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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.

Structural basis of the interspecies interaction between the chaperone DnaK(Hsp70) and the co-chaperone GrpE of archaea and bacteria

100%

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

2007

vol. 54

issue 2

245-252

Hsp70s are chaperone proteins that are conserved in evolution and present in all prokaryotic and eukaryotic organisms. In the archaea, which form a distinct kingdom, the Hsp70 chaperones have been found in some species only, including Methanosarcina mazei. Both the bacterial and archaeal Hsp70(DnaK) chaperones cooperate with a GrpE co-chaperone which stimulates the ATPase activity of the DnaK protein. It is currently believed that the archaeal Hsp70 system was obtained by the lateral transfer of chaperone genes from bacteria. Our previous finding that the DnaK and GrpE proteins of M. mazei can functionally cooperate with the Escherichia coli GrpE and DnaK supported this hypothesis. However, the cooperation was surprising, considering the very low identity of the GrpE proteins (26%) and the relatively low identity of the DnaK proteins (56%). The aim of this work was to investigate the molecular basis of the observed interspecies chaperone interaction. Infrared resolution-enhanced spectra of the M. mazei and E. coli DnaK proteins were almost identical, indicating high similarity of their secondary structures, however, some small differences in band position and in the intensity of amide I' band components were observed and discussed. Profiles of thermal denaturation of both proteins were similar, although they indicated a higher thermostability of the M. mazei DnaK compared to the E. coli DnaK. Electrophoresis under non-denaturing conditions demonstrated that purified DnaK and GrpE of E. coli and M. mazei formed mixed complexes. Protein modeling revealed high similarity of the 3-dimensional structures of the archaeal and bacterial DnaK and GrpE proteins.

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2 Acta Biochimica Polonica

2 2007

2 Banecki B.

2 Kotlarz A.

2 Lipińska B.

2 Macario A. J. L.

2 Skórko-Glonek J.

2 Tanfani F.

2 Żmijewski M. A.

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

Structural basis of the interspecies interaction between the chaperone DnaK(Hsp70) and the co-chaperone GrpE of archaea and bacteria