues of AA-, AB-crystallin and Hsp25, respectively, are highly flexible [36Ϫ38]. These Cterminal extensions are assumed to be important for the solubility of the complexes formed between partially unfolded hydrophobic substrates and the sHsp, and hence for their chaperoning capacity [23,38]. A notable feature of Hsp20 is that it has a considerably shorter C-terminal region, just 14 residues, as compared to 26Ϫ33 for the A-crystallins and Hsp25, taking position 174 [34] as the last residue of the C-terminal domain (see partial alignment in Fig.…”