Crystal Structure of Human Protein N-Terminal Glutamine Amidohydrolase, an Initial Component of the N-End Rule Pathway

Park, Mi Seul; Bitto, E.; Kim, Kyung Rok; Bingman, C.A.; Miller, Mitchell D.; Kim, Hyun-Jung; Han, Byung Woo; Phillips, George N.

doi:10.1371/journal.pone.0111142

Cited by 10 publications

(29 citation statements)

References 29 publications

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“…Moreover, the "recognition" residues of hNtaq1 are entirely distinct from those of yNta1. In yet another contrast with yNta1, two water molecules are proposed to be involved in the catalytic cycle of hNtaq1 (21). The enzymatic mechanism of yNta1 is essentially similar to that of N-carbamyl-D-amino acid amidohydrolase (33) (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The initially deposited structure of hNtaq1 (PDB ID code 3C9Q) was misinterpreted vis-a-vis the electron density of a bound peptide. Later refinements (PDB ID code 4W79) identified the "peptide" as an N-terminal region of the neighboring hNtaq1 molecule in the crystalline lattice, bearing the sequence Ser-Glu-Gly-, i.e., this sequence is not an actual substrate of hNtaq1 (21). In addition, hNtaq1 carries different catalytic triad residues (Cys28-His81-Asp97) that form a different spatial orientation, in comparison with the Glu63-Lys136-Cys187 catalytic triad of yNta1 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Specificities of some enzymes in the Arg/N-end rule pathway are now partially understood through high-resolution structural studies (16)(17)(18)(19)(20)(21)(22). Interestingly, there is a marked difference in the first step of the Arg/N-end rule pathway between fungi on the one hand and animals and plants on the other hand.…”

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confidence: 99%

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Structural basis for dual specificity of yeast N-terminal amidase in the N-end rule pathway

Kim

Lee

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The first step of the hierarchically organized Arg/N-end rule pathway of protein degradation is deamidation of the N-terminal glutamine and asparagine residues of substrate proteins to glutamate and aspartate, respectively. These reactions are catalyzed by the N-terminal amidase (Nt-amidase) Nta1 in fungi such as Saccharomyces cerevisiae, and by the glutamine-specific Ntaq1 and asparagine-specific Ntan1 Nt-amidases in mammals. To investigate the dual specificity of yeast Nta1 (yNta1) and the importance of second-position residues in Asn/Gln-bearing N-terminal degradation signals (N-degrons), we determined crystal structures of yNta1 in the apo state and in complex with various N-degron peptides. Both an Asn-peptide and a Gln-peptide fit well into the hollow active site pocket of yNta1, with the catalytic triad located deeper inside the active site. Specific hydrogen bonds stabilize interactions between N-degron peptides and hydrophobic peripheral regions of the active site pocket. Key determinants for substrate recognition were identified and thereafter confirmed by using structure-based mutagenesis. We also measured affinities between yNta1 (wild-type and its mutants) and specific peptides, and determined K M and k cat for peptides of each type. Together, these results elucidate, in structural and mechanistic detail, specific deamidation mechanisms in the first step of the N-end rule pathway.dual specificity | nitrilase superfamily | N-end rule | Nta1

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Structural basis for dual specificity of yeast N-terminal amidase in the N-end rule pathway

Kim

Lee

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…In the mammalian Arg/N-degron pathway, destabilizing N-terminal residues can be classified into primary, secondary, and tertiary degron [21]. Two tertiary degrons, Asn and Gln, are hydrolyzed into the secondary degron Asp and Glu by N-terminal asparagine amidohydrolase 1 (NTAN1) [22][23][24][25] and N-terminal glutamine amidohydrolase 1 (NTAQ1) [26][27][28], respectively. The other tertiary degron, N-terminal cysteine (Nt-Cys), is oxidized into a secondary destabilizing residue [29][30][31], and recently a family of cysteine oxidases was reported to catalyze the oxidation of Nt-Cys in plants [32].…”

