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Recognition of DNA by the helix-turn-helix global regulatory protein Lrp is modulated by the amino terminus.

ABSTRACT: The AsnC/Lrp family of regulatory proteins links bacterial and archaeal transcription patterns to metabolism. In Escherichia coli, Lrp regulates approximately 400 genes, over 200 of them directly. In earlier studies, lrp genes from Vibrio cholerae, Proteus mirabilis, and E. coli were introduced into the same E. coli background and yielded overlapping but significantly different regulons. These differences were seen despite amino acid sequence identities of 92% (Vibrio) and 98% (Proteus) to E. coli Lrp, including complete conservation of the helix-turn-helix motifs. The N-terminal region contains many of the sequence differences among these Lrp orthologs, which led us to investigate its role in Lrp function. Through the generation of hybrid proteins, we found that the N-terminal diversity is responsible for some of the differences between orthologs in terms of DNA binding (as revealed by mobility shift assays) and multimerization (as revealed by gel filtration, dynamic light scattering, and analytical ultracentrifugation). These observations indicate that the N-terminal tail plays a significant role in modulating Lrp function, similar to what is seen for a number of other regulatory proteins.

SUBMITTER: Hart BR

PROVIDER: S-EPMC3147536 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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Recognition of DNA by the helix-turn-helix global regulatory protein Lrp is modulated by the amino terminus.

Hart Benjamin R BR Mishra Pankaj K PK Lintner Robert E RE Hinerman Jennifer M JM Herr Andrew B AB Blumenthal Robert M RM

Journal of bacteriology 20110603 15

The AsnC/Lrp family of regulatory proteins links bacterial and archaeal transcription patterns to metabolism. In Escherichia coli, Lrp regulates approximately 400 genes, over 200 of them directly. In earlier studies, lrp genes from Vibrio cholerae, Proteus mirabilis, and E. coli were introduced into the same E. coli background and yielded overlapping but significantly different regulons. These differences were seen despite amino acid sequence identities of 92% (Vibrio) and 98% (Proteus) to E. co ...[more]

PMID: 21642464

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Project description:Sequence-specific DNA-binding domains (DBDs) are conserved in all domains of life. These proteins carry out a variety of cellular functions, and there are a number of distinct structural domains already described that allow for sequence-specific DNA binding, including the ubiquitous helix-turn-helix (HTH) domain. In the facultative pathogen Vibrio cholerae, the chitin sensor ChiS is a transcriptional regulator that is critical for the survival of this organism in its marine reservoir. We recently showed that ChiS contains a cryptic DBD in its C terminus. This domain is not homologous to any known DBD, but it is a conserved domain present in other bacterial proteins. Here, we present the crystal structure of the ChiS DBD at a resolution of 1.28 Å. We find that the ChiS DBD contains an HTH domain that is structurally similar to those found in other DNA-binding proteins, like the LacI repressor. However, one striking difference observed in the ChiS DBD is that the canonical tight turn of the HTH is replaced with an insertion containing a β-sheet, a variant which we term the helix-sheet-helix. Through systematic mutagenesis of all positively charged residues within the ChiS DBD, we show that residues within and proximal to the ChiS helix-sheet-helix are critical for DNA binding. Finally, through phylogenetic analyses we show that the ChiS DBD is found in diverse proteobacterial proteins that exhibit distinct domain architectures. Together, these results suggest that the structure described here represents the prototypical member of the ChiS-family of DBDs.IMPORTANCE Regulating gene expression is essential in all domains of life. This process is commonly facilitated by the activity of DNA-binding transcription factors. There are diverse structural domains that allow proteins to bind to specific DNA sequences. The structural basis underlying how some proteins bind to DNA, however, remains unclear. Previously, we showed that in the major human pathogen Vibrio cholerae, the transcription factor ChiS directly regulates gene expression through a cryptic DNA-binding domain. This domain lacked homology to any known DNA-binding protein. In the current study, we determined the structure of the ChiS DNA-binding domain (DBD) and found that the ChiS-family DBD is a cryptic variant of the ubiquitous helix-turn-helix (HTH) domain. We further demonstrate that this domain is conserved in diverse proteins that may represent a novel group of transcriptional regulators.

Project description:BackgroundMolecular interactions between proteins and DNA molecules underlie many cellular processes, including transcriptional regulation, chromosome replication, and nucleosome positioning. Computational analyses of protein-DNA interactions rely on experimental data characterizing known protein-DNA interactions structurally and biochemically. While many databases exist that contain either structural or biochemical data, few integrate these two data sources in a unified fashion. Such integration is becoming increasingly critical with the rapid growth of structural and biochemical data, and the emergence of algorithms that rely on the synthesis of multiple data types to derive computational models of molecular interactions.DescriptionWe have developed an integrated affinity-structure database in which the experimental and quantitative DNA binding affinities of helix-turn-helix proteins are mapped onto the crystal structures of the corresponding protein-DNA complexes. This database provides access to: (i) protein-DNA structures, (ii) quantitative summaries of protein-DNA binding affinities using position weight matrices, and (iii) raw experimental data of protein-DNA binding instances. Critically, this database establishes a correspondence between experimental structural data and quantitative binding affinity data at the single basepair level. Furthermore, we present a novel alignment algorithm that structurally aligns the protein-DNA complexes in the database and creates a unified residue-level coordinate system for comparing the physico-chemical environments at the interface between complexes. Using this unified coordinate system, we compute the statistics of atomic interactions at the protein-DNA interface of helix-turn-helix proteins. We provide an interactive website for visualization, querying, and analyzing this database, and a downloadable version to facilitate programmatic analysis.ConclusionsThis database will facilitate the analysis of protein-DNA interactions and the development of programmatic computational methods that capitalize on integration of structural and biochemical datasets. The database can be accessed at http://ProteinDNA.hms.harvard.edu.

Dataset Information

Recognition of DNA by the helix-turn-helix global regulatory protein Lrp is modulated by the amino terminus.

Publications

Recognition of DNA by the helix-turn-helix global regulatory protein Lrp is modulated by the amino terminus.

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