Site-directed spin labeling study of subunit interactions in the alpha-crystallin domain of small heat-shock proteins. Comparison of the oligomer symmetry in alphaA-crystallin, HSP 27, and HSP 16.3.
Site-directed spin labeling was used to investigate quaternary interactions along a conserved sequence in the alpha-crystallin domain of alphaA-crystallin, heat-shock protein 27 (HSP 27), and Mycobacterium tuberculosis heat-shock protein (HSP 16.3). In previous work, it was demonstrated that this sequence in alphaA-crystallin and HSP 27 forms a beta-strand involved in subunit contacts. In this study, the symmetry and geometry of the resulting interface were investigated. For this purpose, the pattern of spin-spin interactions was analyzed, and the number of interacting spins was determined in alphaA-crystallin and HSP 27. The results reveal a 2-fold symmetric interface consisting of two beta-strands interacting near their N termini in an antiparallel fashion. Remarkably, subunit interactions along this interface persist when the alpha-crystallin domains are expressed in isolation. Because this domain in alphaA-crystallin forms dimers and tetramers, it is inferred that interactions along this interface mediate the formation of a basic dimeric unit. In contrast, in HSP 16.3, spin-spin interactions are observed at only one site near the C terminus of the sequence. Furthermore, cysteine substitutions at residues flanking the N terminus resulted in the dissociation of the oligomeric structure. Analysis of the spin-spin interactions and size exclusion chromatography indicates a 3-fold symmetric interface. Taken together, our results demonstrate that subunit interactions in the alpha-crystallin domain of mammalian small heat-shock proteins assemble a basic building block of the oligomeric structure. Sequence divergence in this domain results in variations in the size and symmetry of the quaternary structure between distant members of the small heat-shock protein family.
Berengian
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AR
National Biomedical ESR Center, Biophysics Research Institute, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Parfenova
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M
Mchaourab
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HS
D000595:Amino Acid Sequence; D003459:Crystallins / Q000737:chemistry* / Q000235:genetics; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis; D011960:Receptors, Estrogen / Q000737:chemistry* / Q000235:genetics; D013113:Spin Labels
D003459:Crystallins; D006360:Heat-Shock Proteins; D011960:Receptors, Estrogen; D013113:Spin Labels