Expression, purification, crystallization and preliminary X-ray analysis of the DNA-binding domain of a<i>Chlamydia trachomatis</i>OmpR/PhoB-subfamily response regulator homolog, ChxR

Hickey, John M.; Hefty, P. Scott; Lamb, Audrey L.

doi:10.1107/s1744309109025184

Cited by 5 publications

(5 citation statements)

References 11 publications

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“…The DNA was visualized and quantified using an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE). The effector domain of ChxR used in this assay was purified as described previously (37).…”

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confidence: 99%

The Atypical Response Regulator Protein ChxR Has Structural Characteristics and Dimer Interface Interactions That Are Unique within the OmpR/PhoB Subfamily

Hickey¹,

Lovell

Battaile

et al. 2011

Journal of Biological Chemistry

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Typically as a result of phosphorylation, OmpR/PhoB response regulators form homodimers through a receiver domain as an integral step in transcriptional activation. Phosphorylation stabilizes the ionic and hydrophobic interactions between monomers. Recent studies have shown that some response regulators retain functional activity in the absence of phosphorylation and are termed atypical response regulators. The two currently available receiver domain structures of atypical response regulators are very similar to their phospho-accepting homologs, and their propensity to form homodimers is generally retained. An atypical response regulator, ChxR, from Chlamydia trachomatis, was previously reported to form homodimers; however, the residues critical to this interaction have not been elucidated. We hypothesize that the intra-and intermolecular interactions involved in forming a transcriptionally competent ChxR are distinct from the canonical phosphorylation (activation) paradigm in the OmpR/PhoB response regulator subfamily. To test this hypothesis, structural and functional studies were performed on the receiver domain of ChxR. Two crystal structures of the receiver domain were solved with the recently developed method using triiodo compound I3C. These structures revealed many characteristics unique to OmpR/PhoB subfamily members: typical or atypical. Included was the absence of two ␣-helices present in all other OmpR/ PhoB response regulators. Functional studies on various dimer interface residues demonstrated that ChxR forms relatively stable homodimers through hydrophobic interactions, and disruption of these can be accomplished with the introduction of a charged residue within the dimer interface. A gel shift study with monomeric ChxR supports that dimerization through the receiver domain is critical for interaction with DNA.

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confidence: 99%

The Atypical Response Regulator Protein ChxR Has Structural Characteristics and Dimer Interface Interactions That Are Unique within the OmpR/PhoB Subfamily

Hickey¹,

Lovell

Battaile

et al. 2011

Journal of Biological Chemistry

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“…The effector domain of ChxR (ChxR Eff ) and full-length ChxR (ChxR) were purified as previously described [17] , [18] . Briefly, each protein was expressed in E. coli BL21(DE3) (Invitrogen, Carlsbad, CA) and initially purified through metal (Co 2+ ) affinity chromatography.…”

Section: Methodsmentioning

confidence: 99%

“…Site-directed Mutagenesis - Mutations were introduced into the full-length ChxR plasmid [17] , [18] using the QuikChange II XL site-directed mutagenesis kit by following the manufacturer’s protocol (Agilent Technologies, La Jolla, CA). All clones were verified by DNA sequencing analysis (ACGT, Inc., Wheeling, IL).…”

Section: Methodsmentioning

confidence: 99%

Atypical Response Regulator ChxR from Chlamydia trachomatis Is Structurally Poised for DNA Binding

et al. 2014

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ChxR is an atypical two-component signal transduction response regulator (RR) of the OmpR/PhoB subfamily encoded by the obligate intracellular bacterial pathogen Chlamydia trachomatis. Despite structural homology within both receiver and effector domains to prototypical subfamily members, ChxR does not require phosphorylation for dimer formation, DNA binding or transcriptional activation. Thus, we hypothesized that ChxR is in a conformation optimal for DNA binding with limited interdomain interactions. To address this hypothesis, the NMR solution structure of the ChxR effector domain was determined and used in combination with the previously reported ChxR receiver domain structure to generate a full-length dimer model based upon SAXS analysis. Small-angle scattering of ChxR supported a dimer with minimal interdomain interactions and effector domains in a conformation that appears to require only subtle reorientation for optimal major/minor groove DNA interactions. SAXS modeling also supported that the effector domains were in a head-to-tail conformation, consistent with ChxR recognizing tandem DNA repeats. The effector domain structure was leveraged to identify key residues that were critical for maintaining protein - nucleic acid interactions. In combination with prior analysis of the essential location of specific nucleotides for ChxR recognition of DNA, a model of the full-length ChxR dimer bound to its cognate cis-acting element was generated.

