Dataset Information

Multicopy Suppressor Analysis of Strains Lacking Cytoplasmic Peptidyl-Prolyl cis/trans Isomerases Identifies Three New PPIase Activities in Escherichia coli That Includes the DksA Transcription Factor.

ABSTRACT: Consistent with a role in catalyzing rate-limiting step of protein folding, removal of genes encoding cytoplasmic protein folding catalysts belonging to the family of peptidyl-prolyl cis/trans isomerases (PPIs) in Escherichia coli confers conditional lethality. To address the molecular basis of the essentiality of PPIs, a multicopy suppressor approach revealed that overexpression of genes encoding chaperones (DnaK/J and GroL/S), transcriptional factors (DksA and SrrA), replication proteins Hda/DiaA, asparatokinase MetL, Cmk and acid resistance regulator (AriR) overcome some defects of ?6ppi strains. Interestingly, viability of ?6ppi bacteria requires the presence of transcriptional factors DksA, SrrA, Cmk or Hda. DksA, MetL and Cmk are for the first time shown to exhibit PPIase activity in chymotrypsin-coupled and RNase T1 refolding assays and their overexpression also restores growth of a ?(dnaK/J/tig) strain, revealing their mechanism of suppression. Mutagenesis of DksA identified that D74, F82 and L84 amino acid residues are critical for its PPIase activity and their replacement abrogated multicopy suppression ability. Mutational studies revealed that DksA-mediated suppression of either ?6ppi or ?dnaK/J is abolished if GroL/S and RpoE are limiting, or in the absence of either major porin regulatory sensory kinase EnvZ or RNase H, transporter TatC or LepA GTPase or Pi-signaling regulator PhoU.

SUBMITTER: Wojtkiewicz P

PROVIDER: S-EPMC7461557 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Multicopy Suppressor Analysis of Strains Lacking Cytoplasmic Peptidyl-Prolyl cis/trans Isomerases Identifies Three New PPIase Activities in Escherichia coli That Includes the DksA Transcription Factor.

Wojtkiewicz Pawel P Biernacka Daria D Gorzelak Patrycja P Stupak Anna A Klein Gracjana G Raina Satish S

International journal of molecular sciences 20200814 16

Consistent with a role in catalyzing rate-limiting step of protein folding, removal of genes encoding cytoplasmic protein folding catalysts belonging to the family of peptidyl-prolyl cis/trans isomerases (PPIs) in Escherichia coli confers conditional lethality. To address the molecular basis of the essentiality of PPIs, a multicopy suppressor approach revealed that overexpression of genes encoding chaperones (DnaK/J and GroL/S), transcriptional factors (DksA and SrrA), replication ...[more]

PMID: 32823955

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Project description:Peptidyl-prolyl isomerases catalyze the conversion between cis and trans isomers of proline. The cyclophilin family of peptidyl-prolyl isomerases is well known for being the target of the immunosuppressive drug cyclosporin, used to combat organ transplant rejection. There is great interest in both the substrate specificity of these enzymes and the design of isoform-selective ligands for them. However, the dearth of available data for individual family members inhibits attempts to design drug specificity; additionally, in order to define physiological functions for the cyclophilins, definitive isoform characterization is required. In the current study, enzymatic activity was assayed for 15 of the 17 human cyclophilin isomerase domains, and binding to the cyclosporin scaffold was tested. In order to rationalize the observed isoform diversity, the high-resolution crystallographic structures of seven cyclophilin domains were determined. These models, combined with seven previously solved cyclophilin isoforms, provide the basis for a family-wide structure:function analysis. Detailed structural analysis of the human cyclophilin isomerase explains why cyclophilin activity against short peptides is correlated with an ability to ligate cyclosporin and why certain isoforms are not competent for either activity. In addition, we find that regions of the isomerase domain outside the proline-binding surface impart isoform specificity for both in vivo substrates and drug design. We hypothesize that there is a well-defined molecular surface corresponding to the substrate-binding S2 position that is a site of diversity in the cyclophilin family. Computational simulations of substrate binding in this region support our observations. Our data indicate that unique isoform determinants exist that may be exploited for development of selective ligands and suggest that the currently available small-molecule and peptide-based ligands for this class of enzyme are insufficient for isoform specificity.

Dataset Information

Multicopy Suppressor Analysis of Strains Lacking Cytoplasmic Peptidyl-Prolyl cis/trans Isomerases Identifies Three New PPIase Activities in Escherichia coli That Includes the DksA Transcription Factor.

Publications

Multicopy Suppressor Analysis of Strains Lacking Cytoplasmic Peptidyl-Prolyl <i>cis/trans</i> Isomerases Identifies Three New PPIase Activities in <i>Escherichia coli</i> That Includes the DksA Transcription Factor.

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