Abstract
The IncP (Incompatibility group P) plasmids are important carriers in the spread of antibiotic resistance across Gram-negative bacteria. Gene expression in the IncP-1 plasmids is stringently controlled by a network of four global repressors, KorA, KorB, TrbA and KorC interacting cooperatively. Intriguingly, KorA and KorB can act as co-repressors at varying distances between their operators, even when they are moved to be on opposite sides of the DNA. KorA is a homodimer with the 101-amino acid subunits, folding into an N-terminal DNA-binding domain and a C-terminal dimerization domain. In this study, we have determined the structures of the free KorA repressor and two complexes each bound to a 20-bp palindromic DNA duplex containing its consensus operator sequence. Using a combination of X-ray crystallography, nuclear magnetic resonance spectroscopy, SAXS and molecular dynamics calculations, we show that the linker between the two domains is very flexible and the protein remains highly mobile in the presence of DNA. This flexibility allows the DNA-binding domains of the dimer to straddle the operator DNA on binding and is likely to be important in cooperative binding to KorB. Unexpectedly, the C-terminal domain of KorA is structurally similar to the dimerization domain of the tumour suppressor p53.
Highlights
The IncP (Incompatibility group P) plasmids are important carriers of antibiotic resistance
The secondary structure of each dimer is similar to the DNA-bound forms (PDB ID: 2W7N [8] and 5CM3, 5CLV, this work), but the orientation of all four copies of the DNA-binding domain (DBD) relative to the C-terminal domain (CTD) differs greatly, both from one another and from their positions in the DNA-bound complexes
The nuclear magnetic resonance (NMR) spectrum of KorA was assigned previously [41], only one signal is seen for each NH group in the 15N-1H HSQC spectrum confirming that the protein is symmetrical in solution on the NMR time scale (Supplementary Figure S2). 15N T1, and T2 relaxation times, and heteronuclear 15N-1H NOEs of the main chain amide groups were measured (Figure 2A)
Summary
The IncP (Incompatibility group P) plasmids are important carriers of antibiotic resistance. The KorA protein from the IncP1 plasmid RK2 binds with KorB at five of its seven operator (OA) sites [2] and they have been shown to act cooperatively at two of these [3] This cooperativity involves the C-terminal domain (CTD) of KorA, which has a 77.4% sequence similarity to that of TrbA (Supplementary Figure S1), that acts cooperatively with KorB [4]. We show that the DBDs significantly change orientation upon DNA binding and that the CTD is mobile, due to the highly flexible linker, both in the free protein and DNA-bound complexes This flexibility is likely to contribute to the cooperativity between KorA and KorB at different distances. Despite the lack of sequence homology, the CTD is structurally similar to the dimerization domain of the tumour suppressor p53
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