PhoP-PhoQ

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1 Introduction
2 PhoP-PhoQ and Virulence
3 3D Structures of PhoP-PhoQ

Introduction

PhoP-PhoQ is a two component regulatory system found in some gram-negative bacteria such as Escherichia coli[1], Salmonella enterica[2], and Yersinia pestis[3]. In a classic two component regulatory system, there exists a sensor kinase and a response regulator^[1]. In the phoP-phoQ system, phoQ acts as the sensor kinase and phoP acts as the response regulator. The purpose of this signal transduction system in bacteria is to modify cellular output in response to environmental signals. In response to particular environmental stimuli, such as a low [Mg²⁺], the sensor kinase, phoQ autophosphorylates. Phosphorylated phoQ then transphosphorylates the response regulator, phoP, which in turn binds DNA and modulates transcription.^[1]

1 PhoP: The Response Regulator
2 Dimerization of Activated phoP
3 Beryllofluoride Coordination
4 Spontaneous Dimerization

PhoP: The Response Regulator

E. coli phoP is a 223 residue protein containing a 120-residue regulatory domain joined by a 5-residue linker to a 98 residue c-terminal DNA binding effector domain.^[2] At a conserved Asp residue (), the regulatory domain can be modified by a phosphoryl group from the protein kinase function of phoQ. Phosphorylation of the regulatory domain modulates the activity of the effector domain to bind DNA and regulate transcription. Phosphorylated, or "activated" phoP binds to "phoP boxes" on bacterial DNA, which consist of two direct hexanucleotide repeats separated by a five nucleotide spacer located at the -35 position.^[2]: (T/G)GTTTA

Dimerization of Activated phoP

The activated phoP regulatory domain dimerizes with two-fold symmetry using the face formed by ^[2]. The phosphoryl group donated from phoQ is used to stabilize the active conformation by inducing dimerization. Due to the absence of an intramolecular domain interface which precludes direct transmission of an activation signal, this function as a dimerization motif seems to be the phosphoryl group's only role.^[2]

Beryllofluoride Coordination

In this structure, (BeF₃^-) is used as a phosphoryl analog to induce the active state conformation. There are numerous bonds that form as a result of BeF₃^- coordination. F₁ of BeF₃^- helps satisfy the octahedral coordination of a present active site ²⁺. The conserved active site lysine, , forms important intramolecular and intermolecular salt bridges, one of which is with F₃ of BeF₃^-. A conserved "switch residue" , involved in the activation of all response regulators, forms a H-bond with F₂ of BeF₃^-. F₂ of BeF₃^- also forms a H-bond with the backbone nitrogen atom of .^[2]

Spontaneous Dimerization

Spontaneous dimerization of the unactivated phoP regulatory domain can be observed in vitro, but is likely due to high concentration of the protein and may not occur in vivo. Although the dimerization of the unactivated phoP regulatory domain results in a homodimer similar to the activated homodimer, it is significantly less stable. Activation by the phosphoryl group helps to stabilize the dimerization interphase. Without the phosphoryl group, the two monomers dimerize in an asymmetric fashion that lends to molecular instability.

PhoP-PhoQ and Virulence

Most known virulence factors have been isolated by designing laboratory conditions that presumably stimulate environmental signals present in host tissues. However, many pathogenic bacteria do not express their virulence factors until specific host signals are detected, signals which are near impossible to accurately reproduce in a laboratory. To overcome this, a new approach entitled IVET (in vivo expression technology) has been developed. IVET uses animal tissue as the selective medium to enrich bacterial virulence factors specifically induced during infection.^[3]

Pathogenic bacteria seldom express virulence genes constitutively, they instead need to be able to express the correct virulence genes in the correct environment. Not all virulence factors confer a selective advantage to the microbe at the same stage of infection. Thus, it is the job of the phoP-phoQ system to modulate virulence gene expression according to the cellular micro-environment.^[1]

A particular and well studied environmental factor relative to the phoP-phoQ system is [Mg²⁺]. In response to low [Mg²⁺], such as would be found inside a macrophage phagosome, phoQ autophosphorylates and transphorphorylates phoP. PhoP then binds to the bacterial DNA and simultaneously activates the expression of pags (phoP activated genes) and represses the expression of prgs (phoP repressed genes). Among the gene products of pags are proteins necessary to survive inside the macrophage, a critical stage of Salmonella Typhirium virulence. Among the gene products of prgs are proteins necessary for invasion and infection of the host, which are less important once in a host macrophage's phagosome.

