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Crystal structure of human protein N-terminal glutamine amidohydrolase, an initial component of the N-end rule pathway.

Park MS, Bitto E, Kim KR, Bingman CA, Miller MD, Kim HJ, Han BW, Phillips GN - PLoS ONE (2014)

Bottom Line: The N-terminus of a symmetry-related Ntaq1 molecule bound in the substrate binding cleft and the active site suggest possible substrate binding mode of hNtaq1.Based on our crystal structure of hNtaq1 and docking study with all the tripeptides with N-terminal glutamine, we propose how the peptide backbone recognition patch of hNtaq1 forms nonspecific interactions with N-terminal peptides of substrate proteins.Upon binding of a substrate with N-terminal glutamine, active site catalytic triad mediates the deamination of the N-terminal residue to glutamate by a mechanism analogous to that of cysteine proteases.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul, Korea.

ABSTRACT
The N-end rule states that half-life of protein is determined by their N-terminal amino acid residue. N-terminal glutamine amidohydrolase (Ntaq) converts N-terminal glutamine to glutamate by eliminating the amine group and plays an essential role in the N-end rule pathway for protein degradation. Here, we report the crystal structure of human Ntaq1 bound with the N-terminus of a symmetry-related Ntaq1 molecule at 1.5 Å resolution. The structure reveals a monomeric globular protein with alpha-beta-alpha three-layer sandwich architecture. The catalytic triad located in the active site, Cys-His-Asp, is highly conserved among Ntaq family and transglutaminases from diverse organisms. The N-terminus of a symmetry-related Ntaq1 molecule bound in the substrate binding cleft and the active site suggest possible substrate binding mode of hNtaq1. Based on our crystal structure of hNtaq1 and docking study with all the tripeptides with N-terminal glutamine, we propose how the peptide backbone recognition patch of hNtaq1 forms nonspecific interactions with N-terminal peptides of substrate proteins. Upon binding of a substrate with N-terminal glutamine, active site catalytic triad mediates the deamination of the N-terminal residue to glutamate by a mechanism analogous to that of cysteine proteases.

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Active site and electrostatic potential surface charge of hNtaq1.(A) Substrate binding cleft of hNtaq1. Carbon in the substrate-mimicking peptide, catalytic triad, α-helices, β-strands, and loops are colored in green, yellow, orange, cyan, and white, respectively. Oxygen, nitrogen, and sulfur atoms are represented as red, blue, and gold, respectively. Two water molecules are shown as red sphere and labeled as W1 and W2. (B) Electron density map from an Fo–Fc omit map calculated without the bound substrate-mimicking peptide. Positive electron density are shown as a green mesh contoured at 2.0 σ, in a stereo view. (C) Electrostatic potential surface and substrate binding cleft region of hNtaq1. Negatively and positively charged surfaces are represented as red and blue shade, respectively. Residues interacting with the substrate-mimicking peptide molecule are labeled.
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pone-0111142-g002: Active site and electrostatic potential surface charge of hNtaq1.(A) Substrate binding cleft of hNtaq1. Carbon in the substrate-mimicking peptide, catalytic triad, α-helices, β-strands, and loops are colored in green, yellow, orange, cyan, and white, respectively. Oxygen, nitrogen, and sulfur atoms are represented as red, blue, and gold, respectively. Two water molecules are shown as red sphere and labeled as W1 and W2. (B) Electron density map from an Fo–Fc omit map calculated without the bound substrate-mimicking peptide. Positive electron density are shown as a green mesh contoured at 2.0 σ, in a stereo view. (C) Electrostatic potential surface and substrate binding cleft region of hNtaq1. Negatively and positively charged surfaces are represented as red and blue shade, respectively. Residues interacting with the substrate-mimicking peptide molecule are labeled.

Mentions: The structure of hNtaq1 shows the precise conformation of the active site and the catalytic triad, Cys28, His81, and Asp97. Previous researches showed that Cys and His residues play a vital role in the activity of Ntaq. In the case of mouse Ntaq Cys30Ala and His83Ala mutants, the mutations almost abolish activities as an N-terminal glutamine amidohydrolase [8]. In the structure of hNtaq1, Asp97 forms hydrogen bonds with His81, Tyr111 and a water molecule 1 (water403 in PDB) and His81 interacts with another water molecule 2 (water444 in PDB) via hydrogen bond. The distances from the sulfhydryl group of Cys28 to water2 and His81 are 3.4 Å and 3.8 Å, respectively. After proper conformational changes that shorten the distance between His81 and Cys28 during a catalytic conversion, the sulfhydryl group of Cys28 is deprived of proton and increases its nucleophilicity for a successive attack on substrates (Figure 2A).


