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

Abstract

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.

Tying up loose ends: the N-degron and C-degron pathways of protein degradation

“…Substrate recognition by UBR family E3 ubiquitin ligases is best understood for UBR1, which harbours two distinct substrate binding sites: one accommodates the positively charged primary type I destabilising residues (R, K and H) [71,72], whilst the second recognises the bulky hydrophobic primary type II destabilising residues (W, Y, F, L, I) [73]. Specificity for the remaining N-terminal residues comes as a result of further N-terminal processing pathways: the tertiary destabilising residues (N and Q) can be deamidated to form the secondary destabilising residues (D and E) [74,75], which are subject to N-terminal arginylation by ATE1 [76]. Oxidised cysteine (C*) is also subject to N-terminal arginylation [ glycine degrons are redundantly targeted by both adaptors, although the ZYG11B degron is shorter, comprising just N-terminal glycine and the following residue, whereas the ZER1 degron extends several residues further into the polypeptide chain and preferentially comprises amino acids with bulky side chains [27].…”

Deamidated Human Triosephosphate Isomerase is a Promising Druggable Target

“…Deamidation causes de novo negative charges into the protein structure by changing Asn to aspartic acid (Asp) or isoaspartic acid (isoAsp) in a nonenzymatic reaction. In addition, in mammals, such reactions can be directed by N-terminal asparagine amidohydrolase 1 (NTAN1) [1]. This modification is believed to be a major pathway to protein turnover but may also induce structural changes that can lead to new functions [2].…”

Five enzymes of the Arg/N-degron pathway form a targeting complex: The concept of superchanneling

“…1). Nevertheless, the human Asn/Nt-amidase hsNTAN1 is not sequelogous to either the human Gln/Nt-amidase hsNTAQ1 or the S. cerevisiae Ntamidase scNTA1, which can deamidate both Nt-Asn and Nt-Gln (47,50,62,72). The absence of sequelogy suggests independent evolutionary origins of the three Nt-amidases.…”

“…1B) (2,59,60). Animals and plants contain two Nt-amidases, the Nt-Asn-specific NTAN1, and the Nt-Gln-specific NTAQ1 (47,50,72). At least in multicellular eukaryotes, Nt-arginylation encompasses not only Nt-Asp and Nt-Glu but also Nt-Cys, after its conditional (oxygen/ NO-dependent) oxidation to Nt-Cys-sulfinate or Nt-Cys-sulfonate ( Fig.…”

“…At least in multicellular eukaryotes, Nt-arginylation encompasses not only Nt-Asp and Nt-Glu but also Nt-Cys, after its conditional (oxygen/ NO-dependent) oxidation to Nt-Cys-sulfinate or Nt-Cys-sulfonate ( Fig. 1B) (44,47,55,(73)(74)(75).…”