Lanz, Martin A.. Conformational change in the C-terminal domain of "B. subtilis" GyrA and in the ATPase-gate of "M. mazei" topoisomerase VI. 2011, PhD Thesis, University of Basel, Faculty of Science.
Official URL: http://edoc.unibas.ch/diss/DissB_9645
The exact position of the GyrA CTD relative to the catalytic core is not known as no full-length high-resolution structure of GyrA or gyrase has been solved. To monitor potential dynamics of a single GyrA CTD and localize the domain with respect to the core of the enzyme, single-molecule FRET (smFRET) experiments were performed. Stable dimeric constructs with one wt and one mutant subunit (carrying solvent-accessible cysteine residues for fluorescent labeling on the GyrA core and on the CTD) were produced in a hetero-dimeric expression system. FRET efficiencies from GyrA constructs labeled at different positions were converted to inter-dye distances, allowing the triangulation of the position of the N-terminal region of the CTD. In absence of DNA and GyrB, the FRET model placed the CTD close to the catalytic core domain, indicating a position suitable to contact a DNA bound to the DNA-gate. Upon addition of GyrB and supercoiled plasmid, the N-terminal region of the CTD is displaced from the core by 2.5 to 2.6 nm (depending on the labeling position of the CTD monitored), indicating an extended conformation of the CTD.
Fluorescence anisotropy titrations suggest that the extended CTD conformation is characterized by simultaneous binding of the DNA to the DNA-gate and the CTDs. However, in the absence of GyrB the CTDs are the main DNA interaction sites of GyrA. Binding of plasmid DNA induces conformational flexibility in the CTDs, which indicates transient bridging interaction of the DNA to both the CTDs and the DNA-gate. Binding of GyrB to GyrA induces only a small movement of the CTDs, probably by spatial interference, indicating that GyrB itself is not responsible for the extended CTD conformation, but rather for the stabilization of the enzyme-DNA complex.
Addition of neg. supercoiled or relaxed plasmid or linear DNAs of 48 to 110 bp to gyrase results in an extended CTD conformation; anisotropy measurements clearly showed that the linear DNAs bind to both the DNA-gate and the CTDs in presence of GyrB. However, a 37bp DNA showed similar KD values for GyrA and GyrA-core in the presence of GyrB. Moreover, binding to gyrase did not result in an extended CTD conformation. These findings indicate that a small interaction between DNA and CTDs (not more than approx. 5 bp) is sufficient to induce the conformational change. Meanwhile, the extended position of the CTD does neither depend on the topology of the substrate nor extensive wrapping or the presence or absence of a T-segment.
In contrast to previous speculations, binding of ADPNP (a non-hydrolysable ATP-analog) to the gyrase-DNA complex did not result in the release of the GyrA CTDs, and no conformational change connected to strand passage could be detected. A cleavage-deficient GyrA mutant exhibits a 1.5-fold higher DNA affinity but retains wild-type like CTD conformations; suggesting that the latter are independent of DNA distortion and cleavage. Deletion of a conserved heptapeptide on the CTD (GyrA-box) abolished the supercoiling activity, but did not alter the DNA affinity or the conformational behavior of the enzyme. Taken together, the present data suggests that the conformational change of the CTDs is mediated by simultaneous binding of DNA to the CTDs and the DNA-gate (a complex which is stabilized by GyrB) and represents an early event in the supercoiling cycle of gyrase.
Topoisomerase VI possesses only two dimerization interfaces compared to the three in conventional type II enzymes, thereby exhibiting less complexity than e.g. gyrase. It catalyzes DNA relaxation and decatenation in an ATP-dependent manner. In smFRET experiments, a fluorescently labeled 50bp DNA was used as a FRET probe for potential DNA-gate dynamics in TopoVI. A reduction in the DNA affinity upon deletion of the active site tyrosine residue inducing DNA cleavage indicated the formation of a covalent protein-DNA intermediate in wild-type TopoVI, although DNA cleavage could not be demonstrated directly; the conformation of the DNA was similar in complexes with wild-type and active-site mutant enzyme. No distortion of the DNA or gate opening could be detected upon addition of ATP and ADPNP.
Conformational changes in the ATPase-gate of TopoVI were monitored in smFRET measurements, using fluorescently labeled enzyme with one dye attached to each nucleotide-binding domain. We have observed four different conformational states in the ATPase-gate: In absence of nucleotide or DNA substrates the ATPase-gate exhibits conformational flexibility, indicating gate opening and closing. Binding of supercoiled plasmid to TopoVI forces the ATPase-gate to open up even wider, although considerable flexibility of the domains is retained; both these states have been proposed on the basis of two crystal structures of intact TopoVI holoenzymes, showing a closed and an open gate conformation. Addition of ADPNP to TopoVI induces a well-defined conformation of the ATPase domains close to each other, indicating dimerization; this is in agreement with crystal structures from isolated TopoVI-B domains which show tight association when bound to ADPNP. Addition of both nucleotide and plasmid immobilizes the domains in a conformation different from the one observed for the closed gate, indicating that the presence of a T-DNA segment in the central cavity induces physical strain to the TopoVI complex; this state has not been proposed so far. During all these conformational changes of the ATPase-gate, the transducer domain linking the two gates barely moves, suggesting that the ATPase domains exhibit a rotational flexibility in absence of nucleotide. TopoVI shows a different conformational behavior than type II topoisomerases (e.g. gyrase) in that it exhibits dynamic opening and closing of the ATPase-gate in absence of ligands. Furthermore it does not show DNA-induced pre-closure, but rather opening of the gate in presence of a supercoiled DNA.
|Committee Members:||Seelig, Joachim|
|Faculties and Departments:||05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Biophysical Chemistry (Klostermeier)|
|Bibsysno:||Link to catalogue|
|Number of Pages:||153 S.|
|Last Modified:||30 Jun 2016 10:42|
|Deposited On:||19 Oct 2011 14:22|
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