Biochemistry zyxwvuts 1995,34, zyxwvu 9801-9808 9801 Nucleotide Binding to the 43-Kilodalton N-Terminal Fragment of the DNA Gyrase B Protein? Janid A. Ali,* George Orphanides,§ and Anthony Maxwell* Department of Biochemistry, University of Leicester, University Road, Leicester LEI 7RH, U.K. Received April zyxwvuts 5, 1995; Revised Manuscript Received May zyxwv 25, 1995@ ABSTRACT: The binding of ADPNP (5'-adenylyl P,y-imidodiphosphate) to the 43-kDa N-terminal fragment of the DNA gyrase B protein is found to stabilize a dimer of the protein. Analysis of the kinetics of binding of ADPNP to the fragment suggests that protein dimers can contain 1 or 2 molecules of bound nucleotide. ATP, ADP, or coumarin drugs inhibit the binding of ADPNP. The rate of dissociation of ADPNP from the 43-kDa protein is found to be very slow and unaffected by the presence of other nucleotides. These data can be accommodated by a scheme in which the 43-kDa monomer forms a short-lived complex with ADPNP that can be converted into long-lived dimer complexes containing either 1 or 2 molecules of bound ADPNP; dimer formation with 2 bound ADPNPs is strongly favored. Coumarin drugs inhibit the binding of ADPNP to the 43-kDa fragment, with novobiocin binding to the protein with a stoichiometry of 1:l and coumermycin binding with a stoichiometry of 0.5:l. DNA gyrase is the bacterial type zyxwvuts I1 topoisomerase which can introduce negative supercoils into DNA using the free energy of ATP hydrolysis [see Reece and Maxwell (1991b) for a recent review]. The enzyme from Escherichia coli consists of two proteins, A and B, of molecular masses 97 and 90 kDa, respectively: the active enzyme is an A2B2 complex. All DNA topoisomerases are able to relax negatively supercoiled DNA but only gyrase can also catalyze the introduction of negative supercoils, in a reaction coupled to ATP hydrolysis. Mechanistic studies have revealed the steps involved in the supercoiling reaction [see Maxwell and Gellert (1986) and Reece and Maxwell (1991b) for reviews]. Briefly, this process involves the wrapping of DNA around the A2B2 complex, cleavage of this DNA in both strands (involving the formation of DNA-protein covalent bonds), and passage of a segment of DNA through this double-stranded break. Resealing of the break results in the introduction of two negative supercoils. Catalytic supercoiling requires the hydrolysis of ATP, but limited supercoiling can be achieved in the presence of the nonhy- drolyzable ATP analogue ADPNP' (5'-adenylyl ,B,y-imido- diphosphate) (Sugino et al., 1978). This result has been interpreted as suggesting that nucleotide binding promotes one round of supercoiling and that hydrolysis is required for the enzyme to turn over. ' This work was funded by the SERC (U.K.). J.A.A. was supported by a CASE studentship from SERC and Glaxo Group Research, and zyxwvuts (3.0. was supported by a CASE studentship from SERC and Zeneca Pharmaceuticals. A.M. is a Lister Institute Jenner Fellow. * Corresponding author. Present address: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110. Present address: Howard Hughes Medical Institute Research Laboratories, Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Denistry of New Jersey, Department of Biochemistry, Piscataway, NJ 08854. @ Abstract published in Advance ACS Abstracts, July 15, 1995. Abbreviations: ADPNP, 5'-adenylyl P,y-imidodiphosphate; DTT, dithiothreitol; GyrA, DNA gyrase A protein; GyrB, DNA gyrase B protein. 0006-2960/95/0434-9801$09.00/0 Both the A and B subunits of gyrase have been shown to contain distinct functional domains. The A protein consists of an N-terminal domain (59-64 kDa) involved in DNA breakage and reunion and a C-terminal domain (33 kDa) involved in DNA-protein interactions (Reece & Maxwell, 1989, 1991a,c). The B protein consists of an N-terminal domain (43 kDa) containing the ATPase activity and a C-terminal domain (47 kDa) involved in interactions with the A protein and DNA (Brown et al., 1979; Gellert et al., 1979; Adachi et al., 1987; Ali et al., 1993). The structure of the 43-kDa N-terminal fragment complexed with ADPNP has been solved to 2.5-A resolution by X-ray crystallography (Wigley et al., 1991). The gyrase supercoiling reaction can be inhibited by a number of compounds, including the quinolone and coumarin groups of antibacterial agents [for reviews see Drlica and Coughlin (1989), RAdl(1990), Reece and Maxwell (1991b), and Maxwell (1992, 1993)l. The quinolones (e.g., nalidixic acid and ciprofloxacin) interrupt the DNA breakage and resealing reaction of gyrase, while the coumarins (e.g., novobiocin and coumermycin A') inhibit the ATPase reac- tion. Early studies suggested that the coumarins might be competitive inhibitors of the ATPase reaction (Sugino et al., 1978; Sugino & Cozzarelli, 1980), but more recent work supports the idea that they may act noncompetitively (Contreras & Maxwell, 1992; Ali et al., 1993; Maxwell, 1993). Sequencing of mutations in gyrB which confer coumarin resistance has suggested that the coumarin-binding site lies in the N-terminal portion of GyrB (del Castillo et al., 1991; Contreras & Maxwell, 1992). Subsequently, a 24- kDa N-terminal fragment of GyrB (residues 2-220) has been cloned and expressed and shown to contain the coumarin- binding site (Gilbert & Maxwell, 1994). The complex of this fragment with the coumarin drug novobiocin has recently been crystallized (Lewis et al., 1994). One of the key questions concerning the mechanism of supercoiling by DNA gyrase is the coupling of ATP hydrolysis to the DNA supercoiling reaction. Although some studies of the ATPase reaction of gyrase have been reported 1995 American Chemical Society