Binding of a chaperonin to the folding intermediates of lactate dehydrogenase

Badcoe, Ian G.; Smith, Corinne J.; Wood, Steven L.; Halsall, David; Holbrook, J. John; Lund, Peter; Clarke, Anthony R.

doi:10.1021/bi00102a010

Cited by 172 publications

(131 citation statements)

References 27 publications

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“…These experiments show that target proteins bind strongly to chaperonin only when the latter is in the ADPbound state. Our data support the notion that the role of ATP hydrolysis is to act as a switch between two conformational states (3,23): one (the ADP-bound form) has the ability to bind unfolded target proteins with high affinity (i.e., less than 0.1 rLM), while the other (the ATP-bound form) cannot. Structural differences between chaperonin forms have already been noted from electron microscopic analyses (16,41).…”

Section: Discussionsupporting

confidence: 85%

Facilitated folding of actins and tubulins occurs via a nucleotide-dependent interaction between cytoplasmic chaperonin and distinctive folding intermediates.

Melki¹,

Cowan²

1994

Mol. Cell. Biol.

107

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In the cytoplasm of eukaryotes, the folding of actins and tubulins is facilitated via interaction with a heteromeric toroidal complex (cytoplasmic chaperonin). The folding reaction consists of the formation of a binary complex between the unfolded target protein and the chaperonin, followed by the ultimate release of the native polypeptide in an ATP-dependent reaction. Here we show that the mitochondrial chaperonin (cpn6O) and the cytoplasmic chaperonin both recognize a range of target proteins with different relative affinities; however, the cytoplasmic chaperonin shows the highest affinity for intermediates derived from unfolded tubulins and actins. These high-affinity actin and tubulin folding intermediates are distinct from the "molten globule" intermediates formed by noncytoskeletal target proteins in that they form relatively slowly. We show that the interaction between cytoplasmic chaperonin and unfolded target proteins depends on the chaperonin being in its ADP-bound state and that the release of the target protein occurs after a transition of the chaperonin to the ATP-bound state. Our data suggest a model in which ATP hydrolysis acts as a switch between conformational forms of the cytoplasmic chaperonin that interact either strongly or weakly with unfolded substrates.The biological function of most, if not all, proteins depends critically upon their three-dimensional structure. Although the information contained in the linear sequence of amino acids is thought to be sufficient. to specify this structure (2, 24), the correct folding of many proteins is not a spontaneous process under physiological conditions; rather, there is a requirement for interaction with a class of proteins or protein complexes known as molecular chaperones (reviewed in references 14, 18, and 39). In particular, one class of chaperones (typified by GroEL in prokaryotes) consist of multisubunit toroidal ring assemblies and are believed to function by providing a sequestered environment in which aberrant folding and aggregation are prevented; the correctly folded polypeptide is ultimately discharged in an Mg-ATP-dependent reaction. These toroidal structures are termed chaperonins (6,10,19,28,34,35,46). Actin and tubulin are the major soluble cytoplasmic proteins in eukaryotic cells and are the subunits from which actin filaments and microtubules are assembled. The folding of actins, tubulins, and actin-and tubulin-related proteins is facilitated by a chaperonin (15-17, 31, 49) that differs from its prokaryotic and mitochondrial homologs in that it is heteromeric: the multisubunit toroidal structure is assembled from eight different (though related) polypeptides (15, 38), one of which is the t-complex polypeptide TCP-1 (27, 42). As is the case for other chaperonins, the cytoplasmic chaperonin forms a stable binary complex with unfolded target polypeptides in the absence of Mg-ATP, and the hydrolysis of Mg-ATP is absolutely required for the generation of folded proteins (15-17). However, an important difference exists between the mecha...

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Section: Discussionsupporting

confidence: 85%

Facilitated folding of actins and tubulins occurs via a nucleotide-dependent interaction between cytoplasmic chaperonin and distinctive folding intermediates.

Melki¹,

Cowan²

1994

Mol. Cell. Biol.

107

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“…In another example, studies of refolding of a thermophilic lactate dehydrogenase (LDH) showed that different intermediate states could be populated upon dilution from various concentrations of guanidine hydrochloride. Only the refolding protein from guanidine concentrations producing either complete denaturation or an early intermediate form, a collapsed, molten globule-like state, interacted with GroEL (Badcoe et al, 1991). By contrast, neither a later intermediate, with more secondary structure, nor the folded monomer was bound by GroEL.…”

Section: Conformations Recognized By Groelmentioning

confidence: 93%

“…The interaction of GroEL with unfolded RUBISCO occurred at a similar rate of 3.5 X IO7 M" s" (Roy et al, 1992). Two early unfolded intermediates of LDH (from Bacillus stearothermophilus) associated considerably more slowly with GroEL, with rate constants of 5 X IO5 M" s-l (Badcoe et al, 1991). The association rates of small unfolded polypeptides with GroEL have also been estimated, ranging from >IO8 M" s-I , approaching diffusion-controlled limits, for barnase (Gray & Fersht, 1993) to 0.2-1 X IO6 M" s-' for apo-a-lactalbumin in the reduced or oxidized molten globule state, respectively (Murai et al, 1995;Katsumata et al, 1996).…”

