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New insights into histidine triad proteins: solution structure of a Streptococcus pneumoniae PhtD domain and zinc transfer to AdcAII.

Bersch B, Bougault C, Roux L, Favier A, Vernet T, Durmort C - PLoS ONE (2013)

Bottom Line: The high resolution NMR structure of t-PhtD shows that Zn(2+) is bound in a tetrahedral site by histidines 83, 86, and 88 as well as by glutamate 63.Comparison of the NMR parameters measured for apo- and Zn(2+)-t-PhtD shows that the loss of Zn(2+) leads to a diminished helical propensity at the C-terminus and increases the local dynamics and overall molecular volume.Structural comparison with the crystal structure of a 55-long fragment of PhtA suggests that Pht proteins are built from short repetitive units formed by three β-strands containing the conserved HxxHxH motif.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie Structurale, Université Grenoble Alpes, Grenoble, France ; Institut de Biologie Structurale, Direction des Sciences du Vivant, Commissariat à l'Energie Atomique et aux Energies Alternatives, Grenoble, France ; Institut de Biologie Structurale, Centre National de la Recherche Scientifique, Grenoble, France.

ABSTRACT
Zinc (Zn(2+)) homeostasis is critical for pathogen host colonization and invasion. Polyhistidine triad (Pht) proteins, located at the surface of various streptococci, have been proposed to be involved in Zn(2+) homeostasis. The phtD gene, coding for a Zn(2+)-binding protein, is organized in an operon with adcAII coding for the extracellular part of a Zn(2+) transporter. In the present work, we investigate the relationship between PhtD and AdcAII using biochemical and structural biology approaches. Immuno-precipitation experiments on purified membranes of Streptococcus pneumoniae (S. pneumoniae) demonstrate that native PhtD and AdcAII interact in vivo confirming our previous in vitro observations. NMR was used to demonstrate Zn(2+) transfer from the Zn(2+)-bound form of a 137 amino acid N-terminal domain of PhtD (t-PhtD) to AdcAII. The high resolution NMR structure of t-PhtD shows that Zn(2+) is bound in a tetrahedral site by histidines 83, 86, and 88 as well as by glutamate 63. Comparison of the NMR parameters measured for apo- and Zn(2+)-t-PhtD shows that the loss of Zn(2+) leads to a diminished helical propensity at the C-terminus and increases the local dynamics and overall molecular volume. Structural comparison with the crystal structure of a 55-long fragment of PhtA suggests that Pht proteins are built from short repetitive units formed by three β-strands containing the conserved HxxHxH motif. Taken together, these results support a role for S. pneumoniae PhtD as a Zn(2+) scavenger for later release to the surface transporter AdcAII, leading to Zn(2+) uptake.

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Comparison of apo- and Zn2+-t-PhtD.(A) Weighted chemical shift difference determined for the amide resonances between the two forms (Δδ  =  [(Δδ1H)2+(0.01 Δδ15N)2]1/2). The insert shows a color-coded projection of the chemical shift differences onto the ribbon structure of Zn2+-t-PhtD. Residues for which no resonance could be identified in the 1H,15N-HSQC spectrum of apo-t-PhtD are shown in orange and proline residues in white. Residues with significant chemical shift differences are identified by their residue numbers. (B) Ratio of transverse (R2) and longitudinal (R1) relaxation rates and (C) {1H}15N-heteronuclear NOE for apo- (red squares) and Zn2+-t-PhtD (black triangles). Secondary structure elements identified in the structure of Zn2+-t-PhtD are indicated on the top of the figure and by grey shadows in each of the panels.
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pone-0081168-g004: Comparison of apo- and Zn2+-t-PhtD.(A) Weighted chemical shift difference determined for the amide resonances between the two forms (Δδ  =  [(Δδ1H)2+(0.01 Δδ15N)2]1/2). The insert shows a color-coded projection of the chemical shift differences onto the ribbon structure of Zn2+-t-PhtD. Residues for which no resonance could be identified in the 1H,15N-HSQC spectrum of apo-t-PhtD are shown in orange and proline residues in white. Residues with significant chemical shift differences are identified by their residue numbers. (B) Ratio of transverse (R2) and longitudinal (R1) relaxation rates and (C) {1H}15N-heteronuclear NOE for apo- (red squares) and Zn2+-t-PhtD (black triangles). Secondary structure elements identified in the structure of Zn2+-t-PhtD are indicated on the top of the figure and by grey shadows in each of the panels.

