Edited by Norma AllewellSmall heat shock proteins (sHsps) are a ubiquitous family of molecular chaperones that suppress the unspecific aggregation of miscellaneous proteins. Multicellular organisms contain a large number of different sHsps, raising questions as to whether they function redundantly or are specialized in terms of substrates and mechanism. To gain insight into this issue, we undertook a comparative analysis of the eight major human sHsps on the aggregation of both model proteins and cytosolic lysates under standardized conditions. We discovered that sHsps, which form large oligomers (HspB1/Hsp27, HspB3, HspB4/␣A-crystallin, and HspB5/␣B-crystallin) are promiscuous chaperones, whereas the chaperone activity of the other sHsps is more substrate-dependent. However, all human sHsps analyzed except HspB7 suppressed the aggregation of cytosolic proteins of HEK293 cells. We identified ϳ1100 heat-sensitive HEK293 proteins, 12% of which could be isolated in complexes with sHsps. Analysis of their biochemical properties revealed that most of the sHsp substrates have a molecular mass from 50 to 100 kDa and a slightly acidic pI (5.4 -6.8). The potency of the sHsps to suppress aggregation of model substrates is correlated with their ability to form stable substrate complexes; especially HspB1 and HspB5, but also B3, bind tightly to a variety of proteins, whereas fewer substrates were detected in complex with the other sHsps, although these were also efficient in preventing the aggregation of cytosolic proteins.

Small heat shock proteins (sHsps) are ubiquitous molecular chaperones that prevent the aggregation of unfolding proteins during proteotoxic stress. In Caenorhabditis elegans, Sip1 is the only sHsp exclusively expressed in oocytes and embryos. Here, we demonstrate that Sip1 is essential for heat shock survival of reproducing adults and embryos. X-ray crystallography and electron microscopy revealed that Sip1 exists in a range of well-defined globular assemblies consisting of two half-spheres, each made of dimeric "spokes." Strikingly, the oligomeric distribution of Sip1 as well as its chaperone activity depend on pH, with a trend toward smaller species and higher activity at acidic conditions such as present in nematode eggs. The analysis of the interactome shows that Sip1 has a specific substrate spectrum including proteins that are essential for embryo development.

Protein kinases are the most prominent group of heat shock protein 90 (Hsp90) clients and are recruited to the molecular chaperone by the kinase-specific cochaperone cell division cycle 37 (Cdc37). The interaction between Hsp90 and nematode Cdc37 is mediated by binding of the Hsp90 middle domain to an N-terminal region of Caenorhabditis elegans Cdc37 (CeCdc37). Here we map the binding site by NMR spectroscopy and define amino acids relevant for the interaction between CeCdc37 and the middle domain of Hsp90. Apart from these distinct Cdc37/ Hsp90 interfaces, binding of the B-Raf protein kinase to the cochaperone is conserved between mammals and nematodes. In both cases, the C-terminal part of Cdc37 is relevant for kinase binding, whereas the N-terminal domain displaces the nucleotide from the kinase. This interaction leads to a cooperative formation of the ternary complex of Cdc37 and kinase with Hsp90. For the mitogen-activated protein kinase extracellular signalregulated kinase 2 (Erk2), we observe that certain features of the interaction with Cdc37⅐Hsp90 are conserved, but the contribution of Cdc37 domains varies slightly, implying that different kinases may utilize distinct variations of this binding mode to interact with the Hsp90 chaperone machinery.The human genome encodes up to 500 kinases, which represent one of the largest families of genes in eukaryotes (1, 2). They regulate essential intracellular processes like proliferation, differentiation, development, stress response, and apoptosis. All protein kinases share a conserved catalytic domain, which switches between an active and an inactive state (3,4). A large number of these protein kinases are dependent on the Hsp90 chaperone system, which modulates their maturation and prevents their degradation (5-8). So far only little structural information of the chaperone-kinase complex is available (9). To facilitate kinase processing by the Hsp90 chaperone machinery, the specific cochaperone Cdc37 is required (9 -11).A well described function of this cochaperone is to slow down the ATPase activity of Hsp90 (12). It is widely expected that Hsp90 and its partner protein Cdc37 bind to the catalytic domain of the kinase (13-16). It remains elusive how the ternary complex consisting of protein kinase, Hsp90 and Cdc37 is structurally organized and how the protein kinase is processed by the chaperone system. Recently Cdc37 was observed to compete with nucleotide binding to the kinase domain of B-Raf (17), but the role of Hsp90 in these structures remains enigmatic. Although Hsp90 is essential for ligand binding in case of steroid hormone receptors the role for B-Raf activation is rather unclear (18). B-Raf is mutated in many human cancers and is known to form complexes with Hsp90 and Cdc37 in cell free systems (19). The interaction with the kinase is also weakened in vivo in presence of an anticancer drug that disrupts nucleotide binding to Hsp90 (20). Given the recent evolution of Hsp90 as a potential cancer target (21-23), it is important to clarify the mechanis...