Three-dimensional structure of α-crystallin domain dimers of human small heat shock proteins HSPB1 and HSPB6.

Small heat shock proteins (sHSPs) are a family of evolutionary conserved ATP-independent chaperones. These proteins share a common architecture defined by a signature α-crystallin domain (ACD) flanked by highly variable N- and C-terminal extensions. The ACD, which has an immunoglobulin-like fold, plays an important role in sHSP assembly. This domain mediates dimer formation of individual protomers, which then may assemble into larger oligomers. In vertebrate sHSPs, the dimer interface is formed by the symmetrical antiparallel pairing of two ß-strands (ß7), generating an extended ß-sheet on one face of the ACD dimer. Recent structural studies of isolated ACDs from a number of vertebrate sHSPs suggest a variability in the register of the ß7/ß7 strand interface, which may, in part, give rise to the polydispersity often associated with the full-length proteins. To further analyze the structure of ACD dimers, we have employed a combination of X-ray crystallography and solution small-angle X-ray scattering (SAXS) to study the ACD-containing fragments of human HSPB1 (HSP27) and HSPB6 (HSP20). Unexpectedly, the obtained crystal structure of the HSPB1 fragment does not reveal the typical ß7/ß7 dimers but, rather, hexamers formed by an asymmetric contact between the ß4 and the ß7 strands from adjacent ACDs. Nevertheless, in solution, both ACDs form stable dimers via the symmetric antiparallel interaction of ß7 strands. Using SAXS, we show that it is possible to discriminate between different putative registers of the ß7/ß7 interface, with the results indicating that, under physiological conditions, there is only a single register of the strands for both proteins.

Phosphorylation by cyclic AMP-dependent protein kinase inhibits chaperone-like activity of human HSP22 in vitro.

Human small heat shock protein with molecular mass 22 kD (HSP22, HspB8) contains two Ser residues (Ser24 and Ser57) in consensus sequence RXS and is effectively phosphorylated by cAMP-dependent protein kinase in vitro. Mutation S24D did not affect, whereas mutations S57D or S24,57D prevented phosphorylation of HSP22 by cAMP-dependent protein kinase thus indicating that Ser57 is the primary site of phosphorylation. Phosphorylation (or mutation) of Ser57 (or Ser24 and Ser57) resulted in changes of the local environment of tryptophan residues and increased HSP22 susceptibility to chymotrypsinolysis. Mutations mimicking phosphorylation decreased dissociation of HSP22 oligomer at low concentration without affecting its quaternary structure at high protein concentration. Mutations S24D, S57D, and especially S24,57D were accompanied by decrease of chaperone-like activity of HSP22 if insulin and rhodanase were used as substrates. Thus, phosphorylation by cAMP-dependent protein kinase affects the structure and decreases chaperone-like activity of HSP22 in vitro.

Structure, properties, and probable physiological role of small heat shock protein with molecular mass 20 kD (Hsp20, HspB6).

This review is devoted to critical analysis of data concerning the structure and functions of small heat shock proteins with apparent molecular mass 20 kD (Hsp20). We describe the structure of Hsp20, its phosphorylation by different protein kinases, interaction of Hsp20 with other small heat shock proteins, and chaperone activity of Hsp20. The distribution of Hsp20 in different animal tissues and the factors affecting expression of Hsp20 are also described. Data on the possible involvement of Hsp20 in regulation of platelet aggregation and glucose transport are presented and analyzed. Special attention is paid to literature data describing probable regulatory effect of Hsp20 on contraction of smooth muscle. Two hypotheses postulating direct effect of Hsp20 on actomyosin interaction or its effect on cytoskeleton are compared and analyzed. The most recent data on the effect of Hsp20 on apoptosis and contractile activity of cardiomyocytes are also presented.