The pivotal role of the β7 strand in the intersubunit contacts of different human small heat shock proteins

Abstract

Human αB-crystallin and small heat shock proteins HspB6 and HspB8 were mutated so that all endogenous Cys residues were replaced by Ser and the single Cys residue was inserted in a position homologous to that of Cys137 of human HspB1, i.e. in a position presumably located in the central part of β7 strand of the α-crystallin domain. The secondary, tertiary, and quaternary structures of thus obtained Cys-mutants as well as their chaperone-like activity were similar to those of their wildtype counterparts. Mild oxidation of Cys-mutants leads to formation of disulfide bond crosslinking neighboring monomers thus indicating participation of the β7 strand in intersubunit interaction. Oxidation weakly affects the secondary and tertiary structure, does not affect the quaternary structure of αB-crystallin and HspB6, and shifts equilibrium between monomer and dimer of HspB8 towards dimer formation. It is concluded that the β7 strand participates in the intersubunit interaction of four human small heat shock proteins (αB-crystallin, HspB1, HspB6, HspB8) having different structure of β2 strand of α-crystallin domain and different length and composition of variable N-and C-terminal tails.

Abnormal interaction of motor neuropathy-associated mutant HspB8 (Hsp22) forms with the RNA helicase Ddx20 (gemin3)

“…The mutation K141E in HspB8 resulted in significant changes in the protein structure and properties that affected also the intersubunit contacts (Kim et al 2006;Kasakov et al 2007). A similar situation was found for the R120G mutation in HspB5 (references in Mymrikov et al 2010). This concept of abnormal sHSP interactions, however, does not explain the molecular mechanisms by which mutations in these sHSPs lead to the slow and selective loss of motor neurons.…”

“…A number of diseaseassociated mutations in HspB8 (K141E and K141N) and HspB1 (S135F, R136W, R140G, and K141Q) are positioned in the β6/β7 strands of their α-crystallin domains (Benndorf 2010). This region was shown to be critical for the homo-dimer formation of several sHSPs, including HspB8 and HspB1, and possibly also for the hetero-dimer formation (Mymrikov et al 2010). The mutation K141E in HspB8 resulted in significant changes in the protein structure and properties that affected also the intersubunit contacts (Kim et al 2006;Kasakov et al 2007).…”

The Chaperone Activity and Substrate Spectrum of Human Small Heat Shock Proteins

“…At the same time, in different studies other human sHsps (e.g. HspB6/Hsp20 and HspB8/Hsp22) were shown to be able to suppress the aggregation of amorphously aggregating model substrates in vitro (23)(24)(25)(26). Recently, an assay using cell lysates and thermal unfolding was introduced to study human HspB1 (27) and HspB5 (28).…”

“…However, different model substrate proteins and different conditions were employed. Although insulin aggregation, one of the commonly used assays, can be performed in a wide range of buffers (19,24,27,29,30) without affecting the aggregation process (19), the impact of variations in the buffer composition on other model substrates and sHsp activity remains elusive. This makes the comparison of data from different studies for a given sHsp or between different sHsps difficult.…”

Influences of temperature, oxidative stress, and phosphorylation on binding of heat shock proteins in skeletal muscle fibers

“…It is also reported that oxidation can modify the structure and effects of HSPs. HSP25 has a single cysteine residue in its ␣-crystallin-like domain (31), and S-thiolation of HSP25 regulates its multimeric aggregate size independently of phosphorylation (10,32). HSP72 has five cysteine residues and can form cross-linked dimers (33), and oxidative modification (specifically, S-glutathionylation) is known to increase its chaperone activity (18).…”