The ansamycin antibiotic geldanamycin, which specifically interacts with the heat shock protein hsp90, was used to study the function of hsp90 in steroid hormone receptors. We observed inhibition of glucocorticoid-specific gene induction in several responsive cell systems. Hormone binding abilities of receptors for glucocorticoid, progestin, androgen, and estrogen were inhibited upon exposing intact cells to geldanamycin. Inhibition was only seen when geldanamycin was applied to cell cultures under growth conditions or was present during in vitro synthesis; presynthesized receptors in cell extracts were not affected. Upon withdrawal of geldanamycin, glucocorticoid binding ability was regained; this was partially independent of de novo protein synthesis. Geldanamycin caused decreased levels of immunoreactive glucocorticoid receptors in wild-type cells with enhanced degradation occurring through the ubiquitin-proteasome pathway. Analysis of receptors from treated cells revealed a heteromeric structure of normal size in which the receptor polypeptide is complexed with normal amounts of hsp90 and the immunophilin p59. These data support the view that hsp90 actively participates in steroid-induced signal transduction, and they suggest that geldanamycin affects receptor action without disrupting hsp90-containing heterocomplexes per se. Nevertheless, complexes synthesized and assembled in vitro in the presence of geldanamycin differ from receptors of cellular origin.Steroid hormone receptors are members of the ever growing family of nuclear receptors (for reviews, see Refs. 1 and 2). They are characterized by sequence homologies that are most prominent within the DNA binding domains. In contrast to most other members of the family, steroid hormone receptors are known to be complexed with heat shock proteins, most notably hsp90, 1 and heat shock proteins are involved in steroidal signal transduction (for reviews, see Refs. 3 and 4). The association with heat shock proteins thus prevents these receptors from interacting with specific DNA recognition sequences while they are not yet complexed with the respective steroidal ligands. On the other hand, hsp90 in association with receptor polypeptides appears to significantly contribute to the receptors' hormone binding ability, as has been established for several steroid hormone receptors (5-9).In recent years, a group of streptomyces antibiotics called benzochinone ansamycins were shown to affect the biological activity of several tyrosine-specific protein kinases (10 -17) that are known to similarly associate with hsp90. Interestingly, geldanamycin and herbimycin A, major representatives of ansamycin antibiotics, were found to directly interact with hsp90 (18). This observation prompted a series of investigations into the effects of these drugs on steroid hormone receptors. In the present study we show that geldanamycin at non-toxic concentrations inhibits hormonal inducibility in several responsive cell systems. Moreover, this drug affects the binding of glucocorticoid, pr...

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The signal recognition particle (SRP) is central to membrane protein targeting; SRP RNA is essential for SRP assembly, elongation arrest, and activation of SRP guanosine triphosphatases. In eukaryotes, SRP function relies on the SRP68-SRP72 heterodimer. We present the crystal structures of the RNA-binding domain of SRP68 (SRP68-RBD) alone and in complex with SRP RNA and SRP19. SRP68-RBD is a tetratricopeptide-like module that binds to a RNA three-way junction, bends the RNA, and inserts an α-helical arginine-rich motif (ARM) into the major groove. The ARM opens the conserved 5f RNA loop, which in ribosome-bound SRP establishes a contact to ribosomal RNA. Our data provide the structural basis for eukaryote-specific, SRP68-driven RNA remodeling required for protein translocation.

Co-translational protein targeting and membrane protein insertion is a fundamental process and depends on the signal recognition particle (SRP). In mammals, SRP is composed of the SRP RNA crucial for SRP assembly and function and six proteins. The two largest proteins SRP68 and SRP72 form a heterodimer and bind to a regulatory site of the SRP RNA. Despite their essential roles in the SRP pathway, structural information has been available only for the SRP68 RNA-binding domain (RBD). Here we present the crystal structures of the SRP68 protein-binding domain (PBD) in complex with SRP72-PBD and of the SRP72-RBD bound to the SRP S domain (SRP RNA, SRP19 and SRP68) detailing all interactions of SRP72 within SRP. The SRP72-PBD is a tetratricopeptide repeat, which binds an extended linear motif of SRP68 with high affinity. The SRP72-RBD is a flexible peptide crawling along the 5e- and 5f-loops of SRP RNA. A conserved tryptophan inserts into the 5e-loop forming a novel type of RNA kink-turn stabilized by a potassium ion, which we define as K+-turn. In addition, SRP72-RBD remodels the 5f-loop involved in ribosome binding and visualizes SRP RNA plasticity. Docking of the S domain structure into cryo-electron microscopy density maps reveals multiple contact sites between SRP68/72 and the ribosome, and explains the role of SRP72 in the SRP pathway.

Co-translational protein targeting to membranes depends on the regulated interaction of two ribonucleoprotein particles (RNPs): the ribosome and the signal recognition particle (SRP). Human SRP is composed of an SRP RNA and six proteins with the SRP GTPase SRP54 forming the targeting complex with the heterodimeric SRP receptor (SRαβ) at the endoplasmic reticulum membrane. While detailed structural and functional data are available especially for the bacterial homologs, the analysis of human SRP was impeded by the unavailability of recombinant SRP. Here, we describe the large-scale production of all human SRP components and the reconstitution of homogeneous SRP and SR complexes. Binding to human ribosomes is determined by microscale thermophoresis for individual components, assembly intermediates and entire SRP, and binding affinities are correlated with structural information available for all ribosomal contacts. We show that SRP RNA does not bind to the ribosome, while SRP binds with nanomolar affinity involving a two-step mechanism of the key-player SRP54. Ultrasensitive binding of SRP68/72 indicates avidity by multiple binding sites that are dominated by the C-terminus of SRP72. Our data extend the experimental basis to understand the mechanistic principles of co-translational targeting in mammals and may guide analyses of complex RNP–RNP interactions in general.