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“…Interestingly, C. trachomatis encodes fewer than 20 TFs despite a complicated developmental cycle (16). Most of these TFs regulate gene expression in response to nutrient and mineral availability (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). In contrast, the transcription repressor HrcA controls response to heat shock (31,32).…”

Section: Introductionmentioning

confidence: 99%

GrgA controlsChlamydia trachomatisgrowth and development by regulating expression of transcription factors Euo and HrcA

Wurihan

Zou

Weber

et al. 2020

Preprint

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ABSTRACTThe obligate intracellular bacterium Chlamydia trachomatis is an important human pathogen whose biphasic developmental cycle consists of an infectious elementary body and a replicative reticulate body. Whereas σ66, the primary sigma factor, is necessary for transcription of most chlamydial genes throughout the developmental cycle, σ28 is required for expression of some late genes. We previously showed that the Chlamydia-specific transcription factor GrgA physically interacts with both of these sigma factors and activates transcription from σ66- and σ28-dependent promoters in vitro. Here, we investigate the organismal functions of GrgA. We show that GrgA overexpression decreased RB proliferation via time-dependent transcriptomic changes. Significantly, σ66-dependent genes that code for two important transcription repressors are among the direct targets of GrgA. One of these repressors is Euo, which prevents the expression of late genes during early phases. The other is HrcA, which regulates gene expression in response to heat shock. The direct regulon of GrgA also includes a σ28-dependent gene that codes for the putative virulence factor PmpI. Conditional overexpression of Euo and HrcA also inhibited chlamydial growth and affected GrgA expression. Transcriptomic studies suggest that GrgA, Euo, and HrcA have distinct but overlapping indirect regulons. Furthermore, overexpression of either GrgA leads to decreased expression of numerous tRNAs. These findings indicate that a GrgA-mediated transcriptional regulatory network controls C. trachomatis growth and development.IMPORTANCEChlamydia trachomatis is the most prevalent sexually transmitted bacterial pathogen worldwide and is a leading cause of preventable blindness in under-developed areas as well as developed countries. Previous studies showed that the novel transcription factor GrgA activated chlamydial gene transcription in vitro, but did not addressed the organismal function of GrgA. Here, we demonstrate growth inhibition in C. trachomatis engineered to conditionally overexpress GrgA. GrgA overexpression immediately increases the expression of two other critical transcription factors (Euo and HrcA) and a candidate virulence factor (PmpI), among several other genes. We also reveal chlamydial growth reduction and transcriptomic changes including decreased GrgA mRNA levels in response to either Euo or HrcA overexpression. Thus, the transcription network controlled by GrgA likely plays a crucial role in chlamydial growth and pathogenesis.

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Expression, purification, crystallization and preliminary X-ray analysis of the DNA-binding domain of aChlamydia trachomatisOmpR/PhoB-subfamily response regulator homolog, ChxR

Cited by 5 publications

References 11 publications

The Atypical Response Regulator Protein ChxR Has Structural Characteristics and Dimer Interface Interactions That Are Unique within the OmpR/PhoB Subfamily

The Atypical Response Regulator Protein ChxR Has Structural Characteristics and Dimer Interface Interactions That Are Unique within the OmpR/PhoB Subfamily

Atypical Response Regulator ChxR from Chlamydia trachomatis Is Structurally Poised for DNA Binding

GrgA controlsChlamydia trachomatisgrowth and development by regulating expression of transcription factors Euo and HrcA

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