Crystal Structures of SlyA Protein, a Master Virulence Regulator of Salmonella, in Free and DNA-bound States

Dolan, Kyle; Duguid, E.M.; He, Chuan

doi:10.1074/jbc.m111.245258

Cited by 81 publications

(108 citation statements)

References 28 publications

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“…With the exception of P62A, these residues will not be considered further based on their lack of effect. The homologous proline in the MepR homologue SlyA of Salmonella enterica (P61) does have functional significance in that the structural analysis of cocrystallized SlyA and cognate DNA revealed that P61 makes van der Waals contacts with nucleobases in the major groove (23,24). Either MepR P62 does not play a similar role, or an alanine can substitute completely for any P62-base contact.…”

Section: Resultsmentioning

confidence: 99%

Functional Consequences of Substitution Mutations in MepR, a Repressor of the Staphylococcus aureus mepA Multidrug Efflux Pump Gene

et al. 2013

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The expression of mepA, encoding the Staphylococcus aureus MepA multidrug efflux protein, is repressed by the MarR homologue MepR. MepR dimers bind differently to operators upstream of mepR and mepA, with affinity being greatest at the mepA operator. MepR substitution mutations may result in mepA overexpression, with A103V most common in clinical strains. Evaluation of the functional consequences of this and other MepR substitutions using a lacZ reporter gene assay revealed markedly reduced repressor activity in the presence of Q18P, F27L, G97E, and A103V substitutions. Reporter data were generally supported by susceptibility and efflux assays, and electrophoretic mobility shift assays (EMSAs) confirmed compromised affinities of MepR F27L and A103V for the mepR and mepA operators. One mutant protein contained two substitutions (T94P and T132M); T132M compensated for the functional defect incurred by T94P and also rescued that of A103V but not F27L, establishing it as a limited-range suppressor. The function of another derivative with 10 substitutions was minimally affected, and this may be an extreme example of suppression involving interactions among several residues. Structural correlations for the observed functional effects were ascertained by modeling mutations onto apo-MepR. It is likely that F27L and A103V affect the protein-DNA interaction by repositioning of DNA recognition helices. Negative functional consequences of MepR substitution mutations may result from interference with structural plasticity, alteration of helical arrangements, reduced protein-cognate DNA affinity, or possibly association of MepR protomers. Structural determinations will provide further insight into the consequences of these and other mutations that affect MepR function, especially the T132M suppressor.

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Section: Resultsmentioning

confidence: 99%

Functional Consequences of Substitution Mutations in MepR, a Repressor of the Staphylococcus aureus mepA Multidrug Efflux Pump Gene

et al. 2013

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“…MarR homologues control pathways that are critical to bacterial physiology such as stress responses (41,42), virulence (43), metabolism (10), and multiple-drug resistance (23,44,45). The activity of a subset of these proteins can be modified through the formation of a disulfide bond (19,41,46).…”

Section: Discussionmentioning

confidence: 99%

Mutational Analysis of the Multiple-Antibiotic Resistance Regulator MarR Reveals a Ligand Binding Pocket at the Interface between the Dimerization and DNA Binding Domains

et al. 2013

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The Escherichia coli regulator MarR represses the multiple-antibiotic resistance operon marRAB and responds to phenolic compounds, including sodium salicylate, which inhibit its activity. Crystals obtained in the presence of a high concentration of salicylate indicated two possible salicylate sites, SAL-A and SAL-B. However, it was unclear whether these sites were physiologically significant or were simply a result of the crystallization conditions. A study carried out on MarR homologue MTH313 suggested the presence of a salicylate binding site buried at the interface between the dimerization and the DNA-binding domains. Interestingly, the authors of the study indicated a similar pocket conserved in the MarR structure. Since no mutagenesis analysis had been performed to test which amino acids were essential in salicylate binding, we examined the role of residues that could potentially interact with salicylate. We demonstrated that mutations in residues shown as interacting with salicylate at SAL-A and SAL-B in the MarR-salicylate structure had no effect on salicylate binding, indicating that these sites were not the physiological regulatory sites. However, some of these residues (P57, R86, M74, and R77) were important for DNA binding. Furthermore, mutations in residues R16, D26, and K44 significantly reduced binding to both salicylate and 2,4-dinitrophenol, while a mutation in residue H19 impaired the binding to 2,4-dinitrophenol only. These findings indicate, as for MTH313, the presence of a ligand binding pocket located between the dimerization and DNA binding domains.

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“…Paradoxically, no overall correlation exists between ΔG SC and Leu phenotype in the four strains. Instead, a Leu + phenotype correlates with the absence of DNA topoisomerase I (TopA − ) other proteins that bind to A + T-rich DNA, including proteins that bind using a winged helix-turn-helix motif (Brennan 1993;Dolan et al 2011;Kenney 2002;Martínez-Hackert and Stock 1997). Here, the protein is interacting with both the major groove (where the DNA base sequence is accessible) and the minor groove (where a specific DNA sequence is not being sought).…”

Section: Protein Binding As a Function Of Dna Topologymentioning

confidence: 99%

DNA supercoiling is a fundamental regulatory principle in the control of bacterial gene expression

Dorman

2016

Biophys Rev

107

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Although it has become routine to consider DNA in terms of its role as a carrier of genetic information, it is also an important contributor to the control of gene expression. This regulatory principle arises from its structural properties. DNA is maintained in an underwound state in most bacterial cells and this has important implications both for DNA storage in the nucleoid and for the expression of genetic information. Underwinding of the DNA through reduction in its linking number potentially imparts energy to the duplex that is available to drive DNA transactions, such as transcription, replication and recombination. The topological state of DNA also influences its affinity for some DNA binding proteins, especially in DNA sequences that have a high A + T base content. The underwinding of DNA by the ATP-dependent topoisomerase DNA gyrase creates a continuum between metabolic flux, DNA topology and gene expression that underpins the global response of the genome to changes in the intracellular and external environments. These connections describe a fundamental and generalised mechanism affecting global gene expression that underlies the specific control of transcription operating through conventional transcription factors. This mechanism also provides a basal level of control for genes acquired by horizontal DNA transfer, assisting microbial evolution, including the evolution of pathogenic bacteria.

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Crystal Structures of SlyA Protein, a Master Virulence Regulator of Salmonella, in Free and DNA-bound States

Cited by 81 publications

References 28 publications

Functional Consequences of Substitution Mutations in MepR, a Repressor of the Staphylococcus aureus mepA Multidrug Efflux Pump Gene

Functional Consequences of Substitution Mutations in MepR, a Repressor of the Staphylococcus aureus mepA Multidrug Efflux Pump Gene

Mutational Analysis of the Multiple-Antibiotic Resistance Regulator MarR Reveals a Ligand Binding Pocket at the Interface between the Dimerization and DNA Binding Domains

DNA supercoiling is a fundamental regulatory principle in the control of bacterial gene expression

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