Hsp90 charged-linker truncation reverses the functional consequences of weakened hydrophobic contacts in the N domain.
Ontology highlight
ABSTRACT:
SUBMITTER: Tsutsumi S
PROVIDER: S-EPMC8459299 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:The heat shock protein 90 (Hsp90) is a dimeric molecular chaperone essential in numerous cellular processes. Its three domains (N, M, and C) are connected via linkers that allow the rearrangement of domains during Hsp90's chaperone cycle. A unique linker, called charged linker (CL), connects the N- and M-domain of Hsp90. We used an integrated approach, combining single-molecule techniques and biochemical and in vivo methods, to study the unresolved structure and function of this region. Here we show that the CL facilitates intramolecular rearrangements on the milliseconds timescale between a state in which the N-domain is docked to the M-domain and a state in which the N-domain is more flexible. The docked conformation is stabilized by 1.1 kBT (2.7 kJ/mol) through binding of the CL to the N-domain of Hsp90. Docking and undocking of the CL affects the much slower intermolecular domain movement and Hsp90's chaperone cycle governing client activation, cell viability, and stress tolerance.
| S-EPMC4273377 | biostudies-literature
Project description:The molecular chaperone Hsp90 supports the functional activity of specific substrate proteins (clients). For client processing, the Hsp90 dimer undergoes a series of ATP-driven conformational rearrangements. Flexible linkers connecting the three domains of Hsp90 are crucial to enable dynamic arrangements. The long charged linker connecting the N-terminal (NTD) and middle (MD) domains exhibits additional functions in vitro and in vivo. The structural basis for these functions remains unclear. Here, we characterize the conformation and dynamics of the linker and NTD-MD domain interactions by NMR spectroscopy. Our results reveal two regions in the linker that are dynamic and exhibit secondary structure conformation. We show that these regions mediate transient interactions with strand β8 of the NTD. As a consequence, this strand detaches and exposes a hydrophobic surface patch, which enables binding to the p53 client. We propose that the charged linker plays an important regulatory role by coupling the Hsp90 NTD-MD arrangement with the accessibility of a client binding site on the NTD.
| S-EPMC8048802 | biostudies-literature
Project description:Hsp90 is an essential and highly conserved modular molecular chaperone whose N and middle domains are separated by a disordered region termed the charged linker. Although its importance has been previously disregarded, because a minimal linker length is sufficient for Hsp90 activity, the evolutionary persistence of extensive charged linkers of divergent sequence in Hsp90 proteins of most eukaryotes remains unexplained. To examine this question further, we introduced human and plasmodium native and length-matched artificial linkers into yeast Hsp90. After evaluating ATPase activity and biophysical characteristics in vitro, and chaperone function in vivo, we conclude that linker sequence affects Hsp90 function, cochaperone interaction, and conformation. We propose that the charged linker, in addition to providing the flexibility necessary for Hsp90 domain rearrangements--likely its original purpose--has evolved in eukaryotes to serve as a rheostat for the Hsp90 chaperone machine.
| S-EPMC3287002 | biostudies-literature
Project description:The ubiquitous molecular chaperone 70-kDa heat shock proteins (Hsp70) play key roles in maintaining protein homeostasis. Hsp70s contain two functional domains: a nucleotide binding domain and a substrate binding domain. The two domains are connected by a highly conserved inter-domain linker, and allosteric coupling between the two domains is critical for chaperone function. The auxiliary chaperone 40-kDa heat shock proteins (Hsp40) facilitate all the biological processes associated with Hsp70s by stimulating the ATPase activity of Hsp70s. Although an overall essential role of the inter-domain linker in both allosteric coupling and Hsp40 interaction has been suggested, the molecular mechanisms remain largely unknown. Previously, we reported a crystal structure of a full-length Hsp70 homolog, in which the inter-domain linker forms a well-ordered β strand. Four highly conserved hydrophobic residues reside on the inter-domain linker. In DnaK, a well-studied Hsp70, these residues are V389, L390, L391, and L392. In this study, we biochemically dissected their roles. The inward-facing side chains of V389 and L391 form extensive hydrophobic contacts with the nucleotide binding domain, suggesting their essential roles in coupling the two functional domains, a hypothesis confirmed by mutational analysis. On the other hand, L390 and L392 face outward on the surface. Mutation of either abolishes DnaK's in vivo function, yet intrinsic biochemical properties remain largely intact. In contrast, Hsp40 interaction is severely compromised. Thus, for the first time, we separated the two essential roles of the highly conserved Hsp70 inter-domain linker: coupling the two functional domains through V389 and L391 and mediating the interaction with Hsp40 through L390 and L392.
