Identification of Substrates of Cytoplasmic Peptidyl-Prolyl Cis/Trans Isomerases and Their Collective Essentiality in Escherichia Coli

• Published in: International journal of molecular sciences 2020-06, Vol.21 (12), p.4212
• Main Authors: Klein, Gracjana, Wojtkiewicz, Pawel, Biernacka, Daria, Stupak, Anna, Gorzelak, Patrycja, Raina, Satish
• Format: Article
• Language: English
• Subjects: Bacteria; Calorimetry; Catalysts; Cell division; Chaperones; Chromosomes; Cytoplasm; Cytoplasm - enzymology; Defects; E coli; Enzymes; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli - growth & development; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Folding; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Genes; heat shock proteins; Homeostasis; Models, Molecular; Mutation; Peptidylprolyl isomerase; Peptidylprolyl Isomerase - chemistry; Peptidylprolyl Isomerase - genetics; Peptidylprolyl Isomerase - metabolism; Polypeptides; PpiB; prolyl isomerase; protein aggregation; Protein Folding; Proteins; RNA polymerase; RpoE sigma factor; Substrate Specificity; Titration
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms21124212

Protein folding often requires molecular chaperones and folding catalysts, such as peptidyl-prolyl isomerases (PPIs). The cytoplasm contains six well-known PPIs, although a requirement of their PPIase activity, the identity of their substrates and relative enzymatic contribution is unknown. Thus, strains lacking all periplasmic and one of the cytoplasmic PPIs were constructed. Measurement of their PPIase activity revealed that PpiB is the major source of PPIase activity in the cytoplasm. Furthermore, viable Δ6 strains could be constructed only on minimal medium in the temperature range of 30-37 °C, but not on rich medium. To address the molecular basis of essentiality of PPIs, proteins that aggregate in their absence were identified. Next, wild-type and putative active site variants of FkpB, FklB, PpiB and PpiC were purified and in pull-down experiments substrates specific to each of these PPIs identified, revealing an overlap of some substrates. Substrates of PpiC were validated by immunoprecipitations using extracts from wild-type and PpiC-H81A strains carrying a 3xFLAG-tag appended to the C-terminal end of the gene on the chromosome. Using isothermal titration calorimetry, RpoE, RseA, S2, and AhpC were established as FkpB substrates and PpiC's PPIase activity was shown to be required for interaction with AhpC.