The multidrug resistance plasmid TP228 replicates at low copy number in Escherichia coli. Stable partitioning of this plasmid is mediated by three essential components: a ParA homologue, ParF; a centromere binding protein, ParG; and a centromere site, parH. ParF and ParG jointly assemble on the parH centromere forming the segrosome complex, and thereby direct intracellular plasmid transport. ParG belongs to the ribbon-helix-helix (RHH) class of dimeric DNA binding proteins. ParG specifically binds the parH site and also is a transcriptional repressor of the parFG genes. Previous studies demonstrated that unstructured N-terminal tails in ParG are not important for dimerization. Instead the tails are implicated in assembly of higher order nucleoprotein complexes essential for transcriptional repression and segrosome assembly, and also influence ParF nucleotide hydrolysis and polymerization. In this study we defined the role of residues in the RHH folded domain that are crucial for ParG dimerization and function. To achieve our goal the two α-helices, the intervening loop, and two C-terminal residues were analyzed fully by alanine scanning mutagenesis. Initially, ParG mutants were constructed and assessed for effects on normal plasmid partition activity and on dimerization. In vivo segregation assays and bacterial two-hybrid studies revealed mutation of residues F49 in α-helix 1 and W71 and L72 in α-helix 2 of ParG each resulted in defective plasmid partition activity and impaired dimerization. In vitro chemical cross-linking of purified proteins ParG-F49A, ParG-W71A and ParG-L72A demonstrated predominant monomeric species whereas wild-type ParG formed dimeric species as noted previously. Multiangle light scattering and sedimentation equilibrium analysis of the mutant proteins showed shifts in molar mass towards monomeric species with increased Kd values for dimerization. Protein-DNA interactions studied by gel retardation assays showed impaired interactions of ParG-F49A, ParG-W71A and ParG-L72A with parH. Results of conserved substitutions at position 71 showed that aromatic substitutions of W71 to Y71 or F71 are tolerated and have no apparent effects on ParG mediated plasmid segregation, but the non-aromatic W71L mutation blocked the segregation. However, a ParG double mutant bearing the ‘reversed’ amino acid pair (W71L-A52Y) retained plasmid segregation activity and behaved like wild-type ParG in dimerization assays in vitro and in vivo. Thus, substitution of W71 by tyrosine or phenylalanine does not disturb the monomer-monomer interface interactions that pack α-helix 2 from one monomer against residues of α-helix 1 and α-helix 2 of the partner monomer. Moreover, the permissible amino acid combinations at interacting positions 52 and 71 in ParG show significant flexibility and reveal key roles for these residues in function and dimerization of ParG. Overall, our in vivo and in vitro interaction studies provide novel information about the role of hydrophobic residues F49, W71 and L72 in ParG dimerization and activity. In the longer term, interference with dimerization by ParG and other centromere binding proteins using artificial ligands may provide a novel strategy for destabilization of antibiotic multiresistance plasmids.