Structural and Functional Defects Caused by Point Mutations in the α-Crystallin Domain of a Bacterial α-Heat Shock Protein

The diverse family of α-crystallin-type small heat shock proteins (α-Hsps or sHsps) is characterised by a central, moderately conserved α-crystallin domain. Oligomerisation followed by dissociation of subparticles is thought to be a prerequisite for chaperone function. We demonstrate that HspH, a bacterial α-Hsp from the soybean-symbiont Bradyrhizobium japonicum, assembles into dynamic complexes freely exchanging subunits with homologous and heterologous complexes. The importance of the α-crystallin domain for oligomerisation and chaperone activity was tested by site-directed mutagenesis of 12 different residues. In contrast to mammalian α-Hsps, the majority of these mutations elicited severe structural and functional defects in HspH. The individual exchange of five amino acid residues throughout the α-crystallin domain was found to compromise oligomerisation to various degrees. Assembly defects resulting in complexes of reduced size correlated with greatly decreased or abolished chaperone activity, reinforcing that complete oligomerisation is required for functionality. Mutation of a highly conserved glycine (G114) at the C-terminal end of the α-crystallin domain specifically impaired chaperone activity without interfering with oligomerisation properties, indicating that this residue is critical for substrate interaction. The structural and functional importance of this and other residues is discussed in the context of a modeled three-dimensional structure of HspH.