Experimental and computational characterization of the dimerization of the PTS-regulation domains of BglG from Escherichia coli

Efrat Ben-Zeev; Liat Fux; Orna Amster-Choder; Miriam Eisenstein

doi:10.1016/j.jmb.2005.01.068

Experimental and computational characterization of the dimerization of the PTS-regulation domains of BglG from Escherichia coli

Efrat Ben-Zeev, Liat Fux, Orna Amster-Choder, Miriam Eisenstein^*

^*Corresponding author for this work

Department of Microbiology and Molecular Genetics

Research output: Contribution to journal › Article › peer-review

13 Scopus citations

Abstract

BglG and LicT are transcriptional antiterminators from Escherichia coli and Bacillus subtilis, respectively, that control the expression of genes and operons involved in transport and catabolism of carbohydrates. Both proteins contain a duplicate conserved domain, the PTS-regulation domain (PRD), and they are regulated by phosphorylation on specific, highly conserved histidine residues located in the PRDs. However, despite their similar function and the high sequence identity, experimental evidence implies different modes of regulation. Thus, BglG must be de-phosphorylated on PRD2 in order to form active dimers, whereas activation of LicT requires de-phosphorylation on PRD1 and phosphorylation on PRD2. Here we address two goals. First, we test in vivo and in silico the effect of point mutations in the PRDs of BglG on the PRD-PRD dimerization. Second, we explore computationally the effect of histidine phosphorylation on PRD dimerization in BglG and LicT. We find excellent correspondence between the experimental and computational measures of the influence of mutations on PRD dimerization in BglG. This establishes that the geometric-electrostatic complementarity scores computed with the program MolFit provide a good measure of the effects of mutations in this system. In addition, it indicates that the dimerization mode of the separately expressed PRDs of BglG is similar to the dimers formed by activated LicT. The computations also show that phosphorylation of the histidine residues in PRD1 of either BglG or LicT leads to a strong electrostatic repulsion. Conversely, the phosphorylation of one histidine residue in PRD2 of LicT leads to improved electrostatic complementarity at the PRD2-PRD2 interface, whereas the corresponding phosphorylation in BglG has negligible contribution. This different conduct may be attributed to a single replacement in the sequence of PRD2 in BglG compared to LicT, Ala262 versus Asp261, respectively.

Original language	English
Pages (from-to)	693-706
Number of pages	14
Journal	Journal of Molecular Biology
Volume	347
Issue number	4
DOIs	https://doi.org/10.1016/j.jmb.2005.01.068
State	Published - 8 Apr 2005

Bibliographical note

Funding Information:
We thank Professor Ephraim Katchalski-Katzir for his advice and support throughout this study. We thank Yifat Weise for help with molecular cloning and β-galactosidase assays. The computational part of this study was supported in part by the Kimmelman center for Biomolecular Structure and Assembly at the Weizmann Institute of Science. The experimental part of this study was supported by a grant from the Israel Science Foundation founded by the Israel Academy of Sciences and Humanities (awarded to O.A.C.).

Keywords

Electrostatic desolvation energy
Geometric-electrostatic docking
Protein recognition
Regulation of BglG
Transcription antitermination

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10.1016/j.jmb.2005.01.068

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title = "Experimental and computational characterization of the dimerization of the PTS-regulation domains of BglG from Escherichia coli",

abstract = "BglG and LicT are transcriptional antiterminators from Escherichia coli and Bacillus subtilis, respectively, that control the expression of genes and operons involved in transport and catabolism of carbohydrates. Both proteins contain a duplicate conserved domain, the PTS-regulation domain (PRD), and they are regulated by phosphorylation on specific, highly conserved histidine residues located in the PRDs. However, despite their similar function and the high sequence identity, experimental evidence implies different modes of regulation. Thus, BglG must be de-phosphorylated on PRD2 in order to form active dimers, whereas activation of LicT requires de-phosphorylation on PRD1 and phosphorylation on PRD2. Here we address two goals. First, we test in vivo and in silico the effect of point mutations in the PRDs of BglG on the PRD-PRD dimerization. Second, we explore computationally the effect of histidine phosphorylation on PRD dimerization in BglG and LicT. We find excellent correspondence between the experimental and computational measures of the influence of mutations on PRD dimerization in BglG. This establishes that the geometric-electrostatic complementarity scores computed with the program MolFit provide a good measure of the effects of mutations in this system. In addition, it indicates that the dimerization mode of the separately expressed PRDs of BglG is similar to the dimers formed by activated LicT. The computations also show that phosphorylation of the histidine residues in PRD1 of either BglG or LicT leads to a strong electrostatic repulsion. Conversely, the phosphorylation of one histidine residue in PRD2 of LicT leads to improved electrostatic complementarity at the PRD2-PRD2 interface, whereas the corresponding phosphorylation in BglG has negligible contribution. This different conduct may be attributed to a single replacement in the sequence of PRD2 in BglG compared to LicT, Ala262 versus Asp261, respectively.",

