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The molecular mechanism of Zinc acquisition by the neisserial outer-membrane transporter ZnuD.

Calmettes C, Ing C, Buckwalter CM, El Bakkouri M, Chieh-Lin Lai C, Pogoutse A, Gray-Owen SD, Pomès R, Moraes TF - Nat Commun (2015)

Bottom Line: Invading bacteria from the Neisseriaceae, Acinetobacteriaceae, Bordetellaceae and Moraxellaceae families express the conserved outer-membrane zinc transporter zinc-uptake component D (ZnuD) to overcome nutritional restriction imposed by the host organism during infection.We also combine X-ray crystallography and molecular dynamics simulations to gain insight into the mechanism of zinc recognition and transport across the bacterial outer-membrane by ZnuD.Because ZnuD is also considered a promising vaccine candidate against N. meningitidis, we use several ZnuD structural intermediates to map potential antigenic epitopes, and propose a mechanism by which ZnuD can maintain high sequence conservation yet avoid immune recognition by altering the conformation of surface-exposed loops.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Toronto, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada.

ABSTRACT
Invading bacteria from the Neisseriaceae, Acinetobacteriaceae, Bordetellaceae and Moraxellaceae families express the conserved outer-membrane zinc transporter zinc-uptake component D (ZnuD) to overcome nutritional restriction imposed by the host organism during infection. Here we demonstrate that ZnuD is required for efficient systemic infections by the causative agent of bacterial meningitis, Neisseria meningitidis, in a mouse model. We also combine X-ray crystallography and molecular dynamics simulations to gain insight into the mechanism of zinc recognition and transport across the bacterial outer-membrane by ZnuD. Because ZnuD is also considered a promising vaccine candidate against N. meningitidis, we use several ZnuD structural intermediates to map potential antigenic epitopes, and propose a mechanism by which ZnuD can maintain high sequence conservation yet avoid immune recognition by altering the conformation of surface-exposed loops.

No MeSH data available.


Related in: MedlinePlus

The extracellular loops contribute to zinc sequestration.Snapshots from Supplementary Movie 1 illustrate the putative flexibility of the extracellular loop 3 during the MD simulations of ZnuD in lipid bilayer in the absence (a) and presence (b) of 100 mM ZnCl2. The extracellular loop 3 contains two clusters (coloured pink and green) previously suggested as putative zinc-binding sites13 due to the high percentage of histidine, aspartic and glutamic acid residues situated there (drawn in stick representation). Histidines, aspartic and glutamic acids are the high frequency residues coordinating zinc ions within non-structural zinc-binding sites51 (structural zinc-binding sites contain His, Asp, Glu and Cys). Cluster #1 (241-HSHEYDDCHAD-251, coloured green) contains two cadmium-binding sites identified within the cadmium co-crystal structure of ZnuD. Cluster #2 (288-HDDDNAHAHTH-298, coloured pink) was shown, through MD simulations, to be a flexible loop that would promote zinc capture by sampling multiple conformational states within the extracellular space. Similarly, the periplasmic zinc-carrier protein, ZnuA, uses a flexible loop with a high ratio of histidine and aspartic acid residues to capture zinc ions52. (c) Cadmium-bound co-crystal structure of ZnuD in the same orientation as a,b; the gold and grey spheres indicate the coordinated cadmium and zinc ions, respectively. (d) The spatial distribution of zinc was computed using VMD volmap50 by averaging post-equilibration frames sampled at an interval of 500 ps on a 1.0-Å3 grid, and overlaid onto the cadmium-bound co-crystal structure. Note the co-localizations of the predicted zinc density with the two His/Asp/Glu clusters and the resolved cadmium/zinc binding sites (see Supplementary Movie 1).
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f3: The extracellular loops contribute to zinc sequestration.Snapshots from Supplementary Movie 1 illustrate the putative flexibility of the extracellular loop 3 during the MD simulations of ZnuD in lipid bilayer in the absence (a) and presence (b) of 100 mM ZnCl2. The extracellular loop 3 contains two clusters (coloured pink and green) previously suggested as putative zinc-binding sites13 due to the high percentage of histidine, aspartic and glutamic acid residues situated there (drawn in stick representation). Histidines, aspartic and glutamic acids are the high frequency residues coordinating zinc ions within non-structural zinc-binding sites51 (structural zinc-binding sites contain His, Asp, Glu and Cys). Cluster #1 (241-HSHEYDDCHAD-251, coloured green) contains two cadmium-binding sites identified within the cadmium co-crystal structure of ZnuD. Cluster #2 (288-HDDDNAHAHTH-298, coloured pink) was shown, through MD simulations, to be a flexible loop that would promote zinc capture by sampling multiple conformational states within the extracellular space. Similarly, the periplasmic zinc-carrier protein, ZnuA, uses a flexible loop with a high ratio of histidine and aspartic acid residues to capture zinc ions52. (c) Cadmium-bound co-crystal structure of ZnuD in the same orientation as a,b; the gold and grey spheres indicate the coordinated cadmium and zinc ions, respectively. (d) The spatial distribution of zinc was computed using VMD volmap50 by averaging post-equilibration frames sampled at an interval of 500 ps on a 1.0-Å3 grid, and overlaid onto the cadmium-bound co-crystal structure. Note the co-localizations of the predicted zinc density with the two His/Asp/Glu clusters and the resolved cadmium/zinc binding sites (see Supplementary Movie 1).

