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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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Proposed catalytic mechanism of hNtaq1.
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pone-0111142-g004: Proposed catalytic mechanism of hNtaq1.

Mentions: Based on our crystal structure of hNtaq1 and docking study with all the possible anticipated substrate tripeptides, we suggest a catalytic mechanism of hNtaq1 as shown in Figure 4. In the first step, nucleophilic sulfhydryl group of Cys28 approaches Cδ of the amide group of the N-terminal glutamine and becomes deprotonated by His81 as shown in Figure 4 step 1. The sulfhydryl group of Cys28 plays a crucial role in the nucleophilic attack on acyl group in the N-terminal glutamine side chain of substrates, which results in formation of a tetrahedral intermediate (Figure 4 step 2). Asp97 facilitates the process by forming a hydrogen bond and electrostatic interactions with His81. The ammonia is released upon productive collapse of the tetrahedral intermediate and a water molecule enters the active site cavity and attacks S-acyl intermediate to convert glutamine to a glutamate (Figure 4 step 3 and 4). As the final step, the glutamate side chain is cleaved from S-acyl of Cys28 (Figure 4 step 5 and 6). In these steps, His81 first acts as a general base activation water for a nucleophilic attack on the S-acyl intermediate, and then upon collapse of the tetrahedral intermediate acts a general acid to protonate the leaving group, i.e. the thiolate of Cys28. The substrate peptide with newly formed N-terminal glutamate is released from the binding cleft at this stage and the enzyme is ready for another round of catalysis (Figure 4 step 7). The proposed reaction mechanism of hNtaq1 and structural information from our study will provide valuable information for understanding the N-end rule pathway and the interaction between hNtaq1 and its protein substrate.


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)

Proposed catalytic mechanism of hNtaq1.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111142-g004: Proposed catalytic mechanism of hNtaq1.
Mentions: Based on our crystal structure of hNtaq1 and docking study with all the possible anticipated substrate tripeptides, we suggest a catalytic mechanism of hNtaq1 as shown in Figure 4. In the first step, nucleophilic sulfhydryl group of Cys28 approaches Cδ of the amide group of the N-terminal glutamine and becomes deprotonated by His81 as shown in Figure 4 step 1. The sulfhydryl group of Cys28 plays a crucial role in the nucleophilic attack on acyl group in the N-terminal glutamine side chain of substrates, which results in formation of a tetrahedral intermediate (Figure 4 step 2). Asp97 facilitates the process by forming a hydrogen bond and electrostatic interactions with His81. The ammonia is released upon productive collapse of the tetrahedral intermediate and a water molecule enters the active site cavity and attacks S-acyl intermediate to convert glutamine to a glutamate (Figure 4 step 3 and 4). As the final step, the glutamate side chain is cleaved from S-acyl of Cys28 (Figure 4 step 5 and 6). In these steps, His81 first acts as a general base activation water for a nucleophilic attack on the S-acyl intermediate, and then upon collapse of the tetrahedral intermediate acts a general acid to protonate the leaving group, i.e. the thiolate of Cys28. The substrate peptide with newly formed N-terminal glutamate is released from the binding cleft at this stage and the enzyme is ready for another round of catalysis (Figure 4 step 7). The proposed reaction mechanism of hNtaq1 and structural information from our study will provide valuable information for understanding the N-end rule pathway and the interaction between hNtaq1 and its protein substrate.

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.

Show MeSH