Revealing biophysical properties of KfrA-type proteins as a novel class of cytoskeletal, coiled-coil plasmid-encoded proteins.

BACKGROUND: 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. RESULTS: This study was focused on two members of the growing family of KfrA-type proteins encoded by the broad-host-range plasmids, R751 of IncP-1ß 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. CONCLUSION: KfrAR751 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.

Dual effect of actin on the accessibility of myosin essential light chain A1 to papain cleavage.

The influence of various amounts of actin on the proteolytic susceptibility of myosin essential light chain (ELC) A1, the binding of isolated A1 light chain and the N-peptide spanning N-terminal sequence of A1 to actin is studied to obtain more information on the role of the N-terminus of A1 light chain in the myosin-actin interaction. Low ratios of actin to myosin (1:1) lead to stimulate cleavage, whereas higher ratios (4:1) lead to protection of A1. Exposure of A1 by actin is especially seen in heavy meromyosin (HMM) and monomeric myosin and this is probably related to the full saturation of actin protomers with myosin heads. The protecting action of actin on A1 cleavage is more pronounced in myosin filaments. Conditions favoring the saturation of myosin regulatory light chain (RLC) with calcium ions instead of magnesium ions promotes the protection of A1. Cross-linking of HMM and actin results in higher yields of A1-actin product at high actin to myosin heads ratios. Isolated A1 light chain is pelleted by actin. A synthetic peptide spanning the N-terminal sequence of A1 can be cross-linked to actin. It is postulated that the protective action of actin on A1 papain cleavage is caused by the binding of the A1 N-terminus to actin. Changes in the RLC phosphorylation level and magnesium-for-calcium exchange in RLC may affect the probability of this interaction.

The shortening of the N-terminus of myosin essential light chain A1 influences the interaction of heavy meromyosin with actin.

The effects resulting from the removal of the N-terminus of heavy meromyosin (HMM) A1 light chain by papain digestion are investigated. The fluorometry of TRITC-phalloidin labelled actin in ghost fibers is used as a tool for sensing conformational changes of rigor complex of phosphorylated and dephosphorylated HMM with actin filament. The experiments were performed both under conditions assuring saturation of RLC with magnesium cation (4 mM EGTA) or calcium cation (0.1 mM CaCl2), and in constant presence of 1 mM magnesium chloride. HMM native and with A1 shortened from the N-terminus is used. As it was observed previously rigor complex of actin filament and native HMM shows sensitivity to the kind of cation saturating RLC and to the phosphorylation status of RLC. In particular, the sin2 theta parameter of actin bound rhodamine-phalloidin fluorescence polarization representing roughly the flexibility of actin filament HMM complex changes significantly with the changes of RLC phosphorylation and cation saturation. Removal of the N-terminus of A1 reduces this sensitivity to cation and phosphorylation both in the case of dephosphorylated and phosphorylated HMM. Our results suggest that the N-terminus of A1 plays significant role in the rigor interaction of myosin heads with actin and is involved in modulatory function of RLC in this interaction.

The possible role of myosin A1 light chain in the weakening of actin-myosin interaction.

The effects resulting from the removal of the N-terminus of myosin A1 by limited papain cleavage are investigated. The myosin and heavy meromyosin K+-ATPase and Ca2+-ATPase activities, and actin-activated ATPase activity of heavy meromyosin (HMM) and subfragment-1, are studied. Myosin and HMM preparations devoid of the A1 N-terminus exhibits lower Ca2+-ATPase activities at low ionic strength whereas no differences in K+- or Ca2+-ATPase activities are observed at high ionic strength. Direct binding of actin to monomeric myosin under K+-activated ATPase conditions is much more effective for myosin containing a shortened A1 light chain. The kinetic constants K(app) for actin and V(max) are calculated from actin-activation curves for HMM and subfragment-1. The kinetic constants for HMM are determined under conditions assuring saturation of regulatory light chains (RLC) either with Mg2+ or Ca2+. The removal of the A1 N-terminus influences the actin-myosin interaction in a Ca2+- and phosphorylation-dependent manner; in most cases, this leads to an increase in affinity. In the case of subfragment-1, the removal of the N-terminus of A1 led to a decrease in affinity. It is reasonable to assume that the intact A1 light chain may cause weakening of the actin-myosin interaction under certain conditions. This weakening may be regulated by RLC phosphorylation and RLC-bound calcium-for-magnesium exchange. Such an effect requires a structural minimum that is present in HMM but not in subfragment-1. Implications of such a role for the A1 N-terminus in the myosin-actin interaction are discussed.