Backbone assignments, and effect of Asn deamidation, of the N-terminal region of the partitioning protein IncC1 from the plasmid RK2

IncC from the low-copy number plasmid RK2, is a member of the ParA family of proteins required for partitioning DNA in many bacteria and plasmids. It is an ATPase that binds DNA and its ParB protein partner, KorB. Together, the proteins move replicated DNA to appropriate cellular positions, so that each daughter cell inherits a copy on cell division. IncC from RK2 is expressed in two forms. IncC2 is homologous to bacterial ParA proteins, while IncC1 has an N-terminal extension of 105 amino acids and is similar in length to ParA homologues in other plasmids. We have been examining the role of this extension, here called IncC NTD. We present its backbone NMR chemical shift assignments and show that it is entirely intrinsically disordered. The assignments were achieved using C-detected, CON-based spectra, complemented by HNN spectra to obtain connectivities from three adjacent amino acids. We also observed evidence of deamidation of the protein at a GNGG sequence, to give isoAsp, giving 2 sets of peaks for residues up to 5 amino acids on either side of the modification. We have assigned resonances from around the position of modification for this form of the protein.

Vibrio cholerae ParE2 toxin modulates its operon transcription by stabilization of an antitoxin DNA ruler

The parDE2 operon of Vibrio cholerae encodes a type II TA system, which is one of three loci in the superintegron of small chromosome II that show modest similarity to the parDE operon of plasmid RK2. ParE2, like plasmid RK2-encoded ParE, inhibits DNA gyrase, an essential topoisomerase that is also the target of quinolone antibacterial agents. Mechanistic understanding on ParE2 toxin inhibition by direct interaction with its cognate antitoxin and transcriptional autoregulation of the TA system are currently lacking. ParD2, the ribbon-helix-helix (RHH) antitoxin, auto-represses the parDE2 promoter. This repression is enhanced by ParE2, which therefore functions as a transcriptional co-repressor. Here we present protein-DNA interaction studies and high-resolution X-ray structures of the ParD2:ParE2 complex and isolated ParD2 antitoxin, revealing the basis of toxin inhibition and autoregulation of the TA operon by conditional cooperativity. Native mass spectrometry, SAXS and MALS studies confirm the presence of different oligomerization states of ParD2 in solution and the role of the DNA-binding hexameric ParD26:ParE22 assembly in transcriptional repression.

The Plasmid RK2 Replication Initiator Protein (TrfA) Binds to the Sliding Clamp β Subunit of DNA Polymerase III: Implication for the Toxicity of a Peptide Derived from the Amino-Terminal Portion of 33-Kilodalton TrfA

ABSTRACT The broad-host-range plasmid RK2 is capable of replication and stable maintenance within a wide range of gram-negative bacterial hosts. It encodes the essential replication initiation protein TrfA, which binds to the host initiation protein, DnaA, at the plasmid origin of replication (oriV). There are two versions of the TrfA protein, 44 and 33 kDa, resulting from alternate in-frame translational starts. We have shown that the smaller protein, TrfA-33, and its 64-residue amino-terminal peptide (designated T1) physically interact with the Escherichia coli β sliding clamp (β2). This interaction appears to be mediated through a QLSLF peptide motif located near the amino-terminal end of TrfA-33 and T1, which is identical to the previously described eubacterial clamp-binding consensus motif. T1 forms a stable complex with β2 and was found to inhibit plasmid RK2 replication in vitro. This specific interaction between T1 and β2 and the ability of T1 to block DNA replication have implications for the previously reported cell lethality caused by overproduction of T1 (P. D. Kim, T. M. Rosche, and W. Firshein, Plasmid 43:214-222, 2000). The toxicity of T1 was suppressed when wild-type T1 was replaced with mutant T1, carrying an LF deletion in the β-binding motif. Previously, T1 toxicity has been shown to be suppressed by Hda, an intermediate regulatory protein which helps prevent overinitiation in E. coli through its interaction with the initiator protein, DnaA, and β2. Our results support a model in which T1 toxicity is caused by T1 binding to β2, especially when T1 is overexpressed, preventing β2 from interacting with host replication proteins such as Hda during the early events of chromosome replication.