Site-directed Spin Labeling Study of Subunit Interactions in the α-Crystallin Domain of Small Heat-shock Proteins

Abstract

Site-directed spin labeling was used to investigate quaternary interactions along a conserved sequence in the ␣-crystallin domain of ␣A-crystallin, heat-shock pro-tein 27 (HSP 27), and Mycobacterium tuberculosis heat-shock protein (HSP 16.3). In previous work, it was dem-onstrated that this sequence in ␣A-crystallin and HSP 27 forms a ␤-strand involved in subunit contacts. In this study, the symmetry and geometry of the resulting in-terface were investigated. For this purpose, the pattern of spin-spin interactions was analyzed, and the number of interacting spins was determined in ␣A-crystallin and HSP 27. The results reveal a 2-fold symmetric interface consisting of two ␤-strands interacting near their N ter-mini in an antiparallel fashion. Remarkably, subunit interactions along this interface persist when the ␣-crystallin domains are expressed in isolation. Because this domain in ␣A-crystallin forms dimers and tetram-ers, it is inferred that interactions along this interface mediate the formation of a basic dimeric unit. In con-trast, in HSP 16.3, spin-spin interactions are observed at only one site near the C terminus of the sequence. Fur-thermore, cysteine substitutions at residues flanking the N terminus resulted in the dissociation of the oligo-meric structure. Analysis of the spin-spin interactions and size exclusion chromatography indicates a 3-fold symmetric interface. Taken together, our results dem-onstrate that subunit interactions in the ␣-crystallin domain of mammalian small heat-shock proteins assem-ble a basic building block of the oligomeric structure. Sequence divergence in this domain results in varia-tions in the size and symmetry of the quaternary struc-ture between distant members of the small heat-shock protein family.