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Structure and dynamics of the membrane attaching nitric oxide transporter nitrophorin 7.

Knipp M, Ogata H, Soavi G, Cerullo G, Allegri A, Abbruzzetti S, Bruno S, Viappiani C, Bidon-Chanal A, Luque FJ - F1000Res (2015)

Bottom Line: However, a chain-like arrangement in the crystal lattice due to a number of head-to-tail electrostatic stabilizing interactions is found in NP7.Fast and ultrafast laser triggered ligand rebinding experiments demonstrate the pH-dependent ligand migration within the cavities and the exit route.Finally, the topological distribution of pockets located around the heme as well as from inner cavities present at the rear of the protein provides a distinctive feature in NP7, so that while a loop gated exit mechanism to the solvent has been proposed for most nitrophorins, a more complex mechanism that involves several interconnected gas hosting cavities is proposed for NP7.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Chemische Energiekonversion, Mülheim an der Ruhr, 45470, Germany.

ABSTRACT
Nitrophorins represent a unique class of heme proteins that are able to perform the delicate transportation and release of the free-radical gaseous messenger nitric oxide (NO) in a pH-triggered manner. Besides its ability to bind to phospholipid membranes, the N-terminus contains an additional Leu-Pro-Gly stretch, which is a unique sequence trait, and the heme cavity is significantly altered with respect to other nitrophorins. These distinctive features encouraged us to solve the X-ray crystallographic structures of NP7 at low and high pH and bound with different heme ligands (nitric oxide, histamine, imidazole). The overall fold of the lipocalin motif is well preserved in the different X-ray structures and resembles the fold of other nitrophorins. However, a chain-like arrangement in the crystal lattice due to a number of head-to-tail electrostatic stabilizing interactions is found in NP7. Furthermore, the X-ray structures also reveal ligand-dependent changes in the orientation of the heme, as well as in specific interactions between the A-B and G-H loops, which are considered to be relevant for the biological function of nitrophorins. Fast and ultrafast laser triggered ligand rebinding experiments demonstrate the pH-dependent ligand migration within the cavities and the exit route. Finally, the topological distribution of pockets located around the heme as well as from inner cavities present at the rear of the protein provides a distinctive feature in NP7, so that while a loop gated exit mechanism to the solvent has been proposed for most nitrophorins, a more complex mechanism that involves several interconnected gas hosting cavities is proposed for NP7.

No MeSH data available.


Related in: MedlinePlus

Local structural details of the heme pocket.(a) Hm, (b) ImH, (c) pH 5.8 and (d) pH 7.8. The numbers represent the bond distances (Å).
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f6: Local structural details of the heme pocket.(a) Hm, (b) ImH, (c) pH 5.8 and (d) pH 7.8. The numbers represent the bond distances (Å).

Mentions: Binding of imidazole and histamine. Similar to other NPs, the binding of Hm is accomplished not only through the coordination of heme iron, but also through the salt bridge of its ethylamine group with the Asp32 carboxylate (Figure 6a). When ImH is bound, the missing ethylamine is compensated by H-bonding to a water molecule, which is then coordinated by Asp32 (Figure 6b). However, in marked contrast to other NPs, the affinity constant for ImH and Hm was markedly decreased. While for NP2 the equilibrium constant (Keq) was found to be 2.5 × 107 M-150, for NP7Keq was 1.0 × 106 M-18. This difference is even more pronounced for Hm, as the equilibrium constants are 1.0 × 108 M-150 and 1.0 × 105 M-1 for NP2 and NP7 [8], respectively. The remarkable difference observed between the two ligands in NP7 as well as between NP2[Hm] and NP7[Hm] is not explained by the structures. However, taking into account that the N-terminus is expected to move into the space between the A-B and G-H loops, the disruption of the Asp32-Hm interaction is feasible. This is further supported by the finding that deletion of the N-terminal residues increases the affinity for Hm (Keq(NP7(Δ1-3)) = 1.3 × 107 M-1). This trend is also found in the enhanced affinity for ImH (Keq(NP7(Δ1-3)) = 3.2 × 107 M-1)8, which compares with the value reported for NP2 (see above).


