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Crystal structures of the UDP-diacylglucosamine pyrophosphohydrase LpxH from Pseudomonas aeruginosa

View Article: PubMed Central - PubMed

ABSTRACT

Lipid A (also known as endotoxin) is the hydrophobic portion of lipopolysaccharides. It is an essential membrane component required for the viability of gram-negative bacteria. The enzymes involved in its biosynthesis are attractive targets for the development of novel antibiotics. LpxH catalyzes the fourth step of the lipid A biosynthesis pathway and cleaves the pyrophosphate bond of UDP-2,3-diacylglucosamine to yield 2,3-diacylglucosamine 1-phosphate (lipid X) and UMP. Here we present the structures of LpxH from Pseudomonas aeruginosa (PaLpxH). PaLpxH consists of two domains: a catalytic domain that is homologous to the metallophosphoesterases and a helical insertion domain. Lipid X was captured in the crevice between these two domains, with its phosphate group facing the dinuclear metal (Mn2+) center and two acyl chains buried in the hydrophobic cavity. The structures reveal that a large conformational change occurs at the lipid X binding site surface upon the binding/release of the product molecule. Based on these observations, we propose a novel model for lipid X embedding, which involves the scissor-like movement of helix α6, resulting in the release of lipid X into the lipid bilayer.

No MeSH data available.


Conformational changes of PaLpxH upon lipid X binding.(a) Stereo view of the superimposed structures of the PaLpxH–Lipid X complex (cyan and yellow) and the apo form (H10N mutant, pink and red). With respect to the PaLpxH–Lipid X complex, the region that changes (α6–α7, residues 146–173) upon lipid X binding is shown in yellow. Lipid X is shown in stick and Mn2+ ions are violet spheres. With respect to the apo form, the region that changes (residues 146–173) is shown in red. H10N is shown in stick. Mn1 is not bound, and Mn2 is shown as a pink sphere. The C-terminal half of α6 and the following loop (residues 161–169 in P212121 with Mn2, 159–165 in P212121 with no Mn2+)) were disordered. Helix α7 became a loop (P212121 with Mn2) or a 310 helix (P212121 with no Mn2+). The H10N mutation does not affect the structure of the catalytic domain, whereas the structure of the α6–α7 region changes. The α6–α7 region is wide open in the apo form, whereas in the EP complex, this gate is closed, possibly by interactions with the product molecule. (b) Detailed views of the variable region; EP complex (left) and apo form (right). The main chain conformation at I171 and I172 changes (psi angles are changed by 180°). In the apo form, the carbonyl oxygen of I172 makes a hydrogen bond with Arg198, and I171 and I172 are positioned closer to the catalytic domain. In the EP complex, the residues in α6–α7 interact with lipid X and are fixed. As a result of the conformational change, I171 and I172 interact with lipid X, not with Arg198.
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f4: Conformational changes of PaLpxH upon lipid X binding.(a) Stereo view of the superimposed structures of the PaLpxH–Lipid X complex (cyan and yellow) and the apo form (H10N mutant, pink and red). With respect to the PaLpxH–Lipid X complex, the region that changes (α6–α7, residues 146–173) upon lipid X binding is shown in yellow. Lipid X is shown in stick and Mn2+ ions are violet spheres. With respect to the apo form, the region that changes (residues 146–173) is shown in red. H10N is shown in stick. Mn1 is not bound, and Mn2 is shown as a pink sphere. The C-terminal half of α6 and the following loop (residues 161–169 in P212121 with Mn2, 159–165 in P212121 with no Mn2+)) were disordered. Helix α7 became a loop (P212121 with Mn2) or a 310 helix (P212121 with no Mn2+). The H10N mutation does not affect the structure of the catalytic domain, whereas the structure of the α6–α7 region changes. The α6–α7 region is wide open in the apo form, whereas in the EP complex, this gate is closed, possibly by interactions with the product molecule. (b) Detailed views of the variable region; EP complex (left) and apo form (right). The main chain conformation at I171 and I172 changes (psi angles are changed by 180°). In the apo form, the carbonyl oxygen of I172 makes a hydrogen bond with Arg198, and I171 and I172 are positioned closer to the catalytic domain. In the EP complex, the residues in α6–α7 interact with lipid X and are fixed. As a result of the conformational change, I171 and I172 interact with lipid X, not with Arg198.

Mentions: Although no major conformational differences are observed in the catalytic domain when comparing the apo and EP complexes, the structure of HI domain varies extensively in the region of α6–α7 (residues 158–172) (Fig. 4). There is no electron density for the C-terminal half of α6 and the following loop region (residues 161–169 in P212121 crystal with one Mn2+, 159–165 in P212121 crystal with no Mn2+) in the apo form, indicating they are highly mobile and disordered. Furthermore, the folding of the protein is completely changed in the subsequent region. In the apo form, α7 (residues 169–172) disappears and a loop (P212121 with one Mn2+) or a short 310 helix at residues 168–170 (P212121 with no Mn2+) is created. As a result, the orientation of the side chains of residues 170–172 is completely different between the two forms. The hydrogen bonds between the side chain of Arg198 and the carbonyl oxygen of Ile171 stabilize the structure in the apo form, whereas they are not present in the EP complex due to an approximate 180° psi rotation at Ile171 and Ile172 (Fig. 4b). As shown in Fig. 3b, a significant structural change in residues 158–172 is important for the recognition of lipid X (and probably also the substrate); the binding site is wide open in the apo form, whereas in the EP complex, two alpha helices (α6 and α7) are formed to assemble a stable EP complex.


