A two-hybrid system based on chimeric operator recognition for studying protein homo/heterodimerization in Escherichia coli

Lallo, Gustavo Di; Castagnoli, Luisa; Ghelardini, P.; Paolozzi, L.

doi:10.1099/00221287-147-6-1651

Cited by 39 publications

(57 citation statements)

References 17 publications

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“…As shown in Fig. 7, which is published as supporting information on the PNAS web site, the fusion constructs repressed the lacZ reporter Ϸ4-to 9-fold over the unrepressed controls, a dynamic range typical of other repressor-based systems (16,19). The chemical modulation of a protein-protein interaction in our RTHS was demonstrated with the FK506-binding protein (FKBP12) and FKBP12-rapamycin-associated protein (FRAP) pairing, whose dimerization depends on the presence of rapamycin (20), a naturally occurring chemical dimerizer.…”

Section: Resultsmentioning

confidence: 99%

A systematic method for identifying small-molecule modulators of protein–protein interactions

Horswill

Savinov

Benkovic

2004

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

110

159

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Discovering small-molecule modulators of protein-protein interactions is a challenging task because of both the generally noncontiguous, large protein surfaces that form these interfaces and the shortage of high-throughput approaches capable of identifying such rare inhibitors. We describe here a robust and flexible methodology that couples disruption of protein-protein complexes to host cell survival. The feasibility of this approach was demonstrated through monitoring a small-molecule-mediated protein-protein association (FKBP12-rapamycin-FRAP) and two cases of dissociation (homodimeric HIV-1 protease and heterodimeric ribonucleotide reductase). For ribonucleotide reductase, we identified cyclic peptide inhibitors from genetically encoded libraries that dissociated the enzyme subunits. A solid-phase synthetic strategy and peptide ELISAs were developed to characterize these inhibitors, resulting in the discovery of cyclic peptides that operate in an unprecedented manner, thus highlighting the strengths of a functional approach. The ability of this method to process large libraries, coupled with the benefits of a genetic selection, allowed us to identify rare, uniquely active small-molecule modulators of protein-protein interactions at a frequency of less than one in 10 million.M any regulatory processes in living organisms are often a consequence of specific protein-protein contacts, and interference with such interactions provides a means to exert control over cellular events. The de novo discovery of small molecules capable of disrupting such protein-protein complexes has been fraught with challenges, yielding very few inhibitors at a low success rate (1-3). These difficulties suggest that large, functionally diverse libraries might be essential for finding unique molecules that are capable of perturbing the intracellular levels of specific protein-protein interactions. The major challenge in sifting through such vast compound pools is the shortage of functional high-throughput assays for detection of the protein complex dissociation (4).Genetic selection is uniquely capable of identifying individual molecules with desired properties from large libraries by using whole cells as reporters and correlating host growth to a desired functional property. Unlike recently popularized affinity-based selections (5), an intracellular genetic selection can directly assay for effects on enzymatic activity or the modulation of a proteinprotein complex, thus bypassing the inherent limitations of in vitro approaches. Additionally, library members must function within the context of the entire host proteome, requiring positive candidates to have an enhanced level of selectivity for their target. This feature represents an important advantage over traditional screen-based methods in drug discovery by allowing both target affinity and selectivity to be simultaneously optimized. The application of a genetic selection to the identification of small-molecule modulators may yield both potent and selective activities as well as uniq...

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Section: Resultsmentioning

confidence: 99%

A systematic method for identifying small-molecule modulators of protein–protein interactions

Horswill

Savinov

Benkovic

2004

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

110

159

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“…Interaction of the two hybrid proteins results in a functional complementation between the T25 and T18 fragments, leading to cAMP synthesis and in turn to transcriptional activation of catabolic operons (such as the lactose operon and the maltose regulon). Importantly, as the BACTH assay involves a cAMP signaling cascade, the interaction between the hybrid proteins does not need to take place near the transcription machinery as is the case with yeast or other bacterial two-hybrid systems (17,19,21,27,31). For this reason, the BACTH system seems to be particularly appropriate for studying interactions among membrane proteins (Fig.…”

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confidence: 99%

Interaction Network amongEscherichia coliMembrane Proteins Involved in Cell Division as Revealed by Bacterial Two-Hybrid Analysis

