MukE and MukF Form Two Distinct High Affinity Complexes

Abstract

The MukBFE complex is essential for chromosome segregation and condensation in Escherichia coli. MukB is functionally related to the structural maintenance of chromosomes (SMC) proteins. Similar to SMCs, MukB requires accessory proteins (MukE and MukF) to form a functional complex for DNA segregation. MukF is a member of the kleisin family, which includes proteins that commonly mediate the interaction between SMCs and other accessory proteins, suggesting that the similarities between the MukBFE and the SMC complexes extend beyond MukB. Although SMCs have been carefully studied, little is known about the roles of their accessory components. In the present work, we characterize the oligomeric states of MukE and MukF using size exclusion chromatography and analytical ultracentrifugation. MukE self-associates to form dimers (K D 18 ؎ 3 M), which in turn interact with the MukF dimer to form two distinct high affinity complexes having 2:2 and 2:4 stoichiometries (F:E). Intermediate complexes are not found, and thus we propose that the equilibrium between these two complexes determines the formation of a functional MukBFE with stoichiometry 2:2:2.

Dynamics of the bacterial SMC complex and SMC-like proteins involved in DNA repair

“…It is clear that the ATPase activity of SMC/MukB and the interaction with the accessory proteins are required for stable association with the chromosome (Mascarenhas et al 2002She et al 2007), but it is unclear whether SMC or MukB constantly interact with ScpAB or MukEF, or whether transient and dynamic interactions exist in vivo. In support of the latter notion, ScpAB and MukEF subcomplexes have been shown to exist in vitro (Yamazoe et al 1999;Mascarenhas et al 2005;Petrushenko et al 2006a;Gloyd et al 2007). It will be important to understand the loading/unloading reactions of the SMC complex at a molecular level to be able to fully understand its real function within the cell.…”

MukBEF, a Chromosomal Organizer

“…MukF serves as a kleisin that interacts with MukB heads and links MukE to the complex [Fennell-Fezzie et al, 2005; Woo et al, 2009]. Both purified and reconstituted MukEF form an elongated complex MukE 4 F 2 [Gloyd et al, 2007; Petrushenko et al, 2006b]. MukF and MukE also form a complex MukE 2 F 2 but only at substoichiometric levels of MukE [Petrushenko et al, 2006b].…”

The Role of MukE in Assembling a Functional MukBEF Complex

“…21 MukE and MukF also form two stable complexes in the absence of MukB, in which either one dimer or two dimers of MukE associate to a dimer of MukF, suggesting that binding and dissociation of the MukE subunit could regulate the opening of the MukBEF ring to either entrap or release DNA. 22 The recent structures of the E. coli MukE:MukF (MukEF) and Haemophilus ducreyi MukBEF complexes have provided insight into the specific inter-subunit interactions that buttress these complexes. 17 The saturated MukEF complex adopts a Y-shaped structure, with the N-terminal winged helix domain (N-WHD; residues 1–103) and the helical bundle (121–292) of MukF accounting for the stem of the Y and the middle region of MukF (292–328) embedded in a MukE dimer accounting for each arm of the Y.…”