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Structural basis for the N-degron specificity of ClpS1 from Arabidopsis thaliana.

ABSTRACT: The N-degron pathway determines the half-life of proteins in both prokaryotes and eukaryotes by precisely recognizing the N-terminal residue (N-degron) of substrates. ClpS proteins from bacteria bind to substrates containing hydrophobic N-degrons (Leu, Phe, Tyr, and Trp) and deliver them to the caseinolytic protease system ClpAP. This mechanism is preserved in organelles such as mitochondria and chloroplasts. Bacterial ClpS adaptors bind preferentially to Leu and Phe N-degrons; however, ClpS1 from Arabidopsis thaliana (AtClpS1) shows a difference in that it binds strongly to Phe and Trp N-degrons and only weakly to Leu. This difference in behavior cannot be explained without structural information due to the high sequence homology between bacterial and plant ClpS proteins. Here, we report the structure of AtClpS1 at 2.0 Å resolution in the presence of a bound N-degron. The key determinants for α-amino group recognition are conserved among all ClpS proteins, but the α3-helix of eukaryotic AtClpS1 is significantly shortened, and consequently, a loop forming a pocket for the N-degron is moved slightly outward to enlarge the pocket. In addition, amino acid replacement from Val to Ala causes a reduction in hydrophobic interactions with Leu N-degron. A combination of the fine-tuned hydrophobic residues in the pocket and the basic gatekeeper at the entrance of the pocket controls the N-degron selectivity of the plant ClpS protein.

SUBMITTER: Kim L

PROVIDER: S-EPMC7888574 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Structural basis for the N-degron specificity of ClpS1 from Arabidopsis thaliana.

Kim Leehyeon L Heo Jiwon J Kwon Do Hoon DH Shin Jin Seok JS Jang Se Hwan SH Park Zee-Yong ZY Song Hyun Kyu HK

Protein science : a publication of the Protein Society 20201230 3

The N-degron pathway determines the half-life of proteins in both prokaryotes and eukaryotes by precisely recognizing the N-terminal residue (N-degron) of substrates. ClpS proteins from bacteria bind to substrates containing hydrophobic N-degrons (Leu, Phe, Tyr, and Trp) and deliver them to the caseinolytic protease system ClpAP. This mechanism is preserved in organelles such as mitochondria and chloroplasts. Bacterial ClpS adaptors bind preferentially to Leu and Phe N-degrons; however, ClpS1 fr ...[more]

PMID: 33368743

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Project description:In plants triosephosphate isomerase (TPI) interconverts glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) during glycolysis, gluconeogenesis, and the Calvin-Benson cycle. The nuclear genome of land plants encodes two tpi genes, one gene product is located in the cytoplasm and the other is imported into the chloroplast. Herein we report the crystal structures of the TPIs from the vascular plant Arabidopsis thaliana (AtTPIs) and address their enzymatic modulation by redox agents. Cytoplasmic TPI (cTPI) and chloroplast TPI (pdTPI) share more than 60% amino acid identity and assemble as (β-α)8 dimers with high structural homology. cTPI and pdTPI harbor two and one accessible thiol groups per monomer respectively. cTPI and pdTPI present a cysteine at an equivalent structural position (C13 and C15 respectively) and cTPI also contains a specific solvent accessible cysteine at residue 218 (cTPI-C218). Site directed mutagenesis of residues pdTPI-C15, cTPI-C13, and cTPI-C218 to serine substantially decreases enzymatic activity, indicating that the structural integrity of these cysteines is necessary for catalysis. AtTPIs exhibit differential responses to oxidative agents, cTPI is susceptible to oxidative agents such as diamide and H2O2, whereas pdTPI is resistant to inhibition. Incubation of AtTPIs with the sulfhydryl conjugating reagents methylmethane thiosulfonate (MMTS) and glutathione inhibits enzymatic activity. However, the concentration necessary to inhibit pdTPI is at least two orders of magnitude higher than the concentration needed to inhibit cTPI. Western-blot analysis indicates that residues cTPI-C13, cTPI-C218, and pdTPI-C15 conjugate with glutathione. In summary, our data indicate that AtTPIs could be redox regulated by the derivatization of specific AtTPI cysteines (cTPI-C13 and pdTPI-C15 and cTPI-C218). Since AtTPIs have evolved by gene duplication, the higher resistance of pdTPI to redox agents may be an adaptive consequence to the redox environment in the chloroplast.

Dataset Information

Structural basis for the N-degron specificity of ClpS1 from Arabidopsis thaliana.

Publications

Structural basis for the N-degron specificity of ClpS1 from Arabidopsis thaliana.

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