Expressed Protein Ligation, a Novel Method for Studying Protein-Protein Interactions in Transcription

Severinov, Konstantin; Muir, Tom W.

doi:10.1074/jbc.273.26.16205

Cited by 192 publications

(178 citation statements)

References 29 publications

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“…As a control for the sensitivity for the chemical shift perturbation method, we measured the effect on the NMR spectra of adding ligands known to bind to region 4.2. Addition of T4 AsiA, a Ϸ90-residue antifactor known to bind to E. coli 70 region 4.2 (19,20), resulted in significant changes in the NMR spectra of ⌬1.1-A* (Fig. 5 A and B and Table 1).…”

Section: Methodsmentioning

confidence: 99%

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Autoregulation of a bacterial σ factor explored by using segmental isotopic labeling and NMR

Camarero

Shekhtman

Campbell

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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115

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Bacterial factors combine with the catalytic core RNA polymerase to direct the process of transcription initiation through sequencespecific interactions with the ؊10 and ؊35 elements of promoter DNA. In the absence of core RNA polymerase, the DNA-binding function of is autoinhibited by its own N-terminal 90 amino acids (region 1.1), putatively by a direct interaction with conserved region 4.2, which binds the ؊35 promoter element. In the present work, this mechanism of autoinhibition was studied by using a combination of NMR spectroscopy and segmental isotopic labeling of a 70 -like subunit from Thermotoga maritima. Our data argue strongly against a high-affinity interaction between these two domains. Instead we suggest that autoinhibition of DNA binding occurs through an indirect steric and͞or electrostatic mechanism. More generally, the present work illustrates the power of segmental isotopic labeling for probing molecular interactions in large proteins by NMR.T he 400-kDa bacterial core RNA polymerase (RNAP) is fully active in RNA polymerization but is incapable of promoter recognition and specific transcription initiation. Promoter recognition, promoter melting to form the open complex, and possibly other functions during transcription initiation, depend on the binding of a factor to the core RNAP (subunit composition ␣ 2 ␤␤Ј ) to form the RNAP holoenzyme (reviewed in ref. 1). The primary factor in Escherichia coli, responsible for the bulk of transcription during exponential growth, is 70 . Sequence comparisons reveal that 70 belongs to a large homologous family of proteins with four regions of highly conserved amino acid sequence (2-5) (Fig. 1).The factors direct the process of transcription initiation by first locating the promoter through sequence-specific recognition of two hexamers of consensus DNA; the Pribnow box or Ϫ10 element, centered at about Ϫ10 with respect to the transcription start site (ϩ1), and the Ϫ35 element (6, 7). Genetic and biophysical evidence strongly suggested that amino acid residues in region 4.2 recognize the Ϫ35 element (8, 9), and this was confirmed by structural studies (10). Nonetheless, most functions of , including sequence-specific DNA binding, manifest themselves only in the context of the RNAP holoenzyme. In the absence of core RNAP, 70 -like factors are unable to recognize promoter DNA in either double-or single-stranded form (11). Specific interactions between N-terminally truncated derivatives of 70 and promoter DNA were detected by using competitive filter-binding assays (11,12), leading to the hypothesis that the latent DNA-binding activity of is inhibited by the N-terminal region 1.1, and that this inhibition is relieved by conformational changes upon binding of to core RNAP. Studies of isolated 70 fragments revealed that region 1.1 inhibited region 4.2 binding to the Ϫ35 element in trans, but not region 2 binding to the Ϫ10 element. These results led to a more detailed model in which region 1.1 directly masks the DNA-binding determinants of region 4.2 (11, 12). I...

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Section: Methodsmentioning

confidence: 99%

“…The ligation junction is in a loop between subregions 4.1 and 4.2 (10), an area that is poorly conserved among 70 -like factors (3,5). Further- more, we have shown previously that a Cys residue is functionally tolerated at this position in E. coli 70 (19).…”

Section: Methodsmentioning

confidence: 99%

Autoregulation of a bacterial σ factor explored by using segmental isotopic labeling and NMR

Camarero

Shekhtman

Campbell

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

115

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“…An additional benefit of the inteins as a fusion partner is that the unique reactivity of the thioester bond can be exploited for a selective modification of the expressed protein. By utilizing this trait, site-selective biotinylation (Lesaicherre et al, 2002), C-terminal amidation (Cottingham et al, 2001), backbone cyclization via a native peptide bond between the N-and the C-terminus of the protein Scott et al, 1999) and addition of non-natural amino acids (Severinov and Muir, 1998) into recombinant proteins has been accomplished.…”

