Biology subjects

Rajasekar, K.

Publications and source records attributed to Rajasekar, K..

3 recordsLinked to original sources

bioRxiv2021

Competitive binding of MatP and topoisomerase IV to the MukB dimerization hinge

SMC complexes have ubiquitous roles in chromosome organisation. In Escherichia coli, the interplay between the SMC complex, MukBEF, and matS-bound MatP in the replication termination region, ter, results in depletion of MukBEF from ter, thus promoting chromosome individualisation by directing replichores to separate cell halves. MukBEF also interacts with topoisomerase IV ParC2E2 heterotetramers, to direct its chromosomal distribution to mirror that of MukBEF, thereby facilitating coordination between chromosome organisation and decatenation by topoisomerase IV. Here we demonstrate that the MukB dimerization hinge binds ParC and MatP with the same dimer to dimer stoichiometry. MatP and ParC have an overlapping binding interface on the MukB hinge, leading to their mutually exclusive binding. Furthermore, the MukB hinge fails to stably associate with matS-bound MatP, while MatP mutants deficient in matS binding are impaired in MukB hinge binding, demonstrating that mats competes with the hinge for MatP binding. Cells expressing MukBEF complexes containing a mutation in the MukB hinge interface for ParC/MatP binding are deficient in ParC binding in vivo, despite having a Muk+ topoisomerase IV+ phenotype. This mutant protein is also impaired in MatP binding in vitro, and cells expressing this variant exhibit a MukBEF cellular localisation consistent with impaired MatP binding.

biochemistry↗

bioRxiv2019

Pal stabilises the bacterial outer membrane during constriction by a mobilisation-and-capture mechanism

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. Recent studies suggest this coordination is through the active accumulation of the peptidoglycan-binding outer membrane lipoprotein Pal at division sites by the Tol system, but the mechanism is unknown. 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.

microbiology↗

bioRxiv2017

Asymmetric MukB ATPases are regulated independently by the N- and C-terminal domains of MukF kleisin

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 dimerization domain interacts functionally with the MukB coiled-coiled neck adjacent to the ATPase head, forming an asymmetric tripartite complex, as in other SMC complexes. Since MukF dimerization is preserved during this interaction, MukF directs the formation of dimer of dimers MukBEF complexes, observed previously in vivo. The MukF N- and C-terminal domains stimulate ATPase independently and additively, consistent with them each targeting only one of the two MukB ATPase active sites in the asymmetric complex. We demonstrate that MukF interaction with the MukB neck turns over during cycles of ATP binding and hydrolysis in vivo and that impairment of this interaction leads to MukBEF release from chromosomes.

biochemistry↗