| S-EPMC5737734 | biostudies-literature
Project description:The ubiquitous molecular chaperone Hsp90 plays a critical role in substrate protein folding and maintenance, but the functional mechanism has been difficult to elucidate. In previous work, a model Hsp90 substrate revealed an activation process in which substrate binding accelerates a large open/closed conformational change required for ATP hydrolysis by Hsp90. While this could serve as an elegant mechanism for conserving ATP usage for productive interactions on the substrate, the structural origin of substrate-catalyzed Hsp90 conformational changes is unknown. Here, we find that substrate binding affects an intrinsically unfavorable rotation of the Hsp90 N-terminal domain (NTD) relative to the middle domain (MD) that is required for closure. We identify an MD substrate binding region on the interior cleft of the Hsp90 dimer and show that a secondary set of substrate contacts drives an NTD orientation change on the opposite monomer. These results suggest an Hsp90 activation mechanism in which cross-monomer contacts mediated by a partially structured substrate prime the chaperone for its functional activity.
| S-EPMC3282117 | biostudies-literature
Project description:The distal end of mouse chromosome 7 (Chr 7) contains a large cluster of imprinted genes. In this region two cis-acting imprinting centers, IC1 (H19 DMR) and IC2 (KvDMR1), define proximal and distal subdomains, respectively. To assess the functional independence of IC1 in the context of Chr 7, we developed a recombinase-mediated chromosome truncation strategy in embryonic stem cells and generated a terminal deletion allele, DelTel7, with a breakpoint in between the two subdomains. We obtained germ line transmission of the truncated Chr 7 and viable paternal heterozygotes, confirming the absence of developmentally required paternally expressed genes distal of Ins2. Conversely, maternal transmission of DelTel7 causes a midgestational lethality, consistent with loss of maternally expressed genes in the IC2 subdomain. Expression and DNA methylation analyses on DelTel7 heterozygotes demonstrate the independent imprinting of IC1 in absence of the entire IC2 subdomain. The evolutionarily conserved linkage between the subdomains is therefore not required for IC1 imprinting on Chr 7. Importantly, the developmental phenotype of maternal heterozygotes is rescued fully by a paternally inherited deletion of IC2. Thus, all the imprinted genes located in the region and required for normal development are silenced by an IC2-dependent mechanism on the paternal allele.
| S-EPMC2223404 | biostudies-literature
Project description:Effective characterization of protein-based therapeutic candidates such as monoclonal antibodies (mAbs) is important to facilitate their successful progression from early discovery and development stages to marketing approval. One challenge relevant to biopharmaceutical development is, understanding how the stability of a protein is affected by the presence of an attached oligosaccharide, termed a glycan. To explore the utility of molecular dynamics simulations as a complementary technique to currently available experimental methods, the Fc fragment was employed as a model system to improve our understanding of protein stabilization by glycan attachment. Long molecular dynamics simulations were performed on three Fc glycoform variants modeled using the crystal structure of a human IgG1 mAb. Two of these three glycoform variants have their glycan carbohydrates partially or completely removed. Structural differences among the glycoform variants during simulations suggest that glycan truncation and/or removal can cause quaternary structural deformation of the Fc as a result of the loss or disruption of a significant number of inter-glycan contacts that are not formed in the human IgG1 crystal structure, but do form during simulations described here. Glycan truncation/removal can also increase the tertiary structural deformation of CH2 domains, demonstrating the importance of specific carbohydrates toward stabilizing individual CH2 domains. At elevated temperatures, glycan truncation can also differentially affect structural deformation in locations (Helix-1 and Helix-2) that are far from the oligosaccharide attachment point. Deformation of these helices, which form part of the FcRn, could affect binding if these regions are unable to refold after temperature normalization. During elevated temperature simulations of the deglycosylated variant, CH2 domains collapsed onto CH3 domains. Observations from these glycan truncation/removal simulations have improved our understanding on how glycan composition can affect mAb stability.
