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, 287 (33), 27731-42

Determinants of Interaction Specificity of the Bacillus Subtilis GlcT Antitermination Protein: Functionality and Phosphorylation Specificity Depend on the Arrangement of the Regulatory Domains

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Determinants of Interaction Specificity of the Bacillus Subtilis GlcT Antitermination Protein: Functionality and Phosphorylation Specificity Depend on the Arrangement of the Regulatory Domains

Sebastian Himmel et al. J Biol Chem.

Abstract

The control of several catabolic operons in bacteria by transcription antitermination is mediated by RNA-binding proteins that consist of an RNA-binding domain and two reiterated phosphotransferase system regulation domains (PRDs). The Bacillus subtilis GlcT antitermination protein regulates the expression of the ptsG gene, encoding the glucose-specific enzyme II of the phosphotransferase system. In the absence of glucose, GlcT becomes inactivated by enzyme II-dependent phosphorylation at its PRD1, whereas the phosphotransferase HPr phosphorylates PRD2. However, here we demonstrate by NMR analysis and mass spectrometry that HPr also phosphorylates PRD1 in vitro but with low efficiency. Size exclusion chromatography revealed that non-phosphorylated PRD1 forms dimers that dissociate upon phosphorylation. The effect of HPr on PRD1 was also investigated in vivo. For this purpose, we used GlcT variants with altered domain arrangements or domain deletions. Our results demonstrate that HPr can target PRD1 when this domain is placed at the C terminus of the protein. In agreement with the in vitro data, HPr exerts a negative control on PRD1. This work provides the first insights into how specificity is achieved in a regulator that contains duplicated regulatory domains with distinct dimerization properties that are controlled by phosphorylation by different phosphate donors. Moreover, the results suggest that the domain arrangement of the PRD-containing antitermination proteins is under selective pressure to ensure the proper regulatory output, i.e. transcription antitermination of the target genes specifically in the presence of the corresponding sugar.