Mentions: To understand the molecular mechanism of zinc-acquisition by ZnuD, we solved several crystal structures of ZnuD in multiple conformations (Fig. 2, Supplementary Fig. 2 and Supplementary Table 1). The structure of the zinc-transporter ZnuD from N. meningitidis was solved using single isomorphous replacement with anomalous signal combining native and seleno-derivative ZnuD crystals that revealed a 22-stranded obstructed pore-architecture belonging to the TbdR family15. Characteristic of other TbdR family members, ZnuD consists of an amino-terminal plug domain (residues 1 to 147) obstructing the lumen of its own carboxy-terminal pore-forming domain (residues 148 to 734). The transport function of TbdR family receptors requires activation of the plug domain via the inner-membrane protein TonB15 (Supplementary Fig. 3), which binds to a conserved Ton-box motif at the amino-terminus of the transporter (residues 10 to 14; disordered peptide unresolved in our three ZnuD structures). ZnuD is the first zinc dedicated TbdR solved to date, as all other TbdR structures that have been solved are related to iron or cobalamin uptake15. Other functions of TbdRs that have been reported include the uptake of nickel in Helicobacter and carbohydrates in Caulobacter22. Uniquely, the ZnuD structures illustrate an arrangement in which the extracellular loops act like a fishing net, facilitating zinc acquisition and sequestration. Indeed, equilibrium MD simulations suggest that the conformation of extracellular loop 3 is substrate-dependent. Although this loop collapses and adopts a partially folded conformation in the presence of 100 mM ZnCl2, it extends into the extracellular solution when zinc is removed from the simulation (Fig. 3 and Supplementary Movie 1). The high affinity of this TbdR for zinc is exemplified by the observation of a zinc ion bound to ZnuD despite no exogenous zinc addition during the purification. Indeed, all three ZnuD intermediate structures presented in this study contain a zinc ion buried in a high-affinity zinc-binding pocket (Fig. 2 and Supplementary Fig. 4).


The molecular mechanism of Zinc acquisition by the neisserial outer-membrane transporter ZnuD.

Calmettes C, Ing C, Buckwalter CM, El Bakkouri M, Chieh-Lin Lai C, Pogoutse A, Gray-Owen SD, Pomès R, Moraes TF - Nat Commun (2015)

The extracellular loops contribute to zinc sequestration.Snapshots from Supplementary Movie 1 illustrate the putative flexibility of the extracellular loop 3 during the MD simulations of ZnuD in lipid bilayer in the absence (a) and presence (b) of 100 mM ZnCl2. The extracellular loop 3 contains two clusters (coloured pink and green) previously suggested as putative zinc-binding sites13 due to the high percentage of histidine, aspartic and glutamic acid residues situated there (drawn in stick representation). Histidines, aspartic and glutamic acids are the high frequency residues coordinating zinc ions within non-structural zinc-binding sites51 (structural zinc-binding sites contain His, Asp, Glu and Cys). Cluster #1 (241-HSHEYDDCHAD-251, coloured green) contains two cadmium-binding sites identified within the cadmium co-crystal structure of ZnuD. Cluster #2 (288-HDDDNAHAHTH-298, coloured pink) was shown, through MD simulations, to be a flexible loop that would promote zinc capture by sampling multiple conformational states within the extracellular space. Similarly, the periplasmic zinc-carrier protein, ZnuA, uses a flexible loop with a high ratio of histidine and aspartic acid residues to capture zinc ions52. (c) Cadmium-bound co-crystal structure of ZnuD in the same orientation as a,b; the gold and grey spheres indicate the coordinated cadmium and zinc ions, respectively. (d) The spatial distribution of zinc was computed using VMD volmap50 by averaging post-equilibration frames sampled at an interval of 500 ps on a 1.0-Å3 grid, and overlaid onto the cadmium-bound co-crystal structure. Note the co-localizations of the predicted zinc density with the two His/Asp/Glu clusters and the resolved cadmium/zinc binding sites (see Supplementary Movie 1).
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Related In: Results  -  Collection

