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

Min Kyung Kim, Sun Joo Oh, Byung Gil Lee, Hyun Kyu Song

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

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 alsomeasured affinities between yNta1 (wild-type and its mutants) and specific peptides, and determined KM and kcat for peptides of each type. Together, these results elucidate, in structural and mechanistic detail, specific deamidationmechanisms in the first step of the N-end rule pathway.

Original languageEnglish
Pages (from-to)12438-12443
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume113
Issue number44
DOIs
Publication statusPublished - 2016 Nov 1

Fingerprint

amidase
Yeasts
Peptides
Catalytic Domain
Asparagine
Glutamine
Amidohydrolases
Aspartic Acid
Mutagenesis
Proteolysis
Saccharomyces cerevisiae
Glutamic Acid
Mammals
Hydrogen
Fungi

Keywords

  • Dual specificity
  • N-end rule
  • Nitrilase superfamily
  • Nta1

ASJC Scopus subject areas

  • General

Cite this

Structural basis for dual specificity of yeast N-terminal amidase in the N-end rule pathway. / Kim, Min Kyung; Oh, Sun Joo; Lee, Byung Gil; Song, Hyun Kyu.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 113, No. 44, 01.11.2016, p. 12438-12443.

Research output: Contribution to journalArticle

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