2005

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Formation of the Escherichia coli division septum is catalyzed by a number of essential proteins (named Fts) that assemble into a ring-like structure at the future division site. Several of these Fts proteins are intrinsic transmembrane proteins whose functions are largely unknown. Although these proteins appear to be recruited to the division site in a hierarchical order, the molecular interactions underlying the assembly of the cell division machinery remain mostly unspecified. In the present study, we used a bacterial two-hybrid system based on interaction-mediated reconstitution of a cyclic AMP (cAMP) signaling cascade to unravel the molecular basis of septum assembly by analyzing the protein interaction network among E. coli cell division proteins. Our results indicate that the Fts proteins are connected to one another through multiple interactions. A deletion mapping analysis carried out with two of these proteins, FtsQ and FtsI, revealed that different regions of the polypeptides are involved in their associations with their partners. Furthermore, we showed that the association between two Fts hybrid proteins could be modulated by the coexpression of a third Fts partner. Altogether, these data suggest that the cell division machinery assembly is driven by the cooperative association among the different Fts proteins to form a dynamic multiprotein structure at the septum site. In addition, our study shows that the cAMP-based two-hybrid system is particularly appropriate for analyzing molecular interactions between membrane proteins.In Escherichia coli, the cell division process, also referred to as cytokinesis, constriction, or septation, is one of the most central yet poorly understood aspects of the bacterial physiology (for reviews, see references 5, 33, and 45). The event takes place at the midcell and starts after the bacterial chromosomal DNA has been duplicated and segregated into two daughter nucleoids. Cell division genes, named fts, have been identified mainly through conditional mutants that form long filamentous cells at nonpermissive temperatures (4, 5). At present, at least fourteen proteins are known to be specifically required for the E. coli cell septation (for reviews, see references 1, 5, 33, 42, and 45). The majority of the Fts proteins are anchored to the cell membrane, and most of them appear to localize to the bacterial septum in a sequential order (for reviews, see references 5, 33, 35, and 40). Fluorescence microscopy studies using immunofluorescence or the green fluorescent protein (GFP) fused to the Fts proteins have revealed that assembly of the septum starts with the positioning of an FtsZ ring in the cell center. The FtsZ ring is stabilized by FtsA and ZipA, which localize to the septum independently of each other but only in the presence of the FtsZ protein. FtsQ follows FtsK, whose localization requires both FtsA and ZipA proteins, in this hierarchical assembly. Then FtsL, FtsB, FtsW, FtsI, FtsN, and AmiC are successively recruited to the FtsZ ring (for reviews, see refere...

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“…The retained proteins were eluted with 100 l of Laemmli buffer (62. 5 In situ digestion. Coomassie blue-stained protein bands were excised from SDS-PAGE gels and washed three times in deionized MilliQ-grade water (10 min each time).…”

Section: Methodsmentioning

confidence: 99%

The Ribosomal Protein L2 Interacts with the RNA Polymerase α Subunit and Acts as a Transcription Modulator in Escherichia coli

et al. 2010

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Identification of interacting proteins in stable complexes is essential to understand the mechanisms that regulate cellular processes at the molecular level. Transcription initiation in prokaryotes requires coordinated proteinprotein and protein-DNA interactions that often involve one or more transcription factors in addition to RNA polymerase (RNAP) subunits. The RNAP ␣ subunit (RNAP␣) is a key regulatory element in gene transcription and functions through direct interaction with other proteins to control all stages of this process. A clear description of the RNAP␣ protein partners should greatly increase our understanding of transcription modulation. A functional proteomics approach was employed to investigate protein components that specifically interact with RNAP␣. A tagged form of Escherichia coli RNAP␣ was used as bait to determine the molecular partners of this subunit in a whole-cell extract. Among other interacting proteins, 50S ribosomal protein L2 (RPL2) was clearly identified by mass spectrometry. The direct interaction between RNAP␣ and RPL2 was confirmed both in vivo and in vitro by performing coimmunoprecipitation and bacterial two-hybrid experiments. The functional role of this interaction was also investigated in the presence of a ribosomal promoter by using a ␤-galactosidase gene reporter assay. The results clearly demonstrated that RPL2 was able to increase ␤-galactosidase expression only in the presence of a specific ribosomal promoter, whereas it was inactive when it was assayed with an unrelated promoter. Interestingly, other ribosomal proteins (L1, L3, L20, and L27) did not have any effect on rRNA expression. The findings reported here strongly suggest that in addition to its role in ribosome assembly the highly conserved RPL2 protein plays a specific and direct role in regulation of transcription.Understanding the mechanism and regulation of transcription in bacteria requires dissection of the specific roles of the individual components of the multiprotein transcription complex, including the multisubunit enzyme RNA polymerase (RNAP). It is well known, in fact, that one of the major factors ensuring correct gene expression in microorganisms is the efficiency with which RNAP recognizes the specific promoters of different genes.The single form of RNAP in Escherichia coli consists of a tetrameric core enzyme (␤␤Ј␣2) that is capable of RNA synthesis and factor-independent termination and a subunit that is responsible for recognition of specific transcription initiation sites. However, although the RNAP subunit is the main determinant for promoter recognition, the ␣ subunit also plays a key role in the stability of the transcription complex (3, 10).The E. coli RNAP ␣ subunit (RNAP␣) consists of two domains, an amino-terminal domain (NTD) and a carboxy-terminal domain (CTD), that are connected by a flexible linker that allows the CTD to interact with promoter elements located different distances from the RNAP binding site (2). The ␣ subunit has at least two functions during gene transcription; ...

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A two-hybrid system based on chimeric operator recognition for studying protein homo/heterodimerization in Escherichia coli

Cited by 39 publications

References 17 publications

A systematic method for identifying small-molecule modulators of protein–protein interactions

A systematic method for identifying small-molecule modulators of protein–protein interactions

Interaction Network amongEscherichia coliMembrane Proteins Involved in Cell Division as Revealed by Bacterial Two-Hybrid Analysis

The Ribosomal Protein L2 Interacts with the RNA Polymerase α Subunit and Acts as a Transcription Modulator in Escherichia coli

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