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PDBsum entry 2xmx
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Antimicrobial protein
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PDB id
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2xmx
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References listed in PDB file
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Key reference
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Title
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Activation of colicin m by the fkpa prolyl cis-Trans isomerase/chaperone.
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Authors
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S.Helbig,
S.I.Patzer,
C.Schiene-Fischer,
K.Zeth,
V.Braun.
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Ref.
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J Biol Chem, 2011,
286,
6280-6290.
[DOI no: ]
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PubMed id
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Abstract
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Colicin M (Cma) is specifically imported into the periplasm of Escherichia coli
and kills the cells. Killing depends on the periplasmic peptidyl prolyl
cis-trans isomerase/chaperone FkpA. To identify the Cma prolyl bonds targeted by
FkpA, we replaced the 15 proline residues individually with alanine. Seven
mutant proteins were fully active; Cma(P129A), Cma(P176A), and Cma(P260A)
displayed 1%, and Cma(P107A) displayed 10% of the wild-type activity.
Cma(P107A), Cma(P129A), and Cma(P260A), but not Cma(P176A), killed cells after
entering the periplasm via osmotic shock, indicating that the former mutants
were translocation-deficient; Cma(P129A) did not bind to the FhuA outer membrane
receptor. The crystal structures of Cma and Cma(P176A) were identical, excluding
inactivation of the activity domain located far from Pro-176. In a new peptidyl
prolyl cis-trans isomerase assay, FkpA isomerized the Cma prolyl bond in peptide
Phe-Pro-176 at a high rate, but Lys-Pro-107 and Leu-Pro-260 isomerized at only
<10% of that rate. The four mutant proteins secreted into the periplasm via a
fused signal sequence were toxic but much less than wild-type Cma. Wild-type and
mutant Cma proteins secreted or translocated across the outer membrane by
energy-coupled import or unspecific osmotic shock were only active in the
presence of FkpA. We propose that Cma unfolds during transfer across the outer
or cytoplasmic membrane and refolds to the active form in the periplasm assisted
by FkpA. Weak refolding of Cma(P176A) would explain its low activity in all
assays. Of the four proline residues identified as being important for Cma
activity, Phe-Pro-176 is most likely targeted by FkpA.
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