Crystal structures of the UDP-diacylglucosamine pyrophosphohydrase LpxH from Pseudomonas aeruginosa
Conformational changes of PaLpxH upon lipid X binding.(a) Stereo view of the superimposed structures of the PaLpxH–Lipid X complex (cyan and yellow) and the apo form (H10N mutant, pink and red). With respect to the PaLpxH–Lipid X complex, the region that changes (α6–α7, residues 146–173) upon lipid X binding is shown in yellow. Lipid X is shown in stick and Mn2+ ions are violet spheres. With respect to the apo form, the region that changes (residues 146–173) is shown in red. H10N is shown in stick. Mn1 is not bound, and Mn2 is shown as a pink sphere. The C-terminal half of α6 and the following loop (residues 161–169 in P212121 with Mn2, 159–165 in P212121 with no Mn2+)) were disordered. Helix α7 became a loop (P212121 with Mn2) or a 310 helix (P212121 with no Mn2+). The H10N mutation does not affect the structure of the catalytic domain, whereas the structure of the α6–α7 region changes. The α6–α7 region is wide open in the apo form, whereas in the EP complex, this gate is closed, possibly by interactions with the product molecule. (b) Detailed views of the variable region; EP complex (left) and apo form (right). The main chain conformation at I171 and I172 changes (psi angles are changed by 180°). In the apo form, the carbonyl oxygen of I172 makes a hydrogen bond with Arg198, and I171 and I172 are positioned closer to the catalytic domain. In the EP complex, the residues in α6–α7 interact with lipid X and are fixed. As a result of the conformational change, I171 and I172 interact with lipid X, not with Arg198.
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Related In: Results  -  Collection

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f4: Conformational changes of PaLpxH upon lipid X binding.(a) Stereo view of the superimposed structures of the PaLpxH–Lipid X complex (cyan and yellow) and the apo form (H10N mutant, pink and red). With respect to the PaLpxH–Lipid X complex, the region that changes (α6–α7, residues 146–173) upon lipid X binding is shown in yellow. Lipid X is shown in stick and Mn2+ ions are violet spheres. With respect to the apo form, the region that changes (residues 146–173) is shown in red. H10N is shown in stick. Mn1 is not bound, and Mn2 is shown as a pink sphere. The C-terminal half of α6 and the following loop (residues 161–169 in P212121 with Mn2, 159–165 in P212121 with no Mn2+)) were disordered. Helix α7 became a loop (P212121 with Mn2) or a 310 helix (P212121 with no Mn2+). The H10N mutation does not affect the structure of the catalytic domain, whereas the structure of the α6–α7 region changes. The α6–α7 region is wide open in the apo form, whereas in the EP complex, this gate is closed, possibly by interactions with the product molecule. (b) Detailed views of the variable region; EP complex (left) and apo form (right). The main chain conformation at I171 and I172 changes (psi angles are changed by 180°). In the apo form, the carbonyl oxygen of I172 makes a hydrogen bond with Arg198, and I171 and I172 are positioned closer to the catalytic domain. In the EP complex, the residues in α6–α7 interact with lipid X and are fixed. As a result of the conformational change, I171 and I172 interact with lipid X, not with Arg198.
Mentions: Although no major conformational differences are observed in the catalytic domain when comparing the apo and EP complexes, the structure of HI domain varies extensively in the region of α6–α7 (residues 158–172) (Fig. 4). There is no electron density for the C-terminal half of α6 and the following loop region (residues 161–169 in P212121 crystal with one Mn2+, 159–165 in P212121 crystal with no Mn2+) in the apo form, indicating they are highly mobile and disordered. Furthermore, the folding of the protein is completely changed in the subsequent region. In the apo form, α7 (residues 169–172) disappears and a loop (P212121 with one Mn2+) or a short 310 helix at residues 168–170 (P212121 with no Mn2+) is created. As a result, the orientation of the side chains of residues 170–172 is completely different between the two forms. The hydrogen bonds between the side chain of Arg198 and the carbonyl oxygen of Ile171 stabilize the structure in the apo form, whereas they are not present in the EP complex due to an approximate 180° psi rotation at Ile171 and Ile172 (Fig. 4b). As shown in Fig. 3b, a significant structural change in residues 158–172 is important for the recognition of lipid X (and probably also the substrate); the binding site is wide open in the apo form, whereas in the EP complex, two alpha helices (α6 and α7) are formed to assemble a stable EP complex.

View Article: PubMed Central - PubMed

ABSTRACT

Lipid A (also known as endotoxin) is the hydrophobic portion of lipopolysaccharides. It is an essential membrane component required for the viability of gram-negative bacteria. The enzymes involved in its biosynthesis are attractive targets for the development of novel antibiotics. LpxH catalyzes the fourth step of the lipid A biosynthesis pathway and cleaves the pyrophosphate bond of UDP-2,3-diacylglucosamine to yield 2,3-diacylglucosamine 1-phosphate (lipid X) and UMP. Here we present the structures of LpxH from Pseudomonas aeruginosa (PaLpxH). PaLpxH consists of two domains: a catalytic domain that is homologous to the metallophosphoesterases and a helical insertion domain. Lipid X was captured in the crevice between these two domains, with its phosphate group facing the dinuclear metal (Mn2+) center and two acyl chains buried in the hydrophobic cavity. The structures reveal that a large conformational change occurs at the lipid X binding site surface upon the binding/release of the product molecule. Based on these observations, we propose a novel model for lipid X embedding, which involves the scissor-like movement of helix α6, resulting in the release of lipid X into the lipid bilayer.

No MeSH data available.