AlphaA-crystallin interacting regions in the small heat shock protein, alphaB-crystallin.
Amino acid sequences of alphaB-crystallin, involved in interaction with alphaA-crystallin, were determined by using peptide scans. Positionally addressable 20-mer overlapping peptides, representing the entire sequence of alphaB-crystallin, were synthesized on a PVDF membrane. The membrane was blocked with albumin and incubated with purified alphaA-crystallin. Probing the membrane with alphaA-crystallin-specific antibodies revealed residues 42-57, 60-71, and 88-123 in alphaB-crystallin to interact with alphaA-crystallin. Residues 42-57 and 60-71 interacted more strongly with alphaA-crystallin than the 88-123 sequence of alphaB-crystallin. Binding of one of the alphaB peptides (42-57) to alphaA-crystallin was also confirmed by gel filtration studies and HPLC analysis. The alphaB-crystallin sequences involved in interaction with alphaA-crystallin were distinct from the chaperone sites reported earlier as binding of the alphaB sequence from residues 42-57 does not alter the chaperone-like function of alphaA-crystallin. To identify the critical residues involved in interaction with alphaA-crystallin, R50G and P51A mutants of alphaB-crystallin were made and tested for their ability to interact with alphaA-crystallin. The oligomeric size and hydrophobicity of the mutants were similar. Circular dichroism studies showed that the P51A mutation increased the alpha-helical content of the protein. While the alphaBR50G mutant showed chaperone-like activity similar to wild-type alphaB, alphaBP51A showed reduced chaperone function. Fluorescence resonance energy transfer studies showed that the P51A mutation decreased the rate of subunit exchange with alphaA by 63%, whereas the R50G mutation reduced the exchange rate by 23%. Similar to wild-type alphaB, alphaB-crystallin peptide (42-57) effectively competed with alphaBP51A and alphaBR50G for interaction with alphaA. Thus, our studies showed that the alphaB-crystallin sequence (42-57) is one of the interacting regions in alphaB and alphaA oligomer formation.
Sreelakshmi
Yellamaraju
Y
Department of Ophthalmology, University of Missouri, Columbia, Missouri 65212, USA.
Santhoshkumar
Puttur
P
Bhattacharyya
Jaya
J
Sharma
K Krishna
KK
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D002942:Circular Dichroism; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D019151:Peptide Library; D010449:Peptide Mapping / Q000379:methods; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D025941:Protein Interaction Mapping; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D020539:Sequence Analysis, Protein; D038202:alpha-Crystallin A Chain / Q000737:chemistry* / Q000378:metabolism; D038203:alpha-Crystallin B Chain / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D019151:Peptide Library; D010455:Peptides; D021122:Protein Subunits; D038202:alpha-Crystallin A Chain; D038203:alpha-Crystallin B Chain