Figures

FIGURE 1.
FIGURE 1.
HPr-dependent PRD1 phosphorylation monitored by NMR. A series of 1H,15N heteronuclear single quantum correlation spectra shows the gradually increasing phosphorylation of isolated PRD1. A, the spectrum of non-phosphorylated PRD1. B–D, spectra of PRD1 phosphorylation mixtures, which contained different compositions of phosphorylation enzymes as indicated in the insets. The phosphorylation effect can be observed by additional and shifted amide resonances. A selection of resonances (Glu-140, Glu-132, and Tyr-141) highlights the spectral changes upon phosphorylation. E, the spectrum of nearly completely phosphorylated PRD1. Phosphorylation of PRD1 was maximal at an equimolar ratio of HPr to PRD1. Folded peaks are plotted with dashed contour lines. EI and EII, enzymes I and II, respectively.
FIGURE 2.
FIGURE 2.
ESI mass spectrum after tryptic digest of phosphorylated PRD1. MS/MS analysis of PRD1 revealed His-170 and/or His-172 as being phosphorylated. The inset shows the MS analysis of peptide AGLCLPEGEIGFIALHIHSALTNRPLSEVNQEFIV derived from PRD1 after hydrolysis of the protein with trypsin endoproteinase (see peptide T9 in supplemental Fig. S2B). The mass of the triply charged peptide was determined as m/z 1362.0109. MS/MS analysis under higher energy collisional dissociation (HCD) conditions showed only a loss of 79 Da (m/z 26.6 as indicated to the right of the signal at m/z 1362.0), generating a peak at m/z 1335.4. The neutral loss of 79 Da is characteristic for HPO3 and strongly suggests His-170 and/or His-172 as phosphorylation site(s). Of note, phosphorylation of serine or threonine residues always results in neutral loss of H3PO4 (98 Da) under collision-induced dissociation conditions. Thus, we conclude that His-170 and/or His-172 is the actual phosphorylation site of this peptide. The listed sequence of the peptide was determined by MS3 experiments on the dephosphorylated peak at m/z 1335.4 (supplemental Fig. S2C).
FIGURE 3.
FIGURE 3.
ESI mass spectrum of phosphorylated PRD1 H172A. A PRD1 H172A phosphorylation mixture shows non-phosphorylated PRD1 H172A (A, 13,178.50 ± 6.36 Da), phosphorylated PRD1 H172A (B, 13,257.53 ± 5.76 Da), and phosphorylated HPr (C, 11,420.13 ± 5.50 Da). Individual charges (z) of A, B, and C are indicated by numbers 7-14. A single phosphate group upon HPr-dependent PRD1 phosphorylation was identified by a mass difference of 79.03 Da.
FIGURE 4.
FIGURE 4.
Size exclusion chromatography elution profiles of PRD1 before and after HPr-dependent phosphorylation. A, elution profiles of non-phosphorylated (solid line) and phosphorylated (dashed line) PRD1 (Superdex 75 16/60). A molar ratio of 1:1 PRD1/HPr was used for phosphorylation. Upon phosphorylation, the elution volume of the non-phosphorylated PRD1 dimer significantly changed from 76 ml to a phosphorylated monomer at 81 ml. B, SDS-PAGE analysis of phosphorylated PRD1. Upon phosphorylation, PRD1 was detected in both peaks (elution volumes of 76 and 81 ml), corresponding to the dimer and monomer. In the SDS-polyacrylamide gel, the band positions of HPr and PRD1 are inverted compared with their molecular masses (lane M). EI, enzyme I.
FIGURE 5.
FIGURE 5.
Size exclusion chromatography elution profiles of RBD-PRD1 before and after HPr-dependent phosphorylation. A, elution profiles of non-phosphorylated (solid line) and phosphorylated (dashed line) RBD-PRD1 (Superdex 75 10/300). Elution volumes and aggregation states are indicated. A molar ratio of 1:1 RBD-PRD1/HPr was used for phosphorylation. Upon phosphorylation, the elution volume of the non-phosphorylated RBD-PRD1 dimer significantly changed from 11.0 ml to a phosphorylated monomer at 11.9 ml. mAU, milli-absorbance units. B and C, SDS-PAGE analysis of non-phosphorylated and phosphorylated RBD-PRD1, respectively. RBD-PRD1 was identified at both elution volumes (11.0 and 11.9 ml), pointing to the dimeric and monomeric states, respectively. M, molecular mass markers; EI, enzyme I.
FIGURE 6.
FIGURE 6.
Comparison of size exclusion chromatography elution profiles of GlcT H218D/H279D before and after HPr-dependent phosphorylation. A, elution profiles of non-phosphorylated (solid line) and phosphorylated (dashed and dotted lines) GlcT H218D/H279D (Superdex 75 10/300). To phosphorylate GlcT H218D/H279D, 1:0.125 (dotted line) and 1:1 (dashed line) ratios of GlcT H218D/H279D to HPr were used. Upon phosphorylation, the elution volume of the non-phosphorylated GlcT H218D/H279D dimer significantly changed from 9.5 ml to a phosphorylated monomer at 10.6 ml. mAU, milli-absorbance units. B and C, SDS-PAGE analysis of non-phosphorylated and phosphorylated (1:1 ratio) GlcT H218D/H279D, respectively. The gels confirm the dimeric and monomeric forms of GlcT H218D/H279D before and after phosphorylation, respectively. M, molecular mass markers; EI, enzyme I.
FIGURE 7.
FIGURE 7.
Regulatory effects of GlcT phosphorylation and domain properties. A–C, three different states of GlcT and their regulatory role in transcription termination/antitermination based on the protein-dependent RNA switch of the ptsG mRNA are shown. HPr- and enzyme II-dependent phosphorylation of PRD1 (A) prevents dimer stabilization and causes GlcT monomerization. This state results in ptsG expression that is equivalent to ∼20–50 units of β-galactosidase (Table 3). Non-phosphorylated GlcT (B) intrinsically binds the ptsG RNA antiterminator (RAT) sequence to prevent transcription termination. This antitermination scenario is enhanced by HPr-dependent phosphorylation of PRD2 (C). These forms of GlcT allow ptsG expression levels that correspond to 300–700 or >800 units of β-galactosidase, respectively (see Table 3). D and E, the impact of phosphorylation of PRD1 is shown for GlcT and a GlcT variant with shuffled PRDs, respectively. The domain organization of the RBD, PRD1, and PRD2 ensures that upon PRD1 phosphorylation, the GlcT dimers dissociate to the inactive monomers. In contrast, the intrinsic ability of PRD2 to form a stable dimer enhances the stability of the shuffled GlcT variant, resulting in constitutive antitermination, irrespective of the PRD1 phosphorylation state.

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