Abstract
BackgroundDNA binding KfrA-type proteins of broad-host-range bacterial plasmids belonging to IncP-1 and IncU incompatibility groups are characterized by globular N-terminal head domains and long alpha-helical coiled-coil tails. They have been shown to act as transcriptional auto-regulators.ResultsThis study was focused on two members of the growing family of KfrA-type proteins encoded by the broad-host-range plasmids, R751 of IncP-1β and RA3 of IncU groups. Comparative in vitro and in silico studies on KfrAR751 and KfrARA3 confirmed their similar biophysical properties despite low conservation of the amino acid sequences. They form a wide range of oligomeric forms in vitro and, in the presence of their cognate DNA binding sites, they polymerize into the higher order filaments visualized as “threads” by negative staining electron microscopy. The studies revealed also temperature-dependent changes in the coiled-coil segment of KfrA proteins that is involved in the stabilization of dimers required for DNA interactions.ConclusionKfrAR751 and KfrARA3 are structural homologues. We postulate that KfrA type proteins have moonlighting activity. They not only act as transcriptional auto-regulators but form cytoskeletal structures, which might facilitate plasmid DNA delivery and positioning in the cells before cell division, involving thermal energy.
Highlights
DNA binding KfrA-type proteins of broad-host-range bacterial plasmids belonging to IncP-1 and IncU incompatibility groups are characterized by globular N-terminal head domains and long alpha-helical coiled-coil tails
Structures of KfrAR751 and KfrARA3 exhibit high alphahelical content To gain information about the structure and properties of KfrA-type proteins, KfrAR751 and KfrARA3, the appropriate ORFs were cloned into pET28M plasmid [28]
The pET28M derivatives, pMAB28.1 T7p-kfrAR751 and pESB6.59 T7p-kfrARA3 were transformed into E.coli BL21(DE3) strain (Supplementary Table S.1)
Summary
DNA binding KfrA-type proteins of broad-host-range bacterial plasmids belonging to IncP-1 and IncU incompatibility groups are characterized by globular N-terminal head domains and long alpha-helical coiled-coil tails. They have been shown to act as transcriptional auto-regulators. The general feature of coiled-coil domains appears to be their ability to act as “cellular velcro” to hold together molecules or subcellular structures. They provide cables and networks in the cytoskeleton, molecular scaffolds for other proteins e.g. ZapB in FtsZ-ring formation [9], and are involved in regulation of transcription. When combined with ATPase or GTPase domains they often function in protein folding (chaperonins) or DNA remodeling (SMC, MukB, nucleases or topoisomerases)
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