| S-EPMC3896604 | biostudies-literature
Project description:Unlike most chaperones, heat-shock protein 90 (Hsp90) interacts with a select group of "client proteins" that regulate essential biological processes. Little is known about how Hsp90 recognizes and binds these proteins. The glucocorticoid receptor (GR) is a well characterized Hsp90 client protein, whose hormone binding, nuclear-cytoplasmic trafficking, and transcriptional activity are regulated by Hsp90. Here, we provide evidence that unliganded and hormone-bound GR interact with two distinct, solvent-exposed hydrophobic sites in the Hsp90 C-terminal domain that contain the sequences "MxxIM" (HM10) and "L/MxxIL" (HM9). Our results indicate that binding of Hsp90 HM10 to unliganded GR stabilizes the unliganded ligand-binding pocket of GR indirectly by promoting an intramolecular interaction between the C-terminal alpha-helix (H12) and a solvent-exposed hydrophobic groove in the GR ligand binding domain. In the presence of hormone, Hsp90 appears to bind the hydrophobic groove of GR directly by mimicking the interactions of GR with transcriptional coactivators. The identified interactions provide insights into the mechanisms that enable Hsp90 to regulate the activity of both unliganded and hormone-bound GR and to sharpen the cellular response to hormone.
| S-EPMC1693689 | biostudies-literature
Project description:The native three-dimensional structure of a single protein is determined by the physicochemical nature of its constituent amino acids. The 20 different types of amino acids, depending on their physicochemical properties, can be grouped into three major classes: hydrophobic, hydrophilic, and charged. The anatomy of the weighted and unweighted networks of hydrophobic, hydrophilic, and charged residues separately for a large number of proteins were studied. Results showed that the average degree of the hydrophobic networks has a significantly larger value than that of hydrophilic and charged networks. The average degree of the hydrophilic networks is slightly higher than that of the charged networks. The average strength of the nodes of hydrophobic networks is nearly equal to that of the charged network, whereas that of hydrophilic networks has a smaller value than that of hydrophobic and charged networks. The average strength for each of the three types of networks varies with its degree. The average strength of a node in a charged network increases more sharply than that of the hydrophobic and hydrophilic networks. Each of the three types of networks exhibits the "small-world" property. Our results further indicate that the all-amino-acids networks and hydrophobic networks are of assortative type. Although most of the hydrophilic and charged networks are of the assortative type, few others have the characteristics of disassortative mixing of the nodes. We have further observed that all-amino-acids networks and hydrophobic networks bear the signature of hierarchy, whereas the hydrophilic and charged networks do not have any hierarchical signature.
| S-EPMC1914426 | biostudies-literature
Project description:The carboxy-terminal domain of RPB1 subunit of RNA Polymerase II (CTD) plays an essential function in the regulation of gene expression and the coordination of co-transcriptional processes. CTD consists of hepta-amino acid repeats varying in number from 52 in humans to 26 in yeast and its length is crucial for spatial organization of transcriptional machinery in the nucleus. We found that the proximal half of the CTD is sufficient to support RNA metabolism and co-transcriptional processing in steady state conditions in human cells. Signal induced transcription, however, is severely impaired upon CTD shortening. Our data suggest that CTD length increased in evolution to allow for spatio-temporal control of gene expression patterns at least in part by facilitating enhancer function.
2021-01-04 | GSE159092 | GEO