Structure and dynamics of the membrane attaching nitric oxide transporter nitrophorin 7.

Knipp M, Ogata H, Soavi G, Cerullo G, Allegri A, Abbruzzetti S, Bruno S, Viappiani C, Bidon-Chanal A, Luque FJ - F1000Res (2015)

Local structural details of the heme pocket.(a) Hm, (b) ImH, (c) pH 5.8 and (d) pH 7.8. The numbers represent the bond distances (Å).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4482215&req=5

f6: Local structural details of the heme pocket.(a) Hm, (b) ImH, (c) pH 5.8 and (d) pH 7.8. The numbers represent the bond distances (Å).
Mentions: Binding of imidazole and histamine. Similar to other NPs, the binding of Hm is accomplished not only through the coordination of heme iron, but also through the salt bridge of its ethylamine group with the Asp32 carboxylate (Figure 6a). When ImH is bound, the missing ethylamine is compensated by H-bonding to a water molecule, which is then coordinated by Asp32 (Figure 6b). However, in marked contrast to other NPs, the affinity constant for ImH and Hm was markedly decreased. While for NP2 the equilibrium constant (Keq) was found to be 2.5 × 107 M-150, for NP7Keq was 1.0 × 106 M-18. This difference is even more pronounced for Hm, as the equilibrium constants are 1.0 × 108 M-150 and 1.0 × 105 M-1 for NP2 and NP7 [8], respectively. The remarkable difference observed between the two ligands in NP7 as well as between NP2[Hm] and NP7[Hm] is not explained by the structures. However, taking into account that the N-terminus is expected to move into the space between the A-B and G-H loops, the disruption of the Asp32-Hm interaction is feasible. This is further supported by the finding that deletion of the N-terminal residues increases the affinity for Hm (Keq(NP7(Δ1-3)) = 1.3 × 107 M-1). This trend is also found in the enhanced affinity for ImH (Keq(NP7(Δ1-3)) = 3.2 × 107 M-1)8, which compares with the value reported for NP2 (see above).

Bottom Line: However, a chain-like arrangement in the crystal lattice due to a number of head-to-tail electrostatic stabilizing interactions is found in NP7.Fast and ultrafast laser triggered ligand rebinding experiments demonstrate the pH-dependent ligand migration within the cavities and the exit route.Finally, the topological distribution of pockets located around the heme as well as from inner cavities present at the rear of the protein provides a distinctive feature in NP7, so that while a loop gated exit mechanism to the solvent has been proposed for most nitrophorins, a more complex mechanism that involves several interconnected gas hosting cavities is proposed for NP7.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Chemische Energiekonversion, Mülheim an der Ruhr, 45470, Germany.

ABSTRACT
Nitrophorins represent a unique class of heme proteins that are able to perform the delicate transportation and release of the free-radical gaseous messenger nitric oxide (NO) in a pH-triggered manner. Besides its ability to bind to phospholipid membranes, the N-terminus contains an additional Leu-Pro-Gly stretch, which is a unique sequence trait, and the heme cavity is significantly altered with respect to other nitrophorins. These distinctive features encouraged us to solve the X-ray crystallographic structures of NP7 at low and high pH and bound with different heme ligands (nitric oxide, histamine, imidazole). The overall fold of the lipocalin motif is well preserved in the different X-ray structures and resembles the fold of other nitrophorins. However, a chain-like arrangement in the crystal lattice due to a number of head-to-tail electrostatic stabilizing interactions is found in NP7. Furthermore, the X-ray structures also reveal ligand-dependent changes in the orientation of the heme, as well as in specific interactions between the A-B and G-H loops, which are considered to be relevant for the biological function of nitrophorins. Fast and ultrafast laser triggered ligand rebinding experiments demonstrate the pH-dependent ligand migration within the cavities and the exit route. Finally, the topological distribution of pockets located around the heme as well as from inner cavities present at the rear of the protein provides a distinctive feature in NP7, so that while a loop gated exit mechanism to the solvent has been proposed for most nitrophorins, a more complex mechanism that involves several interconnected gas hosting cavities is proposed for NP7.

No MeSH data available.


Related in: MedlinePlus