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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

Electron density maps in ligand-bound NP7.2Fo-Fc electron density map (contoured at 1σ) of the heme pocket of (a) NP7 at pH 5.8, (b) NP7 at pH 7.8, (c) NP7[NO], (d) NP7[ImH], (e) NP7[Hm], and (f) NP7[Gly-Gly-Gly].
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f4: Electron density maps in ligand-bound NP7.2Fo-Fc electron density map (contoured at 1σ) of the heme pocket of (a) NP7 at pH 5.8, (b) NP7 at pH 7.8, (c) NP7[NO], (d) NP7[ImH], (e) NP7[Hm], and (f) NP7[Gly-Gly-Gly].

Mentions: In the crystal structure of NP7[ImH] the electron density map clearly reflects the presence of hemeA orientation, as expected for NP7 (see above). However, to our surprise, the NP7 structure that crystallized with Gly–Gly–Gly has hemeB orientation (Table 1 andFigure 4). Thus, it may be concluded that the insertion of this ligand leads to a widening of the heme pocket, which may allow the heme to turn. Since the structures of NP7[NO], NP7[Hm] and unliganded NP7 at pH 5.8 were derived from crystal soaking and displacement of the Gly–Gly–Gly ligand, the heme consequently was found in the hemeB orientation as the crystal lattice does not allow the cofactor to turn. However for NP7[NO],A orientation was demonstrated by circular dichroism spectroscopy30. Caution is needed regarding the presence ofB orientation in unliganded NP7 at pH 7.8, since it could not be properly assigned due to the loosely occupied heme (Table 1).


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)

Electron density maps in ligand-bound NP7.2Fo-Fc electron density map (contoured at 1σ) of the heme pocket of (a) NP7 at pH 5.8, (b) NP7 at pH 7.8, (c) NP7[NO], (d) NP7[ImH], (e) NP7[Hm], and (f) NP7[Gly-Gly-Gly].
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Electron density maps in ligand-bound NP7.2Fo-Fc electron density map (contoured at 1σ) of the heme pocket of (a) NP7 at pH 5.8, (b) NP7 at pH 7.8, (c) NP7[NO], (d) NP7[ImH], (e) NP7[Hm], and (f) NP7[Gly-Gly-Gly].
Mentions: In the crystal structure of NP7[ImH] the electron density map clearly reflects the presence of hemeA orientation, as expected for NP7 (see above). However, to our surprise, the NP7 structure that crystallized with Gly–Gly–Gly has hemeB orientation (Table 1 andFigure 4). Thus, it may be concluded that the insertion of this ligand leads to a widening of the heme pocket, which may allow the heme to turn. Since the structures of NP7[NO], NP7[Hm] and unliganded NP7 at pH 5.8 were derived from crystal soaking and displacement of the Gly–Gly–Gly ligand, the heme consequently was found in the hemeB orientation as the crystal lattice does not allow the cofactor to turn. However for NP7[NO],A orientation was demonstrated by circular dichroism spectroscopy30. Caution is needed regarding the presence ofB orientation in unliganded NP7 at pH 7.8, since it could not be properly assigned due to the loosely occupied heme (Table 1).

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