PDBsum entry: 1ei1

Isomerase

PDB id: 1ei1

Contents

Protein chains

391 a.a. *

Ligands

SO4 ×3

ANP ×2

GOL ×2

Waters ×505

* Residue conservation analysis

PDB id:

1ei1

Name:

Isomerase

Title:

Dimerization of e. Coli DNA gyrase b provides a structural m for activating the atpase catalytic center

Structure:

DNA gyrase b. Chain: a, b. Fragment: n-terminal 43 kda fragment. Synonym: gyrb. Engineered: yes. Mutation: yes

Source:

Escherichia coli. Organism_taxid: 562. Expressed in: escherichia coli. Expression_system_taxid: 562. Pn43-y5s). Other_details: pn43-y5s (tac promoter, beta lactamase gene, gene, pbr322 background)

Biol. unit:

Dimer (from PQS)

Resolution:

2.30Å R-factor: 0.166 R-free: 0.266

Authors:

L.Brino,A.Urzhumtsev,Et Al,P.Oudet,D.Moras

Key ref:

L.Brino et al. (2000). Dimerization of Escherichia coli DNA-gyrase B provides a structural mechanism for activating the ATPase catalytic center. J Biol Chem, 275, 9468-9475. PubMed id: 10734094 DOI: 10.1074/jbc.275.13.9468

Date:

23-Feb-00 Release date: 31-Mar-00

Protein chains

P0AES6  (GYRB_ECOLI) -  DNA gyrase subunit B

Seq: 804 a.a.

Struc: 391 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.5.99.1.3 - Dna topoisomerase (ATP-hydrolyzing).
• Reaction: ATP-dependent breakage, passage and rejoining of double-stranded DNA.

 Gene Ontology (GO) functional annotation 

• Cellular component: chromosome (1 term)
• Biological process: DNA topological change (1 term)
• Biochemical function: DNA binding (3 terms)

DOI no: 10.1074/jbc.275.13.9468

J Biol Chem 275:9468-9475 (2000)

PubMed id: 10734094

Dimerization of Escherichia coli DNA-gyrase B provides a structural mechanism for activating the ATPase catalytic center.

L.Brino, A.Urzhumtsev, M.Mousli, C.Bronner, A.Mitschler, P.Oudet, D.Moras.

ABSTRACT

DNA-gyrase exhibits an unusual ATP-binding site that is formed as a result of gyrase B subunit dimerization, a structural transition that is also essential for DNA capture during the topoisomerization cycle. Previous structural studies on Escherichia coli DNA-gyrase B revealed that dimerization is the result of a polypeptidic exchange involving the N-terminal 14 amino acids. To provide experimental data that dimerization is critical for ATPase activity and enzyme turnover, we generated mutants with reduced dimerization by mutating the two most conserved residues of the GyrB N-terminal arm (Tyr-5 and Ile-10 residues). Our data demonstrate that the hydrophobic Ile-10 residue plays an important role in enzyme dimerization and the nucleotide-protein contact mediated by Tyr-5 side chain residue helps the dimerization process. Analysis of ATPase activities of mutant proteins provides evidence that dimerization enhances the ATP-hydrolysis turnover. The structure of the Y5S mutant of the N-terminal 43-kDa fragment of E. coli DNA GyrB subunit indicates that Tyr-5 residue provides a scaffold for the ATP-hydrolysis center. We describe a channel formed at the dimer interface that provides a structural mechanism to allow reactive water molecules to access the gamma-phosphate group of the bound ATP molecule. Together, these results demonstrate that dimerization strongly contributes to the folding and stability of the catalytic site for ATP hydrolysis. A role for the essential Mg(2+) ion for the orientation of the phosphate groups of the bound nucleotide inside the reactive pocket was also uncovered by superposition of the 5'-adenylyl beta-gamma-imidodiphosphate (ADPNP) wild-type structure to the salt-free ADPNP structure.

Selected figure(s)

Figure 5.
Fig. 5. The tunnel in a gyrase monomer at the phosphate end of the ADPNP. A, side chains of amino acids making front "walls" of the tunnel are indicated. All shown solvent molecules, indicated by balls in magenta (S1 to S4), have temperature factor varied from 15 to 32 Å2. B, interactions with the solvent molecule S1 located near the -phosphate group. Note the interactions of the main chain nitrogens of the sequence 114-120 with the ADPNP molecule fixing the orientation and neutralizing charge of the three phosphate groups. S1 indicates the potential reactive water molecule interacting with O- 1 of the ADPNP (2.6 Å distance), with O- 2 of Glu-42 (2.8 Å), with N- 2 of Gln-335 (3.1 Å), with O- 1 of Gln-335 (2.6 Å), and with O of Gln-335 (3.4 Å). Note also close contacts between O- 1 of Glu-42 and N- 2 of His-38 (2.9 Å), O- 1 of Gln-335, and N- 1 of His-116 (2.9 Å), between N- of Lys-337 and O of Gly-113 (2.8 Å), between N- of Lys-337 and O- 2 of ADPNP (2.7 Å), between N- 2 of Gln-335 and O of Tyr-26 (2.8 Å), between N- 2 of Gln-335 and O- 1 of Glu-42 (3.2 Å). Contacts are shown by small balls in yellow, light blue, and green.

Figure 6.
Fig. 6. Conformational changes near the Mg2+-binding site. ATP/ADPNP, protein conformations, and main interactions are shown near the Mg2+-binding site. ATP is shown in magenta and ADPNP in green (O and N atoms are indicated in red and blue balls, respectively). Protein is shown for the Y5S mutant fragment; Asn-47 and Gln-335 side chains and Leu-115 main chain of the wild-type protein are shown in gray. In the wild-type complex, Mg2+ (big orange ball) interacts with O- 2, O- 1, and O- 1 of the phosphate groups, Asn-46 O- 1 and two water molecules (big red balls). O- 2 interacts with the sugar and O- 3 interacts with Leu-115 main chain NH group. Small light blue balls represent all interactions. In the case of the Y5S mutant fragment complexed with salt-free ADPNP, the orientation of the - phosphates is completely different. The place of Mg2+ is empty, but there are two water molecules (small magenta balls indicated as SA and SB) at positions very close to the original Mg2+-bound water molecules; one of them interacts with O- 1, and the second interacts with Asn-46 O- 1. O- 1 interacts now with the sugar and O- 2 with a new water molecule, SC. After slight rearrangement of Leu-115, its main chain NH group interacts only with O- 1. Small yellow balls indicate all interactions. S1 is the first solvent molecule in the water channel leading to the protein surface. Note the significant change in the Gln-335 conformation. Interaction of the adenine base and surrounding water molecules are not shown.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2000, 275, 9468-9475) copyright 2000.

Figures were selected by the author.