Coiled coils consist of bundles of two or more alpha-helices that are aligned in a parallel or an antiparallel relative orientation. The designed peptides, Acid-p1 and Base-p1, associate in solution to form a parallel, heterodimeric two-stranded coiled coil [O'Shea, E. K., Lumb, K. J., and Kim, P. S. (1993) Curr. Biol. 3, 658]. The buried interface of this complex is formed by hydrophobic Leu residues, with the exception of an Asn residue from each strand that is positioned to engage in a buried polar interaction. Substitution of these buried Asn residues by Leu residues results in a loss of structural uniqueness, as evidenced by a lack of a particular helix orientation in the Acid-Base coiled-coil complex [Lumb, K. J., and Kim, P. S. (1995) Biochemistry 34, 8642]. Here, we alter the positions of the Asn residues in the Acid and Base peptides such that a buried polar interaction is only expected to occur when the helices are in an antiparallel orientation. The resulting peptides, Acid-a1 and Base-a1, associate to form a helical heterodimer, as shown by circular dichroism (CD) and equilibrium sedimentation centrifugation. The helix orientation preference has been measured using covalently linked, disulfide-containing heterodimers in which the constituent peptides are constrained to interact in either a parallel or an antiparallel orientation. Although both the parallel and antiparallel heterodimers form stable, helical structures, the antiparallel heterodimer is the predominant species at equilibrium when the heterodimers are allowed to undergo thiol-disulfide exchange. In addition, the antiparallel heterodimer is more stable to chemical denaturation than the parallel counterpart by approximately 2.3 kcal/mol. These results demonstrate that a single buried polar interaction in the interface between the helices of a coiled coil is sufficient to determine the relative orientation of its constituent helices.

In contrast to the current state of knowledge in the field of eukaryotic chromosome segregation, relatively little is known about the mechanisms coordinating the appropriate segregation of bacterial chromosomes. In Escherichia coli , the MukB/E/F complex and topoisomerase IV (Topo IV) are both crucial players in this process. Topo IV removes DNA entanglements following the replication of the chromosome, whereas MukB, a member of the structural maintenance of chromosomes protein family, serves as a bacterial condensin. We demonstrate here a direct physical interaction between the dimerization domain of MukB and the C-terminal domain of the ParC subunit of Topo IV. In addition, we find that MukB alters the activity of Topo IV in vitro. Finally, we isolate a MukB mutant, D692A, that is deficient in its interaction with ParC and show that this mutant fails to rescue the temperature-sensitive growth phenotype of a mukB - strain. These results show that MukB and Topo IV are linked physically and functionally and indicate that the activities of these proteins are not limited to chromosome segregation but likely also play a key role in the control of higher-order bacterial chromosome structure.

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