Protein diversity confers specificity in plasmid segregation

The ParG segregation protein (8.6 kDa) of multidrug resistance plasmid TP228 is a homodimeric DNA-binding factor. The ParG dimer consists of intertwined C-terminal domains that adopt a ribbon-helix-helix architecture and a pair of flexible, unstructured N-terminal tails. A variety of plasmids possess partition loci with similar organizations to that of TP228, but instead of ParG homologs, these plasmids specify a diversity of unrelated, but similarly sized, partition proteins. These include the proteobacterial pTAR, pVT745, and pB171 plasmids. The ParG analogs of these plasmids were characterized in parallel with the ParG homolog encoded by the pseudomonal plasmid pVS1. Like ParG, the four proteins are dimeric. No heterodimerization was detectable in vivo among the proteins nor with the prototypical ParG protein, suggesting that monomer-monomer interactions are specific among the five proteins. Nevertheless, as with ParG, the ParG analogs all possess significant amounts of unordered amino acid residues, potentially highlighting a common structural link among the proteins. Furthermore, the ParG analogs bind specifically to the DNA regions located upstream of their homologous parF-like genes. These nucleoprotein interactions are largely restricted to cognate protein-DNA pairs. The results reveal that the partition complexes of these and related plasmids have recruited disparate DNA-binding factors that provide a layer of specificity to the macromolecular interactions that mediate plasmid segregation.     

DNA binding properties of protein TrwA, a possible structural variant of the Arc repressor superfamily

Conjugative DNA processing of plasmid R388 requires the concerted action of two proteins, the relaxase-helicase TrwC and the relaxase enhancer TrwA. TrwA can be aligned with DNA binding proteins belonging to the ribbon-helix-helix (RHH) protein family. To further analyse TrwA function, the structural domains of the protein have been identified and dissected by limited proteolysis. Two stable domains were found that resulted to be, according to DNA binding experiments and oligomerization analysis, an N-terminal DNA binding domain and a C-terminal tetramerization domain. Using the three-dimensional structure of the Arc repressor as a guide, it was possible to model TrwA DNA binding site with atomic detail. As a result, TrwA polar amino acids Q8, R10 and S12, contained in the polar face of a putative N-terminal beta-strand, were found to be directly involved in DNA binding, in a manner analogous to RHH proteins. In this respect, TrwA seemed to be a new member of the RHH family. However, secondary structure analyses underscored the existence of a substantial difference in the architecture of the TrwA-oriT complex when compared to the Arc repressor-operator complex.     

Bacterial DNA segregation dynamics mediated by the polymerizing protein ParF

Prokaryotic DNA segregation most commonly involves members of the Walker-type ParA superfamily. Here we show that the ParF partition protein specified by the TP228 plasmid is a ParA ATPase that assembles into extensive filaments in vitro. Polymerization is potentiated by ATP binding and does not require nucleotide hydrolysis. Analysis of mutations in conserved residues of the Walker A motif established a functional coupling between filament dynamics and DNA partitioning. The partner partition protein ParG plays two separable roles in the ParF polymerization process. ParF is unrelated to prokaryotic polymerizing proteins of the actin or tubulin families, but is a homologue of the MinD cell division protein, which also assembles into filaments. The ultrastructures of the ParF and MinD polymers are remarkably similar. This points to an evolutionary parallel between DNA segregation and cytokinesis in prokaryotic cells, and reveals a potential molecular mechanism for plasmid and chromosome segregation mediated by the ubiquitous ParA-type proteins.     

Architecture of the ParF*ParG protein complex involved in prokaryotic DNA segregation

The mechanism by which low copy number plasmids are segregated at cell division involves the concerted action of two plasmid-encoded proteins that assemble on a centromere-like site. This study explores the topology of the DNA segregation machinery specified by the parFG locus of TP228, a partition system which is phylogenetically distinct from more well-characterized archetypes. A variety of genetic, biochemical and biophysical strategies revealed that the ParG protein is dimeric. ParF, which is more closely related to the cell division regulator MinD than to the prototypical ParA partition protein of plasmid P1, is instead multimeric and its polymeric state appears to be modulated by ATP which correlates with the proposed ATP-binding activity of ParF. ParG interacts in a sequence-specific manner with the DNA region upstream of the parFG locus and this binding is modulated by ParF. Intriguingly, the ParF and ParG proteins form at least two types of discrete complex in the absence of this region suggesting that the assembly dynamics of these proteins onto DNA is intricate.     

The affinity-enhancing roles of flexible linkers in two-domain DNA-binding proteins.

Recently many attempts have been made to design high-affinity DNA-binding proteins by linking two domains. Here a theory for guiding these designs is presented. Flexible linkers may play three types of roles: (a) linking domains which by themselves are unfolded and bind to DNA only as a folded dimer (as in a designed single-chain Arc repressor), (b) connecting domains which can separately bind to DNA (as in the Oct-1 POU domain), and (c) linking a DNA-binding domain with a dimerization domain (as in the lambda repressor). In (a), the linker keeps the protein as a folded dimer so that it is always DNA-binding-competent. In (b), the linker is predicted to enhance DNA-binding affinity over those of the individual domains (with dissociation constants K(A) and K(B)) by p(d(0))/K(B) or p(d(0))/K(A), where p(d(0)) = (3/4pil(p)bL)(3/2) exp(-3d(0)(2)/4l(p)bL)(1 - 5l(p)/4bL +...) is the probability density for the end-to-end vector of the linker with L residues to have a distance d(0). In (c), the linker is predicted to enhance the binding affinity by K(d)(C)/p(d(0)), where K(d)(C) is the dimer dissociation constant for the dimerization domain. The predicted affinity enhancements are found to be actually reached by the Oct-1 POU domain and lambda repressor. However, there is room for improvement in many of the recently designed proteins. The theoretical limits presented should provide a useful guide for current efforts of designing DNA-binding proteins.     

Design, synthesis and in vitro evaluation of novel bivalent S-adenosylmethionine analogues

In optimal cases, bivalent ligands can bind with exceptionally high affinity to their protein targets. However, designing optimised linkers, that orient the two binding groups perfectly, is challenging, and yet crucial in both fragment-based ligand design and in the discovery of bisubstrate enzyme inhibitors. To further our understanding of linker design, a series of novel bivalent S-adenosylmethionine (SAM) analogues were designed with the aim of interacting with the MetJ dimer in a bivalent sense (1:1 ligand/MetJ dimer). A range of ligands was synthesised and analyzed for ability to promote binding of the Escherichia coli methionine repressor, MetJ, to its operator DNA. Binding of bivalent SAM analogues to the MetJ homodimer in the presence of operator DNA was evaluated by fluorescence anisotropy and the effect of linker length and structure was investigated. The most effective bivalent ligand identified had a flexible linker, and promoted the DNA-protein interaction at 21-times lower concentration than the corresponding monovalent control compound.