Section: Conformations Recognized By Groelmentioning

confidence: 94%

“…An analogous calculation has been made for the complex of E. coli DHFR with GroEL, with an estimate of <85 nM (Clark et al, 1996). The early unfolded intermediates of LDH formed complexes with GroEL with apparent dissociation constants of -7 nM; the last, molten globule-like, folding intermediate of LDH bound less well by a factor of 5-6 (Badcoe et al, 1991;Staniforth et al, 1994a). For this substrate, the dissociation constants in the presence of ATP or AMP-PNP were also measured and found to be increased to 120 nM and 70 nM, respectively, for the early forms (Staniforth et al, 1994a).…”

Section: Conformations Recognized By Groelmentioning

confidence: 99%

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GroEL‐Mediated protein folding

1997

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I. Architecture of GroEL and GroES and the reaction pathwayA. Architecture of the chaperonins B. Reaction pathway of GroEL-GroES-mediated folding 3. 4. 5.Role of hydrophobicity in recognition Homologous proteins with differing recognition-differences in primary structure versus effects on folding Concluding remarksKeywords: chaperonin; GroES; Hsp60; kinetic partitioning; mechanism of action; X-ray structureThe early studies of Anfinsen and co-workers established that the 1973). Under physiologic conditions inside the cell, however, the primary structure of a protein contains all the information necesfolding process for many proteins, particularly those with multisary to direct the native secondary and tertiary fold (Anfinsen, domain structures, is prone to producing a variety of misfolded species and aggregates. Recent studies indicate that a specialized

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“…The GroEL chaperonin is overexpressed by bacteria on cell stress and frequently co-purifies with recombi- nant fusion proteins. The binding of ATP to GroEL has been shown to induce a conformational change from a tight-binding form of the protein to a weak-binding form (24). To remove the co-purifying GroEL we included an ATP washing step in the purification procedure (25).…”

Section: Purification Of a Gst-he2mentioning

confidence: 99%

DNA Binding and Bending by the Human Papillomavirus Type 16 E2 Protein

Thain¹,

Webster

Emery³

et al. 1997

Journal of Biological Chemistry

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The human papillomavirus (HPV) 16 E2 protein (hE2) binds to four sites present upstream of the P97 promoter and regulates transcription of the viral E6 and E7 oncogenes. We have determined the relative binding constants for the interaction of the full-length hE2 protein with these sites. Our results show that hE2 binds tightly to site 4, less tightly to sites 1 and 2, and weakly to site 3. Similar results have previously been obtained using a C-terminal fragment of the hE2 protein suggesting that the C-terminal domain is the sole determinant of DNA binding affinity and specificity. Using circular permutation assays we show that binding of the hE2 protein induces the formation of a significant DNA bend and that the hE2-induced DNA bend angle is the same at both tight and weak hE2-binding sites. An alignment of the four hE2-binding sites from the HPV 16 genome suggests that this protein recognizes an extended binding site when compared with the bovine papillomavirus E2 protein. Here we show that the hE2 protein binds tightly to sites containing an A:T or a G:C base pair at position 7 of its binding site but weakly to sites containing either C:G or T:A at this position. Using site-directed mutagenesis we demonstrate that an arginine at position 304 of the hE2 protein is responsible for the recognition of specific base pairs at this position.The recognition of specific DNA sequences by transcription factors is often the first step in the regulation of gene expression. An understanding of how these proteins recognize their target sequences, and the effect that this has on DNA conformation, is central to the question of how genes are controlled. We are studying the human papillomavirus E2 protein, a sequence-specific DNA-binding protein involved in the regulation of viral gene expression and DNA replication. Papillomaviruses infect epithelial cells and induce the formation of benign hyperproliferative lesions or warts. Over 70 distinct types of human papillomavirus (HPV) 1 have been described. Some of these viral types produce lesions that have the potential to undergo malignant transformation. HPV 16 and HPV 18, for example, are thought to play a primary role in the development of cervical cancer (for a review, see Ref. 1). The products of the viral E6 and E7 genes form complexes with the cellular tumor suppressor proteins p53 and Rb, respectively. These interactions bring about a change in cell growth rate and promote cell immortalization (reviewed in Ref.2). In HPV 16, transcription of the E6 and E7 genes is under the control of a single promoter (P97) that lies immediately upstream of the E6 gene (3). The activity of the P97 promoter is regulated by a variety of cellular transcription factors and by the viral E2 protein (4 -6).Much early work concentrated on the bovine papillomavirus (BPV) E2 protein. The BPV E2 protein (bE2) binds as a dimer to 12 inverted repeats (consensus sequence 5Ј-ACCGN 4 CGGT-3Ј) present upstream of the BPV early genes and activates transcription (7,8). Binding of bE2 protein to DNA is coo...

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Binding of a chaperonin to the folding intermediates of lactate dehydrogenase

Cited by 172 publications

References 27 publications

Facilitated folding of actins and tubulins occurs via a nucleotide-dependent interaction between cytoplasmic chaperonin and distinctive folding intermediates.

Facilitated folding of actins and tubulins occurs via a nucleotide-dependent interaction between cytoplasmic chaperonin and distinctive folding intermediates.

GroEL‐Mediated protein folding

DNA Binding and Bending by the Human Papillomavirus Type 16 E2 Protein

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