The interaction between αA- and αB-crystallin is sequence-specific

Abstract

Purpose: We have previously shown that residue 42-57 (TSLSPFYLRPPSFLRA; recognition sequence 1 or RS-1) and residue 60-71 (WFDTGLSEMRLE; recognition sequence 2 or RS-2) in αB-crystallin play a role in oligomerization and subunit interaction with αA-crystallin. When we created multiple mutations in αB-crystallin in RS-1 and RS-2 at S53(T), F54(G), L55(G), W60(R), and F61(N), we found that these mutations destabilized the protein, and the protein precipitated. When the individual mutations were created at F54, W60, and F61 in αB-crystallin, protein stability was not affected, but the mutations had an effect on oligomerization and subunit interaction with αA-crystallin. To find out whether the sequence specificity of these residues is important for the overall function of αB-crystallin, we inverted the 54-60 sequence such that 54FLRAPSW60 became 54WSPARLF60 using site-directed mutagenesis. We studied the effect of inversion on oligomerization and subunit interaction with αA-crystallin. Methods: Mutations were introduced using site-directed mutagenesis and the mutant protein, expressed in Escherichia coli BL21(DE3)pLysS cells, was purified by ion-exchange and gel filtration chromatography. The mutation was confirmed by mass spectrometry. The structure and hydrophobicity were analyzed by spectroscopic methods. The chaperone-like activities of wild-type and mutant proteins were compared using alcohol dehydrogenase and citrate synthase. Subunit exchange between αA- and αB-crystallin was monitored by fluorescence resonance energy transfer (FRET). For this purpose, purified αB- and αBinvert-crystallin were labeled with Alexa fluor 350 whereas Alexa fluor 488 was used to label αA-crystallin. Results: The inversion of residues 54-60 led to homooligomers that were 38% smaller in size than their wild-type counterparts. The inversion also reduced the tryptophan fluorescence intensity by 50%, as compared to that of wild-type αB-crystallin. This suggests that Trp54 is less exposed than Trp60. Inversion of residues did not affect the total hydrophobicity in αB-crystallin. Secondary structural analysis revealed a slight increase in the α-helical content of αBinvert-crystallin protein as compared to wild-type αB-crystallin. Except for an increase in the ellipticity of the αBinvert-crystallin mutant, no change was observed in the tertiary structure, as compared with that of wild-type αB-crystallin. Chaperone-like function was similar in the αBinvert-crystallin mutant and wild-type αB-crystallin. The inversion of residues decreased the subunit exchange rate with αA-crystallin by two fold. Conclusions: This study establishes for the first time that proper orientation of residues contributing to RS-1 and RS-2 sites in αB-crystallin is important for homooligomerization and optimal subunit interaction with αA-crystallin. © 2006 Molecular Vision.