αA-crystallin interacting regions in the small heat shock protein, αB-crystallin

Abstract

Amino acid sequences of αB-crystallin, involved in interaction with αA-crystallin, were determined by using peptide scans. Positionally addressable 20-mer overlapping peptides, representing the entire sequence of αB-crystallin, were synthesized on a PVDF membrane. The membrane was blocked with albumin and incubated with purified αA-crystallin. Probing the membrane with αA-crystallin-specific antibodies revealed residues 42-57, 60-71, and 88-123 in αB-crystallin to interact with αA-crystallin. Residues 42-57 and 60-71 interacted more strongly with αA-crystallin than the 88-123 sequence of αB-crystallin. Binding of one of the αB peptides (42-57) to αA-crystallin was also confirmed by gel filtration studies and HPLC analysis. The αB-crystallin sequences involved in interaction with αA-crystallin were distinct from the chaperone sites reported earlier as binding of the αB sequence from residues 42-57 does not alter the chaperone-like function of αA-crystallin. To identify the critical residues involved in interaction with αA-crystallin, R50G and P51A mutants of αB-crystallin were made and tested for their ability to interact with αA-crystallin. The oligomeric size and hydrophobicity of the mutants were, similar. Circular dichroism studies showed that the P51A mutation increased the α-helical content of the protein. While the αBR50G mutant showed chaperone-like activity similar to wild-type αB, αBP51A showed reduced chaperone function. Fluorescence resonance energy transfer studies showed that the P51A mutation decreased the rate of subunit exchange with αA by 63%, whereas the R50G mutation reduced the exchange rate by 23%. Similar to wild-type αB, αB-crystallin peptide (42-57) effectively competed with αBP51A and αBR50G for interaction with αA. Thus, our studies showed that the αB-crystallin sequence (42-57) is one of the interacting regions in αB and αA oligomer formation.