<i>myo</i>
            -Inositol and
            <scp>d</scp>
            -Ribose Ligand Discrimination in an ABC Periplasmic Binding Protein

Herrou, Julien; Crosson, Sean

doi:10.1128/jb.00116-13

Cited by 16 publications

(22 citation statements)

References 69 publications

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“…No shift of T m was observed for IbpA, confirming that these molecules had a specific stabilization effect on YehZ. As a positive control, we also tested IbpA stabilization by myo-inositol, which is known to be its bona fide ligand (42). In the presence of myo-inositol, the IbpA T m shifted by 13°C, demonstrating protein stabilization/binding by myo-inositol.…”

Section: Figmentioning

confidence: 73%

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Conserved ABC Transport System Regulated by the General Stress Response Pathways of Alpha- and Gammaproteobacteria

et al. 2017

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Brucella abortus E1 is an EcfG family sigma factor that regulates the transcription of dozens of genes in response to diverse stress conditions and is required for maintenance of chronic infection in a mouse model. A putative ATPbinding cassette transporter operon, bab1_0223-bab1_0226, is among the most highly activated gene sets in the E1 regulon. The proteins encoded by the operon resemble quaternary ammonium-compatible solute importers but are most similar in sequence to the broadly conserved YehZYXW system, which remains largely uncharacterized. Transcription of yehZYXW is activated by the general stress sigma factor S in Enterobacteriaceae, which suggests a functional role for this transport system in bacterial stress response across the classes Alphaproteobacteria and Gammaproteobacteria. We present evidence that B. abortus YehZYXW does not function as an importer of known compatible solutes under physiological conditions and does not contribute to the virulence defect of a E1 -null strain. The sole in vitro phenotype associated with genetic disruption of this putative transport system is reduced growth in the presence of high Li ϩ ion concentrations. A crystal structure of B. abortus YehZ revealed a class II periplasmic binding protein fold with significant structural homology to Archaeoglobus fulgidus ProX, which binds glycine betaine. However, the structure of the YehZ ligand-binding pocket is incompatible with high-affinity binding to glycine betaine. This is consistent with weak measured binding of YehZ to glycine betaine and related compatible solutes. We conclude that YehZYXW is a conserved, stress-regulated transport system that is phylogenetically and functionally distinct from quaternary ammonium-compatible solute importers.IMPORTANCE Brucella abortus E1 regulates transcription in response to stressors encountered in its mammalian host and is necessary for maintenance of chronic infection in a mouse model. The functions of the majority of genes regulated by E1 remain undefined. We present a functional/structural analysis of a conserved putative membrane transport system (YehZYXW) whose expression is strongly activated by E1 . Though annotated as a quaternary ammonium osmolyte uptake system, experimental physiological studies and measured ligand-binding properties of the periplasmic binding protein (PBP), YehZ, are inconsistent with this function. A crystal structure of B. abortus YehZ provides molecular insight into differences between bona fide quaternary ammonium osmolyte importers and YehZ-related proteins, which form a distinct phylogenetic and functional group of PBPs.

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Section: Figmentioning

confidence: 73%

“…6A). To confirm that stabilization was specific to YehZ, we also tested these molecules on purified IbpA, a C. crescentus PBP we previously characterized (42). No shift of T m was observed for IbpA, confirming that these molecules had a specific stabilization effect on YehZ.…”

Section: Figmentioning

confidence: 80%

Conserved ABC Transport System Regulated by the General Stress Response Pathways of Alpha- and Gammaproteobacteria

et al. 2017

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“…Analyses of ChpT by CD spectroscopy, ITC, and SAXS were performed using previously published methods (59,68,69) and are described in detail in Supporting Information. SAXS data on ChpT were collected at the Advanced Photon Source beamline 18-ID (BioCAT).…”

Section: Methodsmentioning

confidence: 99%

Structural asymmetry in a conserved signaling system that regulates division, replication, and virulence of an intracellular pathogen

