N- and C-Terminal Motifs in Human αB Crystallin Play an Important Role in the Recognition, Selection, and Solubilization of Substrates

The functions of the interactive sequences in human αB crystallin that are involved in chaperone activity and complex assembly of small heat shock proteins need to be characterized to understand the mechanisms of action on unfolding and misfolding proteins. Protein pin arrays identified the hydrophobic N-terminal sequence (41STSLSPFYLRPPSFLRAP58) and the polar C-terminal sequence (155PERTIPITREE165) as interactive domains in human αB crystallin, which were then deleted to evaluate their importance in complex assembly and chaperone activity. Size exclusion chromatography determined that the complexes formed by the deletion mutants, Δ41−58 and Δ155−165, were larger and more polydisperse than the wild-type (wt) αB crystallin complex. In chaperone assays, the Δ41−58 mutant was as effective as wt αB crystallin in protecting partially unfolded βL crystallin and alcohol dehydrogenase (ADH) and significantly less effective than wt αB crystallin in protecting unfolded citrate synthase (CS) from aggregation. Chaperone activity did not correlate with complex size but corresponded with the amount of substrate protein unfolding. The results confirmed the importance of N-terminal residues 41−58 in selective interactions with completely unfolded substrates. Poor solubility and limited or no chaperone activity for the three substrates characterized the Δ155−165 deletion mutant, which demonstrated the importance of C-terminal residues 155−165 in maintaining the solubility of unfolded substrates in a manner independent of the amount of substrate protein unfolding. The results presented in this report established that interactive domains in the N- and C-termini of human αB crystallin are important for the recognition, selection, and solubility of unfolding substrate proteins.