Crystal structure of human protein N-terminal glutamine amidohydrolase, an initial component of the N-end rule pathway.

Park MS, Bitto E, Kim KR, Bingman CA, Miller MD, Kim HJ, Han BW, Phillips GN - PLoS ONE (2014)

Active site and electrostatic potential surface charge of hNtaq1.(A) Substrate binding cleft of hNtaq1. Carbon in the substrate-mimicking peptide, catalytic triad, α-helices, β-strands, and loops are colored in green, yellow, orange, cyan, and white, respectively. Oxygen, nitrogen, and sulfur atoms are represented as red, blue, and gold, respectively. Two water molecules are shown as red sphere and labeled as W1 and W2. (B) Electron density map from an Fo–Fc omit map calculated without the bound substrate-mimicking peptide. Positive electron density are shown as a green mesh contoured at 2.0 σ, in a stereo view. (C) Electrostatic potential surface and substrate binding cleft region of hNtaq1. Negatively and positively charged surfaces are represented as red and blue shade, respectively. Residues interacting with the substrate-mimicking peptide molecule are labeled.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4214742&req=5

pone-0111142-g002: Active site and electrostatic potential surface charge of hNtaq1.(A) Substrate binding cleft of hNtaq1. Carbon in the substrate-mimicking peptide, catalytic triad, α-helices, β-strands, and loops are colored in green, yellow, orange, cyan, and white, respectively. Oxygen, nitrogen, and sulfur atoms are represented as red, blue, and gold, respectively. Two water molecules are shown as red sphere and labeled as W1 and W2. (B) Electron density map from an Fo–Fc omit map calculated without the bound substrate-mimicking peptide. Positive electron density are shown as a green mesh contoured at 2.0 σ, in a stereo view. (C) Electrostatic potential surface and substrate binding cleft region of hNtaq1. Negatively and positively charged surfaces are represented as red and blue shade, respectively. Residues interacting with the substrate-mimicking peptide molecule are labeled.
Mentions: The structure of hNtaq1 shows the precise conformation of the active site and the catalytic triad, Cys28, His81, and Asp97. Previous researches showed that Cys and His residues play a vital role in the activity of Ntaq. In the case of mouse Ntaq Cys30Ala and His83Ala mutants, the mutations almost abolish activities as an N-terminal glutamine amidohydrolase [8]. In the structure of hNtaq1, Asp97 forms hydrogen bonds with His81, Tyr111 and a water molecule 1 (water403 in PDB) and His81 interacts with another water molecule 2 (water444 in PDB) via hydrogen bond. The distances from the sulfhydryl group of Cys28 to water2 and His81 are 3.4 Å and 3.8 Å, respectively. After proper conformational changes that shorten the distance between His81 and Cys28 during a catalytic conversion, the sulfhydryl group of Cys28 is deprived of proton and increases its nucleophilicity for a successive attack on substrates (Figure 2A).

Bottom Line: The N-terminus of a symmetry-related Ntaq1 molecule bound in the substrate binding cleft and the active site suggest possible substrate binding mode of hNtaq1.Based on our crystal structure of hNtaq1 and docking study with all the tripeptides with N-terminal glutamine, we propose how the peptide backbone recognition patch of hNtaq1 forms nonspecific interactions with N-terminal peptides of substrate proteins.Upon binding of a substrate with N-terminal glutamine, active site catalytic triad mediates the deamination of the N-terminal residue to glutamate by a mechanism analogous to that of cysteine proteases.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul, Korea.

ABSTRACT
The N-end rule states that half-life of protein is determined by their N-terminal amino acid residue. N-terminal glutamine amidohydrolase (Ntaq) converts N-terminal glutamine to glutamate by eliminating the amine group and plays an essential role in the N-end rule pathway for protein degradation. Here, we report the crystal structure of human Ntaq1 bound with the N-terminus of a symmetry-related Ntaq1 molecule at 1.5 Å resolution. The structure reveals a monomeric globular protein with alpha-beta-alpha three-layer sandwich architecture. The catalytic triad located in the active site, Cys-His-Asp, is highly conserved among Ntaq family and transglutaminases from diverse organisms. The N-terminus of a symmetry-related Ntaq1 molecule bound in the substrate binding cleft and the active site suggest possible substrate binding mode of hNtaq1. Based on our crystal structure of hNtaq1 and docking study with all the tripeptides with N-terminal glutamine, we propose how the peptide backbone recognition patch of hNtaq1 forms nonspecific interactions with N-terminal peptides of substrate proteins. Upon binding of a substrate with N-terminal glutamine, active site catalytic triad mediates the deamination of the N-terminal residue to glutamate by a mechanism analogous to that of cysteine proteases.

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