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The DnaK chaperones from the archaeon Methanosarcina mazei and the bacterium Escherichia coli have different substrate specificities

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Żmijewski M. A., Skórko-Glonek J., Tanfani F., Banecki B., Kotlarz A., Macario A. J. L., Lipińska B.

2007

vol. 54

issue 3

509-522

Hsp70 (DnaK) is a highly conserved molecular chaperone present in bacteria, eukaryotes, and some archaea. In a previous work we demonstrated that DnaK from the archaeon Methanosarcina mazei (DnaKMm) and the DnaK from the bacterium Escherichia coli (DnaKEc) were functionally similar when assayed in vitro but DnaKMm failed to substitute for DnaKEc in vivo. Searching for the molecular basis of the observed DnaK species specificity we compared substrate binding by DnaKMm and DnaKEc. DnaKMm showed a lower affinity for the model peptide (a-CALLQSRLLS) compared to DnaKEc. Furthermore, it was unable to negatively regulate the E. coli σ32 transcription factor level under heat shock conditions and poorly bound purified σ32, which is a native substrate of DnaKEc. These observations taken together indicate differences in substrate specificity of archaeal and bacterial DnaKs. Structural modeling of DnaKMm showed some structural differences in the substrate-binding domains of DnaKMm and DnaKEc, which may be responsible, at least partially, for the differences in peptide binding. Size-exclusion chromatography and native gel electrophoresis revealed that DnaKMm was found preferably in high molecular mass oligomeric forms, contrary to DnaKEc. Oligomers of DnaKMm could be dissociated in the presence of ATP and a substrate (peptide) but not ADP, which may suggest that monomer is the active form of DnaKMm.

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1 Acta Biochimica Polonica

1 2007

1 Banecki B.

1 Kotlarz A.

1 Lipińska B.

1 Macario A. J. L.

1 Skórko-Glonek J.

1 Tanfani F.

1 Żmijewski M. A.

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The DnaK chaperones from the archaeon Methanosarcina mazei and the bacterium Escherichia coli have different substrate specificities