Mentions: The final structure calculation was performed with Aria/CNS using 2574 distance restraints and 186 TALOS+-derived dihedral angle restraints after inclusion of the Zn2+ coordination geometry. The final structural ensemble comprises 20 water-refined structures and is shown in Fig. 3 with an emphasis on the Zn2+ site. The backbone is very well defined with a root mean square deviation (rmsd) of 0.56 Å (residues 51–158) with respect to the mean structure. Zn2+-t-PhtD contains two nearly perpendicular β-sheets (sheet I: residues I70–T75, G78–H83, H86–Y90, I99–S101; sheet II: residues I118–E121, Y126–V130, K133–Y137) flanked by an N-terminal and a C-terminal helix (residues P56–E63 and K150–K157, respectively), the latter folding back to the top of β-sheet I. The two β-sheets are connected through a long loop that appears ordered throughout the structural ensemble. An additional short stretch of α-helical structure is formed by residues E102–L104. The Zn2+ coordinating residues are located at the end of the N-terminal helix (E63) and in the first β-sheet (H83, strand 2, H86 and H88 strand 3). Residues S40 to K50 do not adopt a well-defined structure within the ensemble, suggesting that this part of the protein is disordered. This was confirmed by the heteronuclear {1H}15N-heteronuclear NOE data (see below, Fig. 4C). Structural statistics are presented in Table 1. Structure calculations of Zn2+-t-PhtD in absence of Zn2+ converged to very similar structures (rmsd for the backbone atoms of the two ensemble averages: 0.79 Å), indicating that the presence of the non-standard residue did not influence the outcome of the structure calculation. Structure calculation of apo-t-PhtD was not attempted due to apo-t-PhtD inherent flexibility (see below).


New insights into histidine triad proteins: solution structure of a Streptococcus pneumoniae PhtD domain and zinc transfer to AdcAII.

Bersch B, Bougault C, Roux L, Favier A, Vernet T, Durmort C - PLoS ONE (2013)

Comparison of apo- and Zn2+-t-PhtD.(A) Weighted chemical shift difference determined for the amide resonances between the two forms (Δδ  =  [(Δδ1H)2+(0.01 Δδ15N)2]1/2). The insert shows a color-coded projection of the chemical shift differences onto the ribbon structure of Zn2+-t-PhtD. Residues for which no resonance could be identified in the 1H,15N-HSQC spectrum of apo-t-PhtD are shown in orange and proline residues in white. Residues with significant chemical shift differences are identified by their residue numbers. (B) Ratio of transverse (R2) and longitudinal (R1) relaxation rates and (C) {1H}15N-heteronuclear NOE for apo- (red squares) and Zn2+-t-PhtD (black triangles). Secondary structure elements identified in the structure of Zn2+-t-PhtD are indicated on the top of the figure and by grey shadows in each of the panels.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842936&req=5