Willett

Herrou

Briegel

et al. 2015

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

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We have functionally and structurally defined an essential protein phosphorelay that regulates expression of genes required for growth, division, and intracellular survival of the global zoonotic pathogen Brucella abortus. Our study delineates phosphoryl transfer through this molecular pathway, which initiates from the sensor kinase CckA and proceeds through the ChpT phosphotransferase to two regulatory substrates: CtrA and CpdR. Genetic perturbation of this system results in defects in cell growth and division site selection, and a specific viability deficit inside human phagocytic cells. Thus, proper control of B. abortus division site polarity is necessary for survival in the intracellular niche. We further define the structural foundations of signaling from the central phosphotransferase, ChpT, to its response regulator substrate, CtrA, and provide evidence that there are at least two modes of interaction between ChpT and CtrA, only one of which is competent to catalyze phosphoryltransfer. The structure and dynamics of the active site on each side of the ChpT homodimer are distinct, supporting a model in which quaternary structure of the 2:2 ChpT-CtrA complex enforces an asymmetric mechanism of phosphoryl transfer between ChpT and CtrA. Our study provides mechanistic understanding, from the cellular to the atomic scale, of a conserved transcriptional regulatory system that controls the cellular and infection biology of B. abortus. More generally, our results provide insight into the structural basis of two-component signal transduction, which is broadly conserved in bacteria, plants, and fungi.rucellosis, caused by Brucella spp., is among the most common zoonotic diseases worldwide (1). These intracellular pathogens are estimated to cause at least 500,000 new human infections each year and, in areas of Africa, Asia, and South America, inflict significant agricultural losses due to decreased livestock production (2, 3). Survival of Brucella within mammals is linked to their ability to infect and survive inside professional phagocytic cells (2). If left untreated in human hosts, Brucella eventually spread to multiple tissue types, which can lead to a range of debilitating chronic sequelae including reticuloendothelial, cardiovascular, gastrointestinal, and neurological damage.Brucella are members of the α-proteobacteria, a diverse class of Gram-negative species adapted for growth across a range of environmental conditions including plant surfaces and roots, aquatic and soil ecosystems, and the interior of mammalian cells (4, 5). Among the central regulatory systems controlling the α-proteobacterial cell cycle is a multistep phosphorelay composed of four proteins: (i) the hybrid sensor histidine kinase (HK) CckA, (ii) the histidine phosphotransferase ChpT, (iii) the DNA-binding response regulator CtrA, and (iv) the phospho-receiver protein CpdR (Fig. 1). Our current understanding of this conserved regulatory system is based largely on studies of the related aquatic bacterium Caulobacter crescentus (6, 7). In Ca...

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“…The substrate-binding protein is the prime determinant of selectivity in ABC-type import systems Berntsson et al, 2010;Eitinger et al, 2011;Herrou and Crosson, 2013;Scheepers et al, 2016). However, a low-affinity substrate-binding site positioned in the TMDs of the maltose/maltodextrin ABC system, a so-called type-I ABC importer (Locher, 2016;Lewinson and Livnat-Levanon, 2017), has been detected genetically and confirmed through crystallographic analysis (Shuman, 1982;Covitz et al, 1994;Boos and Shuman, 1998;Oldham et al, 2007;2013;Oldham and Chen, 2011;Chen, 2013).…”

Section: Introductionmentioning

confidence: 99%

From substrate specificity to promiscuity: hybrid ABC transporters for osmoprotectants

Teichmann

Chen

Hoffmann

et al. 2017

Molecular Microbiology

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The ABC-transporters OpuB and OpuC from Bacillus subtilis function as osmoprotectant import systems. Their structural genes have most likely evolved through a duplication event but the two transporters are remarkably different in their substrate profile. OpuB possesses narrow substrate specificity, while OpuC is promiscuous. We assessed the functionality of hybrids between these two ABC-transporters by reciprocally exchanging the coding regions for the OpuBC and OpuCC substrate-binding proteins between the corresponding opuB and opuC operons. Substantiating the critical role of the binding protein in setting the substrate specificity of ABC transporters, OpuB::OpuCC turned into a promiscuous system, while OpuC::OpuBC now exhibited narrow substrate specificity. Both hybrid transporters possessed a high affinity for their substrates but the transport capacity of the OpuB::OpuCC system was moderate due to the synthesis of only low amounts of the xenogenetic OpuCC protein. Suppressor mutations causing single amino acid substitutions in the GbsR repressor controlling the choline to glycine betaine biosynthesis pathway greatly improved OpuB::OpuCC-mediated compatible solute import through transcriptional up-regulation of the hybrid opuB::opuCC operon. Collectively, we demonstrate for the first time that one can synthetically switch the substrate specificity of a given ABC transporter by combining its core components with a xenogenetic ligand-binding protein.

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myo -Inositol and d -Ribose Ligand Discrimination in an ABC Periplasmic Binding Protein

Cited by 16 publications

References 69 publications

Conserved ABC Transport System Regulated by the General Stress Response Pathways of Alpha- and Gammaproteobacteria

Conserved ABC Transport System Regulated by the General Stress Response Pathways of Alpha- and Gammaproteobacteria

Structural asymmetry in a conserved signaling system that regulates division, replication, and virulence of an intracellular pathogen

From substrate specificity to promiscuity: hybrid ABC transporters for osmoprotectants

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