Dataset Information

Close and Allosteric Opening of the Polypeptide-Binding Site in a Human Hsp70 Chaperone BiP.

ABSTRACT: Binding immunoglobulin protein (BiP), an essential and ubiquitous Hsp70 chaperone in the ER, plays a key role in protein folding and quality control. BiP contains two functional domains: a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD). NBD binds and hydrolyzes ATP; the substrates for SBD are extended polypeptides. ATP binding allosterically accelerates polypeptide binding and release. Although crucial to the chaperone activity, the molecular mechanisms of polypeptide binding and allosteric coupling of BiP are poorly understood. Here, we present crystal structures of an intact human BiP in the ATP-bound state, the first intact eukaryotic Hsp70 structure, and isolated BiP-SBD with a peptide substrate bound representing the ADP-bound state. These structures and our biochemical analysis demonstrate that BiP has a unique NBD-SBD interface that is highly conserved only in eukaryotic Hsp70s found in the cytosol and ER to fortify its ATP-bound state and promote the opening of its polypeptide-binding pocket.

SUBMITTER: Yang J

PROVIDER: S-EPMC4680848 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Close and Allosteric Opening of the Polypeptide-Binding Site in a Human Hsp70 Chaperone BiP.

Yang Jiao J Nune Melesse M Zong Yinong Y Zhou Lei L Liu Qinglian Q

Structure (London, England : 1993) 20151119 12

Binding immunoglobulin protein (BiP), an essential and ubiquitous Hsp70 chaperone in the ER, plays a key role in protein folding and quality control. BiP contains two functional domains: a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD). NBD binds and hydrolyzes ATP; the substrates for SBD are extended polypeptides. ATP binding allosterically accelerates polypeptide binding and release. Although crucial to the chaperone activity, the molecular mechanisms of polypeptide bindi ...[more]

PMID: 26655470

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Project description:BackgroundHuman BiP is traditionally regarded as a major endoplasmic reticulum (ER) chaperone performing a number of well-described functions in the ER. In recent years it was well established that this molecule can also be located in other cell organelles and compartments, on the cell surface or be secreted. Also novel functions were assigned to this protein. Importantly, BiP protein appears to be involved in cancer and rheumatoid arthritis progression, autoimmune inflammation and tissue damage, and thus could potentially be used for therapeutic purposes. In addition, a growing body of evidence indicates BiP as a new therapeutic target for the treatment of neurodegenerative diseases. Increasing importance of this protein and its involvement in critical human diseases demands new source of high quality native recombinant human BiP for further studies and potential application. Here we introduce yeast Saccharomyces cerevisiae as a host for the generation of human BiP protein.ResultsExpression of a full-length human BiP precursor in S. cerevisiae resulted in a high-level secretion of mature recombinant protein into the culture medium. The newly discovered ability of the yeast cells to recognize, correctly process the native signal sequence of human BiP and secrete this protein into the growth media allowed simple one-step purification of highly pure recombinant BiP protein with yields reaching 10 mg/L. Data presented in this study shows that secreted recombinant human BiP possesses native amino acid sequence and structural integrity, is biologically active and without yeast-derived modifications. Strikingly, ATPase activity of yeast-derived human BiP protein exceeded the activity of E. coli-derived recombinant human BiP by a 3-fold.ConclusionsS. cerevisiae is able to correctly process and secrete human BiP protein. Consequently, resulting recombinant BiP protein corresponds accurately to native analogue. The ability to produce large quantities of native recombinant human BiP in yeast expression system should accelerate the analysis and application of this important protein.

Project description:BiP overexpression improves leaf water relations during droughts and delays drought-induced leaf senescence. However, whether BiP controls cellular homeostasis under drought conditions or simply delays dehydration-induced leaf senescence as the primary cause for water stress tolerance remains to be determined. To address this issue, we examined the drought-induced transcriptomes of BiP-overexpressing lines and wild-type (WT) lines under similar leaf water potential (ψw) values. In the WT leaves, a ψw reduction of -1.0 resulted in 1339 up-regulated and 2710 down-regulated genes; in the BiP-overexpressing line 35S::BiP-4, only 334 and 420 genes were induced and repressed, respectively, at a similar leaf ψw = -1.0 MPa. This level of leaf dehydration was low enough to induce a repertory of typical drought-responsive genes in WT leaves but not in 35S::BiP-4 dehydrated leaves. The responders included hormone-related genes, functional and regulatory genes involved in drought protection and senescence-associated genes. The number of differentially expressed genes in the 35S::BiP-4 line approached the wild type number at a leaf ψw = -1.6 MPa. However, N-rich protein (NRP)- mediated cell death signaling genes and unfolded protein response (UPR) genes were induced to a much lower extent in the 35S::BiP-4 line than in the WT even at ψw = -1.6 MPa. The heatmaps for UPR, ERAD (ER-associated degradation protein system), drought-responsive and cell death-associated genes revealed that the leaf transcriptome of 35S::BiP-4 at ψw = -1.0 MPa clustered together with the transcriptome of well-watered leaves and they diverged considerably from the drought-induced transcriptome of the WT (ψw = -1.0, -1.7 and -2.0 MPa) and 35S::BiP-4 leaves at ψw = -1.6 MPa. Taken together, our data revealed that BiP-overexpressing lines requires a much higher level of stress (ψw = -1.6 MPa) to respond to drought than that of WT (ψw = -1.0). Therefore, BiP overexpression maintains cellular homeostasis under water stress conditions and thus ameliorates endogenous osmotic stress.

Dataset Information

Close and Allosteric Opening of the Polypeptide-Binding Site in a Human Hsp70 Chaperone BiP.

Publications

Close and Allosteric Opening of the Polypeptide-Binding Site in a Human Hsp70 Chaperone BiP.

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