Backbone Assignment of Gyrase-B P24 Fragment and its Interaction with the Inibitor GR122222

DNA topoisomerases are ubiquitous enzymes that control the topological state of DNA in cells. There are several classes of topoisomerases, each with distinct properties. Among them, bacterial DNA-gyrase is able to introduce supercoils into DNA in a reaction coupled to hydrolysis of ATP. Gyrase is essential in all bacteria but it is not found in eukaryotes and is therefore a good target for antibiotics. Gyrase consists of 2 subunits GyrA and GyrB, the active enzyme being an A2B2 complex. The ATPase reaction takes place in the B subunit and binding sites of different inhibitors have been localised in the N-terminal 24kDa fragment of GyrB (P24).The X-ray structures of this fragment complexed with different ligands have been published but structural studies in solution are scarce. NMR provides useful tools to study the interactions of a protein and small substrates in solution but the first, demanding step required for this approach is the assignment of resonances, which must be achieved at least in part. In the present work, the fragment P24 was investigated by heteronuclear NMR both in the free form and as a complex with the cyclothialidine GR122222. Backbone assignment for the 2 forms was obtained using TROSY-based triple resonance experiments on a triply labelled sample (100%15N, 100%13C, 75%2H) of P24 from E. coli. Few peaks were not detected in 15N HSQC spectrum because of line broadening probably due to conformational exchange. These peaks are mainly located in a flexible region comprising the P-loop motif (GXXGXG) implicated in nucleotide binding. Chemical shift data together with analysis of NOESY connectivities lead to a preliminary definition of secondary structural elements in solution. An initial characterization of the binding site was performed on the basis of binding-induced changes of backbone chemical shifts.