Immunoglobulin Binding Protein (BiP) is a chaperone and molecular motor belonging to the Hsp70 family, involved in the regulation of important biological processes such as synthesis, folding and translocation of proteins in the Endoplasmic Reticulum. BiP has two highly conserved domains: the N‐terminal Nucleotide‐Binding Domain (NBD), and the C‐terminal Substrate‐Binding Domain (SBD), connected by a hydrophobic linker. ATP binds and it is hydrolyzed to ADP in the NBD, and BiP's extended polypeptide substrates bind in the SBD. Like many molecular motors, BiP function depends on both structural and catalytic properties that may contribute to its performance. One novel approach to study the mechanical properties of BiP considers exploring the changes in the viscoelastic behavior upon ligand binding, using a technique called nano‐rheology. This technique is essentially a traditional rheology experiment, in which an oscillatory force is directly applied to the protein under study, and the resulting average deformation is measured. Our results show that the folded state of the protein behaves like a viscoelastic material, getting softer when it binds nucleotides‐ ATP, ADP, and AMP‐PNP‐, but stiffer when binding HTFPAVL peptide substrate. Also, we observed that peptide binding dramatically increases the affinity for ADP, decreasing it dissociation constant (
The 70 kDa heat shock proteins (Hsp70) are a family of molecular chaperones involved in protein folding, aggregate prevention, and protein disaggregation. They consist of the substrate‐binding domain (SBD) that binds client substrates, and the nucleotide‐binding domain (NBD), whose cycles of nucleotide hydrolysis and exchange underpin the activity of the chaperone. To characterize the structure–function relationships that link the binding state of the NBD to its conformational behavior, we analyzed the dynamics of the NBD of the Hsp70 chaperone from
- PAR ID:
- 10239301
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Proteins: Structure, Function, and Bioinformatics
- Volume:
- 83
- Issue:
- 2
- ISSN:
- 0887-3585
- Page Range / eLocation ID:
- p. 282-299
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract K D) around 1000 times, demonstrating allosteric coupling between SBD and NBD domains. -
Our early efforts to find a covalent inhibitor of mortalin, a member of the 70
kD heat shock protein (Hsp70) family, led us to solve the structure of the mortalin nucleotide‐binding domain (NBD ) in complex with N6‐propargyladenosine‐5′‐diphosphate. The acquired structure emphasizes the ability of the nucleotide‐binding pocket to accommodate modifiedADP compounds. A library ofADP analogs modified at either the 2‐ or N6‐positions of adenosine was screened against the mortalin‐NBD . Competitive inhibition and binding assays of the analogs demonstrate that modifications at the 2‐ or N6‐positions have potential to bind and inhibit mortalin uniquely compared to other Hsp70 homologs, and that modifications at the 2‐position confer the greatest selectivity in binding and inhibition of the mortalin‐NBD . -
Abstract Many proteins must interact with molecular chaperones to achieve their native state in the cell. Yet, how chaperone binding‐site characteristics affect the folding process is poorly understood. The ubiquitous Hsp70 chaperone system prevents client‐protein aggregation by holding unfolded conformations and by unfolding misfolded states. Hsp70 binding sites of client proteins comprise a nonpolar core surrounded by positively charged residues. However, a detailed analysis of Hsp70 binding sites on a proteome‐wide scale is still lacking. Further, it is not known whether proteins undergo some degree of folding while chaperone bound. Here, we begin to address the above questions by identifying Hsp70 binding sites in 2258
Escherichia coli (E. coli ) proteins. We find that most proteins bear at least one Hsp70 binding site and that the number of Hsp70 binding sites is directly proportional to protein size. Aggregation propensity upon release from the ribosome correlates with number of Hsp70 binding sites only in the case of large proteins. Interestingly, Hsp70 binding sites are more solvent‐exposed than other nonpolar sites, in protein native states. Our findings show that the majority ofE. coli proteins are systematically enabled to interact with Hsp70 even if this interaction only takes place during a fraction of the protein lifetime. In addition, our data suggest that some conformational sampling may take place within Hsp70‐bound states, due to the solvent exposure of some chaperone binding sites in native proteins. In all, we propose that Hsp70‐chaperone‐binding traits have evolved to favor Hsp70‐assisted protein folding devoid of aggregation. -
Abstract Chaperones are a large family of proteins crucial for maintaining cellular protein homeostasis. One such chaperone is the 70 kDa heat shock protein (Hsp70), which plays a crucial role in protein (re)folding, stability, functionality, and translocation. While the key events in the Hsp70 chaperone cycle are well established, a relatively small number of distinct substrates were repetitively investigated. This is despite Hsp70 engaging with a plethora of cellular proteins of various structural properties and folding pathways. Here we analyzed novel Hsp70 substrates, based on tandem repeats of NanoLuc (Nluc), a small and highly bioluminescent protein with unique structural characteristics. In previous mechanical unfolding and refolding studies, we have identified interesting misfolding propensities of these Nluc‐based tandem repeats. In this study, we further investigate these properties through in vitro bulk experiments. Similar to monomeric Nluc, engineered Nluc dyads and triads proved to be highly bioluminescent. Using the bioluminescence signal as the proxy for their structural integrity, we determined that heat‐denatured Nluc dyads and triads can be efficiently refolded by the
E. coli Hsp70 chaperone system, which comprises DnaK, DnaJ, and GrpE. In contrast to previous studies with other substrates, we observed that Nluc repeats can be efficiently refolded by DnaK and DnaJ, even in the absence of GrpE co‐chaperone. Taken together, our study offers a new powerful substrate for chaperone research and raises intriguing questions about the Hsp70 mechanisms, particularly in the context of structurally diverse proteins. -
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