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Show All Figures
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f3: The extracellular loops contribute to zinc sequestration.Snapshots from Supplementary Movie 1 illustrate the putative flexibility of the extracellular loop 3 during the MD simulations of ZnuD in lipid bilayer in the absence (a) and presence (b) of 100 mM ZnCl2. The extracellular loop 3 contains two clusters (coloured pink and green) previously suggested as putative zinc-binding sites13 due to the high percentage of histidine, aspartic and glutamic acid residues situated there (drawn in stick representation). Histidines, aspartic and glutamic acids are the high frequency residues coordinating zinc ions within non-structural zinc-binding sites51 (structural zinc-binding sites contain His, Asp, Glu and Cys). Cluster #1 (241-HSHEYDDCHAD-251, coloured green) contains two cadmium-binding sites identified within the cadmium co-crystal structure of ZnuD. Cluster #2 (288-HDDDNAHAHTH-298, coloured pink) was shown, through MD simulations, to be a flexible loop that would promote zinc capture by sampling multiple conformational states within the extracellular space. Similarly, the periplasmic zinc-carrier protein, ZnuA, uses a flexible loop with a high ratio of histidine and aspartic acid residues to capture zinc ions52. (c) Cadmium-bound co-crystal structure of ZnuD in the same orientation as a,b; the gold and grey spheres indicate the coordinated cadmium and zinc ions, respectively. (d) The spatial distribution of zinc was computed using VMD volmap50 by averaging post-equilibration frames sampled at an interval of 500 ps on a 1.0-Å3 grid, and overlaid onto the cadmium-bound co-crystal structure. Note the co-localizations of the predicted zinc density with the two His/Asp/Glu clusters and the resolved cadmium/zinc binding sites (see Supplementary Movie 1).
Mentions: To understand the molecular mechanism of zinc-acquisition by ZnuD, we solved several crystal structures of ZnuD in multiple conformations (Fig. 2, Supplementary Fig. 2 and Supplementary Table 1). The structure of the zinc-transporter ZnuD from N. meningitidis was solved using single isomorphous replacement with anomalous signal combining native and seleno-derivative ZnuD crystals that revealed a 22-stranded obstructed pore-architecture belonging to the TbdR family15. Characteristic of other TbdR family members, ZnuD consists of an amino-terminal plug domain (residues 1 to 147) obstructing the lumen of its own carboxy-terminal pore-forming domain (residues 148 to 734). The transport function of TbdR family receptors requires activation of the plug domain via the inner-membrane protein TonB15 (Supplementary Fig. 3), which binds to a conserved Ton-box motif at the amino-terminus of the transporter (residues 10 to 14; disordered peptide unresolved in our three ZnuD structures). ZnuD is the first zinc dedicated TbdR solved to date, as all other TbdR structures that have been solved are related to iron or cobalamin uptake15. Other functions of TbdRs that have been reported include the uptake of nickel in Helicobacter and carbohydrates in Caulobacter22. Uniquely, the ZnuD structures illustrate an arrangement in which the extracellular loops act like a fishing net, facilitating zinc acquisition and sequestration. Indeed, equilibrium MD simulations suggest that the conformation of extracellular loop 3 is substrate-dependent. Although this loop collapses and adopts a partially folded conformation in the presence of 100 mM ZnCl2, it extends into the extracellular solution when zinc is removed from the simulation (Fig. 3 and Supplementary Movie 1). The high affinity of this TbdR for zinc is exemplified by the observation of a zinc ion bound to ZnuD despite no exogenous zinc addition during the purification. Indeed, all three ZnuD intermediate structures presented in this study contain a zinc ion buried in a high-affinity zinc-binding pocket (Fig. 2 and Supplementary Fig. 4).

Bottom Line: Invading bacteria from the Neisseriaceae, Acinetobacteriaceae, Bordetellaceae and Moraxellaceae families express the conserved outer-membrane zinc transporter zinc-uptake component D (ZnuD) to overcome nutritional restriction imposed by the host organism during infection.We also combine X-ray crystallography and molecular dynamics simulations to gain insight into the mechanism of zinc recognition and transport across the bacterial outer-membrane by ZnuD.Because ZnuD is also considered a promising vaccine candidate against N. meningitidis, we use several ZnuD structural intermediates to map potential antigenic epitopes, and propose a mechanism by which ZnuD can maintain high sequence conservation yet avoid immune recognition by altering the conformation of surface-exposed loops.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Toronto, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada.

ABSTRACT
Invading bacteria from the Neisseriaceae, Acinetobacteriaceae, Bordetellaceae and Moraxellaceae families express the conserved outer-membrane zinc transporter zinc-uptake component D (ZnuD) to overcome nutritional restriction imposed by the host organism during infection. Here we demonstrate that ZnuD is required for efficient systemic infections by the causative agent of bacterial meningitis, Neisseria meningitidis, in a mouse model. We also combine X-ray crystallography and molecular dynamics simulations to gain insight into the mechanism of zinc recognition and transport across the bacterial outer-membrane by ZnuD. Because ZnuD is also considered a promising vaccine candidate against N. meningitidis, we use several ZnuD structural intermediates to map potential antigenic epitopes, and propose a mechanism by which ZnuD can maintain high sequence conservation yet avoid immune recognition by altering the conformation of surface-exposed loops.

No MeSH data available.


Related in: MedlinePlus