Requirement of periplasmic chaperones in protein import
Zusammenfassung der Projektergebnisse
Periplasmic chaperones aid in folding and prevent misfolding of newly synthesized proteins destined for export. We have discovered that a chaperone is required for import of a protein. The chaperone is FkpA, a peptidyl prolyl cis-trans isomerase and the protein is colicin M (Cma), a bacterial toxin that is imported into the periplasm of sensitive cells where it inhibits murein (peptidoglycan) biosynthesis by cleaving the C55-lipid carrier from the murein precursor. FkpA is essential for killing by imported Cma and for exported Cma secreted from the cytoplasm with an artificial signal sequence. In both cases Cma must unfold for crossing membranes, the outer membrane for import, the cytoplasmic membrane for export and FkpA is required for refolding. Cma bound to the receptor FhuA is trypsin sensitive in contrast to isolated Cma. FkpA accelerates refolding of in vitro denatured Cma. A mutation analysis of the fifteen P-bonds in Cma identified proline P176 as the most likely residue which is cis-trans isomerized. P176 is exposed on the surface of the Cma crystal structure which was determined for wild-type Cma and the inactive P176A mutant. Among P bonds tested in synthetic peptides F175-P176 is isomerized with the highest rate. Of six inactive FkpA point mutants with amino acid replacements all are located in the isomerase domain. This data without reasonable doubt identify FkpA as essential for Cma in vivo activity and suggest that Cma is unfolded during import and refolded by FkpA through cis-trans isomerization of the F175-P176 bond. These results show for the first time a function of a periplasmic chaperone in protein import, demonstrate an activity for FkpA and identify a natural proline bond that is isomerized. Since Cma is unique in that it represents a novel phosphatase in sequence, structure, and substrate specificity, the residues involved in catalysis were determined by mutant analysis. D226 is the most likely directly involved in catalysis, surrounded by residues that contribute to the hydrolytic activity. We found that cells producing Cma are protected by an immunity protein that is encoded by the cmi gene adjacent to cma. Imported Cma is inactivated in the periplasm where Cmi resides anchored to the cytoplasmic membrane by its hydrophobic N terminus. Cmi does not protect cells against newly synthesized Cma in the cytoplasm. Cmi binds weakly to exported Cma and cell-bound Cma and presumably recognizes the unfolded Cma after Cma import across the outer membrane. To obtain structural insights into Cma inactivation by Cmi the crystal structure of Cmi was determined. Two disulfide bridges interlink two monomers to a dimer mediated by a 3D domain swap. We identified another novel Cma resistance gene, designated cbrA. In contrast to fkpA whose deletion confers Cma resistance, cbrA must be overexpressed under stress conditions which also induce cma transcription. We showed that cbrA encodes a flavin adenine dinucleotide protein. The resistance mechanism has not been determined.
Projektbezogene Publikationen (Auswahl)
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Crystal structure of colicin M, a novel phosphatase specifically imported by Escherichia coli. J. Biol. Chem. 283:25324-25331 (2008)
Zeth, K., Römer, C, Patzer, SI., Braun, V.
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Periplasmic chaperone FkpA is essential for imported colicin M toxicity. Mol. Microbiol. 69:926-937 (2008)
Hullmann J., Patzer, S.I, Römer, C, Hantke, K., Braun, V.
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Activation of colicin M by the FkpA prolyl-cis-trans isomerase/chaperone. J. Biol. Chem. 286:6280-6290 (2011)
Helbig, S., Patzer, S.I., Schiene-Fischer, C., Zeth, K., Braun, V.
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Expression, purification, and crystallization of the Cmi immunity protein from Escherichia coli. Acta Crystal. Sect. F., 67517-520(2011)
Römer, R., Patzer, S.I., Albrecht, R., Zeth, K., Braun, V.
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Mapping functional domains of colicin M. J. Bacteriol. 193:815-821 (2011)
Helbig, S., Braun, V.
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CbrA is a flavin adenine dinucleotide protein that modifies the Escherichia coli outer membrane and confers specific resistance to colicin M. J. Bacteriol. 194/18: (2012)
Helbig S., Hantke, K., Ammelburg, M., Braun, V.
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The crystal structure of the dimeric colicin M immunity protein displays a 3D domain swap. J. Struct. Biol. 178:45-53 (2012)
Uson, I., Patzer, S.I., Rodriguez, D.D., Braun, V., Zeth, K.