Section: Recombinant Protein Expression Using Self-splicing Inteinsmentioning

confidence: 99%

Gelatinase-mediated migration and invasion of cancer cells

Björklund

Koivunen

2005

Biochimica et Biophysica Acta (BBA) - Reviews on Cancer

465

609

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“…S3), whereby only the NTD portion of ClpB is isotopically enriched with 13 CH 3 -methyl groups, whereas the rest of the molecule is fully deuterated (NMR invisible). The optimized protocol (see Materials and Methods for details) enabled us to selectively methyl label only isolated NTDs [ 2 H, 13 CH 3 -ILVM] and then reassemble the full-length ClpB protein using the expressed protein ligation protocol (33,34). Samples produced in this manner have allowed us to obtain very high-quality NMR spectra for the single NTD domain in the context of the full 580-kDa hexameric protein so that detailed studies can be undertaken (see below).…”

Section: Nmr Characterization Of a Segmentally Labeled 580-kda Hexamericmentioning

confidence: 99%

ClpB N-terminal domain plays a regulatory role in protein disaggregation

Rosenzweig

Farber

Vėlyvis

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

114

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ClpB/Hsp100 is an ATP-dependent disaggregase that solubilizes and reactivates protein aggregates in cooperation with the DnaK/ Hsp70 chaperone system. The ClpB-substrate interaction is mediated by conserved tyrosine residues located in flexible loops in nucleotide-binding domain-1 that extend into the ClpB central pore. In addition to the tyrosines, the ClpB N-terminal domain (NTD) was suggested to provide a second substrate-binding site; however, the manner in which the NTD recognizes and binds substrate proteins has remained elusive. Herein, we present an NMR spectroscopy study to structurally characterize the NTD-substrate interaction. We show that the NTD includes a substrate-binding groove that specifically recognizes exposed hydrophobic stretches in unfolded or aggregated client proteins. Using an optimized segmental labeling technique in combination with methyl-transverse relaxation optimized spectroscopy (TROSY) NMR, the interaction of client proteins with both the NTD and the pore-loop tyrosines in the 580-kDa ClpB hexamer has been characterized. Unlike contacts with the tyrosines, the NTD-substrate interaction is independent of the ClpB nucleotide state and protein conformational changes that result from ATP hydrolysis. The NTD interaction destabilizes client proteins, priming them for subsequent unfolding and translocation. Mutations in the NTD substrate-binding groove are shown to have a dramatic effect on protein translocation through the ClpB central pore, suggesting that, before their interaction with substrates, the NTDs block the translocation channel. Together, our findings provide both a detailed characterization of the NTD-substrate complex and insight into the functional regulatory role of the ClpB NTD in protein disaggregation.T he heat shock protein ClpB (Escherichia coli) or Hsp100 (eukaryotes) is the main protein disaggregase in bacteria, yeast, plants, and mitochondria of all eukaryotic cells, and it is essential for cell survival during severe stress (1-4). Recovery of functional proteins from aggregates by ClpB requires the synergistic interaction with a second molecular chaperone, DnaK (1). Through its cochaperone, DnaJ, DnaK initially binds to the aggregates, leading to the exposure of peptide segments that can be recognized by ClpB (5, 6). DnaK then recruits ClpB to the site of aggregation through direct physical interaction (7, 8), transferring the aggregate to ClpB. Using the energy derived from ATP hydrolysis, ClpB unravels the aggregate by threading single polypeptide chains, one at a time, through the central pore of its hexameric ring (9). Once released from the aggregate, the unfolded polypeptides can either refold spontaneously or fold with the help of additional cellular chaperones.Like other Hsp100 proteins, ClpB forms a hexameric ring, with each protomer comprising an N-terminal domain (NTD) and two nucleotide binding domains (NBD1 and NBD2) separated by a unique regulatory coil-coil domain (10) essential for DnaK binding (7, 11) ( Fig. 1 A and B). Both NBDs contain Walk...

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Expressed Protein Ligation, a Novel Method for Studying Protein-Protein Interactions in Transcription

Cited by 192 publications

References 29 publications

Autoregulation of a bacterial σ factor explored by using segmental isotopic labeling and NMR

Autoregulation of a bacterial σ factor explored by using segmental isotopic labeling and NMR

Gelatinase-mediated migration and invasion of cancer cells

ClpB N-terminal domain plays a regulatory role in protein disaggregation

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