The Escherichia coli SMC complex, MukBEF, acts in chromosome segregation. MukBEF shares the distinctive architecture of other SMC complexes, with one prominent difference; unlike other kleisins, MukF forms dimers through its N-terminal domain. We show that a 4-helix bundle adjacent to the MukF dimerisation domain interacts functionally with the MukB coiled-coiled ‘neck’ adjacent to the ATPase head. We propose that this interaction leads to an asymmetric tripartite complex, as in other SMC complexes. Since MukF dimerisation is preserved during this interaction, MukF directs the formation of dimer of dimer MukBEF complexes, observed previously in vivo. The MukF N- and C-terminal domains stimulate MukB ATPase independently and additively. We demonstrate that impairment of the MukF interaction with MukB in vivo leads to ATP hydrolysis-dependent release of MukBEF complexes from chromosomes.

Coordination of outer membrane constriction with septation is critical to faithful division in Gram-negative bacteria and vital to the barrier function of the membrane. This coordination requires the recruitment of the peptidoglycan-binding outer-membrane lipoprotein Pal at division sites by the Tol system. Here, we show that Pal accumulation at Escherichia coli division sites is a consequence of three key functions of the Tol system. First, Tol mobilises Pal molecules in dividing cells, which otherwise diffuse very slowly due to their binding of the cell wall. Second, Tol actively captures mobilised Pal molecules and deposits them at the division septum. Third, the active capture mechanism is analogous to that used by the inner membrane protein TonB to dislodge the plug domains of outer membrane TonB-dependent nutrient transporters. We conclude that outer membrane constriction is coordinated with cell division by active mobilisation-and-capture of Pal at division septa by the Tol system.

RLIP76 (RalBP1) is a multidomain protein that interacts with multiple small G protein families: Ral via a specific binding domain, and Rho and R-Ras via a GTPase activating domain. RLIP76 interacts with endocytosis proteins and has also been shown to behave as a membrane ATPase that transports chemotherapeutic agents from the cell. We have determined the structure of the Ral-binding domain of RLIP76 and show that it comprises a coiled-coil motif. The structure of the RLIP76-RalB complex reveals a novel mode of binding compared to the structures of RalA complexed with the exocyst components Sec5 and Exo84. RLIP76 interacts with both nucleotide-sensitive regions of RalB, and key residues in the interface have been identified using affinity measurements of RalB mutants. Sec5, Exo84, and RLIP76 bind Ral proteins competitively and with similar affinities in vitro.

Redox-regulated effector systems that counteract oxidative stress are essential for all forms of life. Here we uncover a new paradigm for sensing oxidative stress centred on the hydrophobic core of a sensor protein. RsrA is an archetypal zinc-binding anti-sigma factor that responds to disulfide stress in the cytoplasm of Actinobacteria. We show that RsrA utilizes its hydrophobic core to bind the sigma factor σR preventing its association with RNA polymerase, and that zinc plays a central role in maintaining this high-affinity complex. Oxidation of RsrA is limited by the rate of zinc release, which weakens the RsrA–σR complex by accelerating its dissociation. The subsequent trigger disulfide, formed between specific combinations of RsrA's three zinc-binding cysteines, precipitates structural collapse to a compact state where all σR-binding residues are sequestered back into its hydrophobic core, releasing σR to activate transcription of anti-oxidant genes.

Ubiquitous Structural Maintenance of Chromosomes (SMC) complexes use a proteinaceous ring-shaped architecture to organize and individualize chromosomes, thereby facilitating chromosome segregation. They utilize cycles of adenosine triphosphate (ATP) binding and hydrolysis to transport themselves rapidly with respect to DNA, a process requiring protein conformational changes and multiple DNA contact sites. By analysing changes in the architecture and stoichiometry of the Escherichia coli SMC complex, MukBEF, as a function of nucleotide binding to MukB and subsequent ATP hydrolysis, we demonstrate directly the formation of dimer of MukBEF dimer complexes, dependent on dimeric MukF kleisin. Using truncated and full length MukB, in combination with MukEF, we show that engagement of the MukB ATPase heads on nucleotide binding directs the formation of dimers of heads-engaged dimer complexes. Complex formation requires functional interactions between the C- and N-terminal domains of MukF with the MukB head and neck, respectively, and MukE, which organizes the complexes by stabilizing binding of MukB heads to MukF. In the absence of head engagement, a MukF dimer bound by MukE forms complexes containing only a dimer of MukB. Finally, we demonstrate that cells expressing MukBEF complexes in which MukF is monomeric are Muk−, with the complexes failing to associate with chromosomes.

The plasmid partition protein KorB has a dual role: it is essential for the correct segregation of the low copy number broad host range RK2 plasmid while also being an important regulator of transcription. KorB belongs to the ParB family of proteins, and partitioning in RK2 has been studied as a simplified model of bacterial chromosome segregation. Structural information on full-length ParB proteins is limited, mainly due to the inability to grow crystals suitable for diffraction studies. We show, using CD and NMR, that KorB has regions of significant intrinsic disorder and hence it adopts a multiplicity of conformations in solution. The biophysical data are consistent with bioinformatic predictions based on the amino acid sequence that the N-terminal region and also the region between the central DNA-binding domain and the C-terminal dimerization domain are intrinsically disordered. We have used small angle x-ray scattering data to determine the ensemble of solution conformations for KorB and selected deletion mutants, based on models of the known domain structures. This conformational range of KorB is likely to be biologically required for DNA partitioning and for binding to a diverse set of partner proteins.

SummaryA central feature of broad host range IncP-1 plasmids is the set of regulatory circuits that tightly control plasmid core functions under steady-state conditions. Cooperativity between KorB and either KorA or TrbA repressor proteins is a key element of these circuits and deletion analysis has implicated the conserved C-terminal domain of KorA and TrbA in this interaction. By NMR we show that KorA and KorB interact directly and identify KorA amino acids that are affected on KorB binding. Studies on mutants showed that tyrosine 84 (or phenylalanine, in some alleles) is dispensable for repressor activity but critical for the specific interaction with KorB in both in vivo reporter gene assays and in vitro electrophoretic mobility shift and co-purification assays. This confirms that direct and specific protein-protein interactions are responsible for the cooperativity observed between KorB and its corepressors and lays the basis for determining the biological importance of this cooperativity.

SummaryRLIP76 is an effector for Ral small GTPases, which in turn lie downstream of the master regulator Ras. Evidence is growing that Ral and RLIP76 play a role in tumorigenesis, invasion, and metastasis. RLIP76 contains both a RhoGAP domain and a Ral binding domain (GBD) and is, therefore, a node between Ras and Rho family signaling. The structure of the RhoGAP-GBD dyad reveals that the RLIP76 RhoGAP domain adopts a canonical RhoGAP domain structure and that the linker between the two RLIP76 domains is structured, fixing the orientation of the two domains and allowing RLIP76 to interact with Rho-family GTPases and Ral simultaneously. However, the juxtaposed domains do not influence each other functionally, suggesting that the RLIP76-Ral interaction controls cellular localization and that the fixed orientation of the two domains orientates the RhoGAP domain with respect to the membrane, allowing it to be perfectly poised to engage its target G proteins.