PDBsum entry: 1aj6

Topoisomerase

PDB id: 1aj6

Contents

Protein chain

194 a.a. *

Ligands

NOV

Waters ×89

* Residue conservation analysis

PDB id:

1aj6

Name:

Topoisomerase

Title:

Novobiocin-resistant mutant (r136h) of the n-terminal 24 kda of DNA gyrase b complexed with novobiocin at 2.3 angstroms

Structure:

Gyrase. Chain: a. Fragment: n-terminal 24 kda. Engineered: yes. Mutation: yes

Source:

Escherichia coli. Organism_taxid: 562. Cellular_location: cytoplasm. Gene: gyrb. Expressed in: escherichia coli. Expression_system_taxid: 562. Other_details: plasmid ptb382 encoding mutant derived from which encodes 24 kda subdomain (supplied by a. Maxwell, lei which in turn was derived from pag111 plasmid which encodes

Resolution:

2.30Å R-factor: 0.206 R-free: 0.290

Authors:

S.A.Weston,A.Tunnicliffe,R.A.Pauptit

Key ref:

G.A.Holdgate et al. (1997). The entropic penalty of ordered water accounts for weaker binding of the antibiotic novobiocin to a resistant mutant of DNA gyrase: a thermodynamic and crystallographic study. Biochemistry, 36, 9663-9673. PubMed id: 9245398 DOI: 10.1021/bi970294+

Date:

15-May-97 Release date: 20-May-98

Protein chain

P0AES6  (GYRB_ECOLI) -  DNA gyrase subunit B

Seq: 804 a.a.

Struc: 194 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

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: 10.1021/bi970294+

Biochemistry 36:9663-9673 (1997)

PubMed id: 9245398

The entropic penalty of ordered water accounts for weaker binding of the antibiotic novobiocin to a resistant mutant of DNA gyrase: a thermodynamic and crystallographic study.

G.A.Holdgate, A.Tunnicliffe, W.H.Ward, S.A.Weston, G.Rosenbrock, P.T.Barth, I.W.Taylor, R.A.Pauptit, D.Timms.

ABSTRACT

Novobiocin is an antibiotic which binds to a 24 kDa fragment from the B subunit of DNA gyrase. Naturally occurring resistance arises from mutation of Arg-136 which hydrogen bonds to the coumarin ring of novobiocin. We have applied calorimetry to characterize the binding of novobiocin to wild-type and R136H mutant 24 kDa fragments. Upon mutation, the Kd increases from 32 to 1200 nM at 300 K. The enthalpy of binding is more favorable for the mutant (DeltaH degrees shifts from -12.1 to -17.5 kcal/mol), and the entropy of binding is much less favorable (TDeltaS degrees changes from -1.8 to -9.4 kcal/mol). Both of these changes are in the direction opposite to that expected if the loss of the Arg residue reduces hydrogen bonding. The change in heat capacity at constant pressure upon binding (DeltaCp) shifts from -295 to -454 cal mol-1 K-1. We also report the crystal structure, at 2.3 A resolution, of a complex between the R136H 24 kDa fragment and novobiocin. Although the change in DeltaCp often would be interpreted as reflecting increased burial of hydrophobic surface on binding, this structure reveals a small decrease. Furthermore, an ordered water molecule is sequestered into the volume vacated by removal of the guanidinium group. There are large discrepancies when the measured thermodynamic parameters are compared to those estimated from the structural data using empirical relationships. These differences seem to arise from the effects of sequestering ordered water molecules upon complexation. The water-mediated hydrogen bonds linking novobiocin to the mutant protein make a favorable enthalpic contribution, whereas the immobilization of the water leads to an entropic cost and a reduction in the heat capacity of the system. Such a negative contribution to DeltaCp, DeltaH degrees , and TDeltaS degrees appears to be a general property of water molecules that are sequestered when ligands bind to proteins.