Section: Introductionmentioning

confidence: 99%

“…In yeast, both Nt-Asn and Nt-Gln are deamidated by N-terminal amidase 1 (yNta1), which has the structural fold of the nitrilase superfamily [46,47]. In multicellular eukaryotes, Nt-Asn and Nt-Gln are deamidated by NTAN1-encoded Nt N -amidase and NTAQ1-encoded Nt Q -amidase, respectively [22][23][24][25][26][27][28]. We have previously determined the crystal structure of human NTAQ1 and proposed a Cys protease-like reaction mechanism [27].…”

Section: Introductionmentioning

confidence: 99%

Structural Analyses on the Deamidation of N-Terminal Asn in the Human N-Degron Pathway

et al. 2020

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The N-degron pathway is a proteolytic system in which a single N-terminal amino acid acts as a determinant of protein degradation. Especially, degradation signaling of N-terminal asparagine (Nt-Asn) in eukaryotes is initiated from its deamidation by N-terminal asparagine amidohydrolase 1 (NTAN1) into aspartate. Here, we have elucidated structural principles of deamidation by human NTAN1. NTAN1 adopts the characteristic scaffold of CNF1/YfiH-like cysteine hydrolases that features an α-β-β sandwich structure and a catalytic triad comprising Cys, His, and Ser. In vitro deamidation assays using model peptide substrates with varying lengths and sequences showed that NTAN1 prefers hydrophobic residues at the second-position. The structures of NTAN1-peptide complexes further revealed that the recognition of Nt-Asn is sufficiently organized to produce high specificity, and the side chain of the second-position residue is accommodated in a hydrophobic pocket adjacent to the active site of NTAN1. Collectively, our structural and biochemical analyses of the substrate specificity of NTAN1 contribute to understanding the structural basis of all three amidases in the eukaryotic N-degron pathway.

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Structural study for substrate recognition of human N‐terminal glutamine amidohydrolase 1 in the arginine N‐degron pathway

Kang,

Park,

Lee

et al. 2024

Protein Science

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The N‐degron pathway determines the half‐life of proteins by selectively destabilizing the proteins bearing N‐degrons. N‐terminal glutamine amidohydrolase 1 (NTAQ1) plays an essential role in the arginine N‐degron (Arg/N‐degron) pathway as an initializing enzyme via the deamidation of the N‐terminal (Nt) glutamine (Gln). However, the Nt‐serine‐bound conformation of hNTAQ1 according to the previously identified crystal structure suggests the possibility of other factors influencing the recognition of Nt residues by hNTAQ1. Hence, in the current study, we aimed to further elucidate the substrate recognition of hNTAQ1; specifically, we explored 12 different substrate‐binding conformations of hNTAQ1 depending on the subsequent residue of Nt‐Gln. Results revealed that hNTAQ1 primarily interacts with the protein Nt backbone, instead of the side chain, for substrate recognition. Here, we report that the Nt backbone of proteins appears to be a key component of hNTAQ1 function and is the main determinant of substrate recognition. Moreover, not all second residues from Nt‐Gln, but rather distinctive and charged residues, appeared to aid in detecting substrate recognition. These new findings define the substrate‐recognition process of hNTAQ1 and emphasize the importance of the subsequent Gln residue in the Nt‐Gln degradation system. Our extensive structural and biochemical analyses provide insights into the substrate specificity of the N‐degron pathway and shed light on the mechanism underlying hNTAQ1 substrate recognition. An improved understanding of the protein degradation machinery could aid in developing therapies to promote overall health through enhanced protein regulation, such as targeted protein therapies.

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Crystal Structure of Human Protein N-Terminal Glutamine Amidohydrolase, an Initial Component of the N-End Rule Pathway

Cited by 10 publications

References 29 publications

Structural basis for dual specificity of yeast N-terminal amidase in the N-end rule pathway

Structural basis for dual specificity of yeast N-terminal amidase in the N-end rule pathway

Structural Analyses on the Deamidation of N-Terminal Asn in the Human N-Degron Pathway

Structural study for substrate recognition of human N‐terminal glutamine amidohydrolase 1 in the arginine N‐degron pathway

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