The function of steroid receptors in the cell depends on the chaperone machinery of Hsp90, as Hsp90 primes steroid receptors for hormone binding and transcriptional activation. Several conserved proteins are known to additionally participate in receptor chaperone assemblies, but the regulation of the process is not understood in detail. Also, it is unknown to what extent the contribution of these cofactors is conserved in other eukaryotes. We here examine the reconstituted C. elegans and human chaperone assemblies. We find that the nematode phosphatase PPH-5 and the prolyl isomerase FKB-6 facilitate the formation of glucocorticoid receptor (GR) complexes with Hsp90. Within these complexes, Hsp90 can perform its closing reaction more efficiently. By combining chemical crosslinking and mass spectrometry, we define contact sites within these assemblies. Compared to the nematode Hsp90 system, the human system shows less cooperative client interaction and a stricter requirement for the co-chaperone p23 to complete the closing reaction of GR•Hsp90•Pp5/Fkbp51/ Fkbp52 complexes. In both systems, hormone binding to GR is accelerated by Hsp90 alone and in the presence of its cofactors. our results show that cooperative complex formation and hormone binding patterns are, in many aspects, conserved between the nematode and human systems. Hsp90 is an ATP-driven molecular machine, highly abundant in the cytosol. It is recruited to client proteins that require energy-intense rearrangements and supports these reactions by performing nucleotide-induced conformational changes 1,2. These changes lead to the compaction of the Hsp90 dimer and the transient formation of a ring-like structure, where the N-terminal domains are dimerized in addition to the chaperone's permanent C-terminal dimerization 3. Only in the bacterial Hsp90 system are these changes performed and controlled by Hsp90 itself. In the eukaryotic systems, client-specific cofactors are present, which tailor the Hsp90 machinery into a client-specific mode and regulate the conformational changes while providing further interaction sites for the clients 4. This has been investigated for the Hsp90-dependent maturation of protein kinases with the help of the cofactor Cdc37 and for the chaperoning of steroid receptors, where a larger set of cofactors participates 5-7. Partly, these reactions have been reconstituted with recombinant proteins to identify interaction mechanisms, but mostly addressing the cofactors' interaction with Hsp90 in the absence of clients. Hsp90-containing protein complexes were first identified in the 1980s 8,9. In these studies, steroid hormone receptors, such as the glucocorticoid, mineralocorticoid or progesterone receptors, were isolated from vertebrate cells and the associated proteins were identified and studied by western blot analyses. Hsp90 and several other proteins were detected in these complexes, leading to the identification of Fkbp51, Fkbp52, Cyp40, Hop, p23, Hip and Pp5 as components of steroid hormone receptor complexes 10-14. The gluco...

The subtilase cytotoxin (SubAB) of Shiga toxin-producing Escherichia coli (STEC) is a member of the AB5 toxin family. In the current study, we analyzed the formation of active homo- and hetero-complexes of SubAB variants in vitro to characterize the mode of assembly of the subunits. Recombinant SubA1-His, SubB1-His, SubA2-2-His, and SubB2-2-His subunits, and His-tag-free SubA2-2 were separately expressed, purified, and biochemically characterized by circular dichroism (CD) spectroscopy, size-exclusion chromatography (SEC), and analytical ultracentrifugation (aUC). To confirm their biological activity, cytotoxicity assays were performed with HeLa cells. The formation of AB5 complexes was investigated with aUC and isothermal titration calorimetry (ITC). Binding of SubAB2-2-His to HeLa cells was characterized with flow cytometry (FACS). Cytotoxicity experiments revealed that the analyzed recombinant subtilase subunits were biochemically functional and capable of intoxicating HeLa cells. Inhibition of cytotoxicity by Brefeldin A demonstrated that the cleavage is specific. All His-tagged subunits, as well as the non-tagged SubA2-2 subunit, showed the expected secondary structural compositions and oligomerization. Whereas SubAB1-His complexes could be reconstituted in solution, and revealed a Kd value of 3.9 ± 0.8 μmol/L in the lower micromolar range, only transient interactions were observed for the subunits of SubAB2-2-His in solution, which did not result in any binding constant when analyzed with ITC. Additional studies on the binding characteristics of SubAB2-2-His on HeLa cells revealed that the formation of transient complexes improved binding to the target cells. Conclusively, we hypothesize that SubAB variants exhibit different characteristics in their binding behavior to their target cells.

The actin cytoskeleton is of profound importance to cell shape, division, and intracellular force generation. Profilins bind to globular (G-)actin and regulate actin filament formation. Although profilins are well-established actin regulators, the distinct roles of the dominant profilin, profilin 1 (PFN1), versus the less abundant profilin 2 (PFN2) remain enigmatic. In this study, we use interaction proteomics to discover that PFN2 is an interaction partner of the actin N-terminal acetyltransferase NAA80, and further confirmed this by analytical ultracentrifugation. Enzyme assays with NAA80 and different profilins demonstrate that PFN2 binding specifically increases the intrinsic catalytic activity of NAA80. NAA80 binds PFN2 through a proline-rich loop, deletion of which abrogates PFN2 binding. Small-angle X-ray scattering shows that NAA80, actin and PFN2 form a ternary complex and that NAA80 has partly disordered regions in the N-terminus and the proline-rich loop, the latter of which is partly ordered upon PFN2 binding. Furthermore, binding of PFN2 to NAA80 via the proline-rich loop promotes binding between the globular domains of actin and NAA80, and thus acetylation of actin. However, the majority of cellular NAA80 is stably bound to PFN2 and not to actin, and we propose that this complex acetylates G-actin before it is incorporated into filaments. In conclusion, we reveal a functionally specific role of PFN2 as a stable interactor and regulator of the actin N-terminal acetyltransferase NAA80, and establish the modus operandi for NAA80-mediated actin N-terminal acetylation, a modification with a major impact on cytoskeletal dynamics. Data are available via ProteomeXchange with identifier PXD021408.