Two component regulatory systems such as phoP-phoQ are obviously an attractive target for future antimicrobial drugs. If the phoP-phoQ can be altered to a dysfunctional state, the relevant bacteria would have decreased pathogenicity and increased suceptibility to nonspecific immune defense.

3D Structures of PhoP-PhoQ

PhoP

3r0j – MtPhoP – Mycobacterium tuberculosis
2pmu – MtPhoP DNA-binding domain
2pkx – EcPhoP regulatory domain (mutant) – Escherichia coli
2pl1 - EcPhoP regulatory domain (mutant) + BeF3
1mvo – PhoP N terminal – Bacillus subtilis

PhoQ

3cgy – StPhoQ catalytic domain + radicicol – Salmonella typhimurium
3cgz - StPhoQ catalytic domain
1yax - StPhoQ sensor domain (mutant)
3bq8 – EcPhoQ
3bqa – EcPhoQ (mutant)
1id0 – EcPhoQ kinase domain

References

↑ ^1.0 ^1.1 ^1.2 Hoch JA. Two-component and phosphorelay signal transduction. Curr Opin Microbiol. 2000 Apr;3(2):165-70. PMID:10745001
↑ ^2.0 ^2.1 ^2.2 ^2.3 ^2.4 Bachhawat P, Stock AM. Crystal structures of the receiver domain of the response regulator PhoP from Escherichia coli in the absence and presence of the phosphoryl analog beryllofluoride. J Bacteriol. 2007 Aug;189(16):5987-95. Epub 2007 Jun 1. PMID:17545283 doi:10.1128/JB.00049-07
↑ Angelichio MJ, Camilli A. In vivo expression technology. Infect Immun. 2002 Dec;70(12):6518-23. PMID:12438320

Proteopedia Page Contributors and Editors (what is this?)

Andrew Brockfield, Michal Harel, Alexander Berchansky

Retrieved from "http://52.214.119.220/wiki/index.php/PhoP-PhoQ"

Category: Topic Page

@@ Line 14: / Line 14: @@
 ----
-E. coli phoP is a 223 residue protein containing a 120-residue regulatory domain joined by a 5-residue linker to a 98 residue c-terminal DNA binding effector domain.<ref name=Bachhawat>PMID:17545283</ref> At a conserved Asp residue, the regulatory domain can be modified by a phosphoryl group from the protein kinase function of phoQ. Phosphorylation of the regulatory domain modulates the activity of the effector domain to bind DNA and regulate transcription. Phosphorylated, or "activated" phoP binds to "phoP boxes" on bacterial DNA, which consist of two direct hexanucleotide repeats separated by a five nucleotide spacer located at the -35 position.<ref name=Bachhawat>PMID:17545283</ref>:
+E. coli phoP is a 223 residue protein containing a 120-residue regulatory domain joined by a 5-residue linker to a 98 residue c-terminal DNA binding effector domain.<ref name=Bachhawat>PMID:17545283</ref> At a conserved Asp residue (<scene name='PhoP-PhoQ/Asp51-bef3/1'>Asp51 in this case</scene>), the regulatory domain can be modified by a phosphoryl group from the protein kinase function of phoQ. Phosphorylation of the regulatory domain modulates the activity of the effector domain to bind DNA and regulate transcription. Phosphorylated, or "activated" phoP binds to "phoP boxes" on bacterial DNA, which consist of two direct hexanucleotide repeats separated by a five nucleotide spacer located at the -35 position.<ref name=Bachhawat>PMID:17545283</ref>:
 (T/G)GTTTA
@@ Line 33: / Line 33: @@
 ----
-Spontaneous dimerization of the unactivated phoP regulatory domain can be observed in vitro, but is likely due to high concentration of the protein and may not occur in vivo. Although the dimerization of the unactivated phoP regulatory domain results in a homodimer similar to the activated homodimer, it is significantly less stable. Activation by the phosphoryl group helps to stabilize the α-4 helix, β-5 sheet and α-5 helix dimerization interphase. Without the phosphoryl group, the two monomers dimerize in an asymmetric fashion that lends to molecular instability.
+Spontaneous dimerization of the unactivated phoP regulatory domain can be observed in vitro, but is likely due to high concentration of the protein and may not occur in vivo. Although the dimerization of the unactivated phoP regulatory domain results in a homodimer similar to the activated homodimer, it is significantly less stable. Activation by the phosphoryl group helps to stabilize the <scene name='Sandbox_Reserved_344/Dimerization_surface/1'>α-4 helix, β-5 sheet and α-5 helix face</scene> dimerization interphase. Without the phosphoryl group, the two monomers dimerize in an asymmetric fashion that lends to molecular instability.
 </StructureSection>
 ===PhoP-PhoQ and Virulence===