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Abstract
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.
Highlights
Site-directed spin labeling was used to investigate quaternary interactions along a conserved sequence in the ␣-crystallin domain of ␣A-crystallin, heat-shock protein 27 (HSP 27), and Mycobacterium tuberculosis heatshock protein (HSP 16.3)
It was demonstrated that this sequence in ␣A-crystallin and heatshock proteins (HSP) 27 forms a -strand involved in subunit contacts
Stretch in HSP 27 is a 2-fold rotational symmetry that results in the hydrogen bonding of the two strands in an antiparallel fashion
Summary
Vol 274, No 10, Issue of March 5, pp. 6305–6314, 1999 Printed in U.S.A. Site-directed Spin Labeling Study of Subunit Interactions in the ␣-Crystallin Domain of Small Heat-shock Proteins. Our results demonstrate that subunit interactions in the ␣-crystallin domain of mammalian small heat-shock proteins assemble a basic building block of the oligomeric structure. We have used site-directed spin labeling [19] to demonstrate the existence of subunit interfaces in the ␣-crystallin domain of ␣A-crystallin and HSP 27 and to determine the folding pattern of a part of this domain in ␣A-crystallin (20 –22) In both proteins, evidence of spatial proximities between single nitroxides introduced along a highly conserved sequence led to the. The data are consistent with the presence of antiparallel -strands related by a 2-fold symmetry The role of this interface in the assembly of a basic multimeric unit is examined within the context of the ␣-crystallin domain expressed in isolation. The results are compared with the recently determined crystal structure of M. jannaschii HSP 16.5 [4]
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