pone-0081168-g004: Comparison of apo- and Zn2+-t-PhtD.(A) Weighted chemical shift difference determined for the amide resonances between the two forms (Δδ  =  [(Δδ1H)2+(0.01 Δδ15N)2]1/2). The insert shows a color-coded projection of the chemical shift differences onto the ribbon structure of Zn2+-t-PhtD. Residues for which no resonance could be identified in the 1H,15N-HSQC spectrum of apo-t-PhtD are shown in orange and proline residues in white. Residues with significant chemical shift differences are identified by their residue numbers. (B) Ratio of transverse (R2) and longitudinal (R1) relaxation rates and (C) {1H}15N-heteronuclear NOE for apo- (red squares) and Zn2+-t-PhtD (black triangles). Secondary structure elements identified in the structure of Zn2+-t-PhtD are indicated on the top of the figure and by grey shadows in each of the panels.
Mentions: The final structure calculation was performed with Aria/CNS using 2574 distance restraints and 186 TALOS+-derived dihedral angle restraints after inclusion of the Zn2+ coordination geometry. The final structural ensemble comprises 20 water-refined structures and is shown in Fig. 3 with an emphasis on the Zn2+ site. The backbone is very well defined with a root mean square deviation (rmsd) of 0.56 Å (residues 51–158) with respect to the mean structure. Zn2+-t-PhtD contains two nearly perpendicular β-sheets (sheet I: residues I70–T75, G78–H83, H86–Y90, I99–S101; sheet II: residues I118–E121, Y126–V130, K133–Y137) flanked by an N-terminal and a C-terminal helix (residues P56–E63 and K150–K157, respectively), the latter folding back to the top of β-sheet I. The two β-sheets are connected through a long loop that appears ordered throughout the structural ensemble. An additional short stretch of α-helical structure is formed by residues E102–L104. The Zn2+ coordinating residues are located at the end of the N-terminal helix (E63) and in the first β-sheet (H83, strand 2, H86 and H88 strand 3). Residues S40 to K50 do not adopt a well-defined structure within the ensemble, suggesting that this part of the protein is disordered. This was confirmed by the heteronuclear {1H}15N-heteronuclear NOE data (see below, Fig. 4C). Structural statistics are presented in Table 1. Structure calculations of Zn2+-t-PhtD in absence of Zn2+ converged to very similar structures (rmsd for the backbone atoms of the two ensemble averages: 0.79 Å), indicating that the presence of the non-standard residue did not influence the outcome of the structure calculation. Structure calculation of apo-t-PhtD was not attempted due to apo-t-PhtD inherent flexibility (see below).

Bottom Line: The high resolution NMR structure of t-PhtD shows that Zn(2+) is bound in a tetrahedral site by histidines 83, 86, and 88 as well as by glutamate 63.Comparison of the NMR parameters measured for apo- and Zn(2+)-t-PhtD shows that the loss of Zn(2+) leads to a diminished helical propensity at the C-terminus and increases the local dynamics and overall molecular volume.Structural comparison with the crystal structure of a 55-long fragment of PhtA suggests that Pht proteins are built from short repetitive units formed by three β-strands containing the conserved HxxHxH motif.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie Structurale, Université Grenoble Alpes, Grenoble, France ; Institut de Biologie Structurale, Direction des Sciences du Vivant, Commissariat à l'Energie Atomique et aux Energies Alternatives, Grenoble, France ; Institut de Biologie Structurale, Centre National de la Recherche Scientifique, Grenoble, France.

ABSTRACT
Zinc (Zn(2+)) homeostasis is critical for pathogen host colonization and invasion. Polyhistidine triad (Pht) proteins, located at the surface of various streptococci, have been proposed to be involved in Zn(2+) homeostasis. The phtD gene, coding for a Zn(2+)-binding protein, is organized in an operon with adcAII coding for the extracellular part of a Zn(2+) transporter. In the present work, we investigate the relationship between PhtD and AdcAII using biochemical and structural biology approaches. Immuno-precipitation experiments on purified membranes of Streptococcus pneumoniae (S. pneumoniae) demonstrate that native PhtD and AdcAII interact in vivo confirming our previous in vitro observations. NMR was used to demonstrate Zn(2+) transfer from the Zn(2+)-bound form of a 137 amino acid N-terminal domain of PhtD (t-PhtD) to AdcAII. The high resolution NMR structure of t-PhtD shows that Zn(2+) is bound in a tetrahedral site by histidines 83, 86, and 88 as well as by glutamate 63. Comparison of the NMR parameters measured for apo- and Zn(2+)-t-PhtD shows that the loss of Zn(2+) leads to a diminished helical propensity at the C-terminus and increases the local dynamics and overall molecular volume. Structural comparison with the crystal structure of a 55-long fragment of PhtA suggests that Pht proteins are built from short repetitive units formed by three β-strands containing the conserved HxxHxH motif. Taken together, these results support a role for S. pneumoniae PhtD as a Zn(2+) scavenger for later release to the surface transporter AdcAII, leading to Zn(2+) uptake.

Show MeSH
Related in: MedlinePlus