keywords = "Electrostatic desolvation energy, Geometric-electrostatic docking, Protein recognition, Regulation of BglG, Transcription antitermination",

author = "Efrat Ben-Zeev and Liat Fux and Orna Amster-Choder and Miriam Eisenstein",

note = "Funding Information: We thank Professor Ephraim Katchalski-Katzir for his advice and support throughout this study. We thank Yifat Weise for help with molecular cloning and β-galactosidase assays. The computational part of this study was supported in part by the Kimmelman center for Biomolecular Structure and Assembly at the Weizmann Institute of Science. The experimental part of this study was supported by a grant from the Israel Science Foundation founded by the Israel Academy of Sciences and Humanities (awarded to O.A.C.).",

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AU - Fux, Liat

AU - Amster-Choder, Orna

AU - Eisenstein, Miriam

N1 - Funding Information: We thank Professor Ephraim Katchalski-Katzir for his advice and support throughout this study. We thank Yifat Weise for help with molecular cloning and β-galactosidase assays. The computational part of this study was supported in part by the Kimmelman center for Biomolecular Structure and Assembly at the Weizmann Institute of Science. The experimental part of this study was supported by a grant from the Israel Science Foundation founded by the Israel Academy of Sciences and Humanities (awarded to O.A.C.).

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N2 - BglG and LicT are transcriptional antiterminators from Escherichia coli and Bacillus subtilis, respectively, that control the expression of genes and operons involved in transport and catabolism of carbohydrates. Both proteins contain a duplicate conserved domain, the PTS-regulation domain (PRD), and they are regulated by phosphorylation on specific, highly conserved histidine residues located in the PRDs. However, despite their similar function and the high sequence identity, experimental evidence implies different modes of regulation. Thus, BglG must be de-phosphorylated on PRD2 in order to form active dimers, whereas activation of LicT requires de-phosphorylation on PRD1 and phosphorylation on PRD2. Here we address two goals. First, we test in vivo and in silico the effect of point mutations in the PRDs of BglG on the PRD-PRD dimerization. Second, we explore computationally the effect of histidine phosphorylation on PRD dimerization in BglG and LicT. We find excellent correspondence between the experimental and computational measures of the influence of mutations on PRD dimerization in BglG. This establishes that the geometric-electrostatic complementarity scores computed with the program MolFit provide a good measure of the effects of mutations in this system. In addition, it indicates that the dimerization mode of the separately expressed PRDs of BglG is similar to the dimers formed by activated LicT. The computations also show that phosphorylation of the histidine residues in PRD1 of either BglG or LicT leads to a strong electrostatic repulsion. Conversely, the phosphorylation of one histidine residue in PRD2 of LicT leads to improved electrostatic complementarity at the PRD2-PRD2 interface, whereas the corresponding phosphorylation in BglG has negligible contribution. This different conduct may be attributed to a single replacement in the sequence of PRD2 in BglG compared to LicT, Ala262 versus Asp261, respectively.

AB - BglG and LicT are transcriptional antiterminators from Escherichia coli and Bacillus subtilis, respectively, that control the expression of genes and operons involved in transport and catabolism of carbohydrates. Both proteins contain a duplicate conserved domain, the PTS-regulation domain (PRD), and they are regulated by phosphorylation on specific, highly conserved histidine residues located in the PRDs. However, despite their similar function and the high sequence identity, experimental evidence implies different modes of regulation. Thus, BglG must be de-phosphorylated on PRD2 in order to form active dimers, whereas activation of LicT requires de-phosphorylation on PRD1 and phosphorylation on PRD2. Here we address two goals. First, we test in vivo and in silico the effect of point mutations in the PRDs of BglG on the PRD-PRD dimerization. Second, we explore computationally the effect of histidine phosphorylation on PRD dimerization in BglG and LicT. We find excellent correspondence between the experimental and computational measures of the influence of mutations on PRD dimerization in BglG. This establishes that the geometric-electrostatic complementarity scores computed with the program MolFit provide a good measure of the effects of mutations in this system. In addition, it indicates that the dimerization mode of the separately expressed PRDs of BglG is similar to the dimers formed by activated LicT. The computations also show that phosphorylation of the histidine residues in PRD1 of either BglG or LicT leads to a strong electrostatic repulsion. Conversely, the phosphorylation of one histidine residue in PRD2 of LicT leads to improved electrostatic complementarity at the PRD2-PRD2 interface, whereas the corresponding phosphorylation in BglG has negligible contribution. This different conduct may be attributed to a single replacement in the sequence of PRD2 in BglG compared to LicT, Ala262 versus Asp261, respectively.

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KW - Geometric-electrostatic docking

KW - Protein recognition

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Experimental and computational characterization of the dimerization of the PTS-regulation domains of BglG from Escherichia coli

Abstract

Bibliographical note

Keywords

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