PDBsum entry 1aj6

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Topoisomerase PDB id
Protein chain
194 a.a. *
Waters ×89
* Residue conservation analysis
PDB id:
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
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+
15-May-97     Release date:   20-May-98    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
P0AES6  (GYRB_ECOLI) -  DNA gyrase subunit B
804 a.a.
194 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 1 residue position (black cross)

 Enzyme reactions 
   Enzyme class: E.C.  - Dna topoisomerase (ATP-hydrolyzing).
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: ATP-dependent breakage, passage and rejoining of double-stranded DNA.
 Gene Ontology (GO) functional annotation 
  GO annot!
  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.
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.

Literature references that cite this PDB file's key reference

  PubMed id Reference
19857499 I.Gómez García, C.E.Stevenson, I.Usón, C.L.Freel Meyers, C.T.Walsh, and D.M.Lawson (2010).
The crystal structure of the novobiocin biosynthetic enzyme NovP: the first representative structure for the TylF O-methyltransferase superfamily.
  J Mol Biol, 395, 390-407.
PDB code: 2wk1
19926848 L.Martínez, A.S.Nascimento, F.M.Nunes, K.Phillips, R.Aparicio, S.M.Dias, A.C.Figueira, J.H.Lin, P.Nguyen, J.W.Apriletti, F.A.Neves, J.D.Baxter, P.Webb, M.S.Skaf, and I.Polikarpov (2009).
Gaining ligand selectivity in thyroid hormone receptors via entropy.
  Proc Natl Acad Sci U S A, 106, 20717-20722.
PDB codes: 3jzb 3jzc
19965760 M.J.Edwards, R.H.Flatman, L.A.Mitchenall, C.E.Stevenson, T.B.Le, T.A.Clarke, A.R.McKay, H.P.Fiedler, M.J.Buttner, D.M.Lawson, and A.Maxwell (2009).
A crystal structure of the bifunctional antibiotic simocyclinone d8, bound to DNA gyrase.
  Science, 326, 1415-1418.
PDB codes: 2wl2 2y3p
19646998 S.H.Mishra, A.M.Spring, and M.W.Germann (2009).
Thermodynamic profiling of HIV RREIIB RNA-zinc finger interactions.
  J Mol Biol, 393, 369-382.  
19923714 V.Venugopal, A.K.Datta, D.Bhattacharyya, D.Dasgupta, and R.Banerjee (2009).
Structure of cyclophilin from Leishmania donovani bound to cyclosporin at 2.6 A resolution: correlation between structure and thermodynamic data.
  Acta Crystallogr D Biol Crystallogr, 65, 1187-1195.
PDB code: 3eov
18939877 A.C.Donnelly, J.R.Mays, J.A.Burlison, J.T.Nelson, G.Vielhauer, J.Holzbeierlein, and B.S.Blagg (2008).
The design, synthesis, and evaluation of coumarin ring derivatives of the novobiocin scaffold that exhibit antiproliferative activity.
  J Org Chem, 73, 8901-8920.  
18991631 A.Donnelly, and B.S.Blagg (2008).
Novobiocin and additional inhibitors of the Hsp90 C-terminal nucleotide-binding pocket.
  Curr Med Chem, 15, 2702-2717.  
  18997325 I.Gómez García, C.L.Freel Meyers, C.T.Walsh, and D.M.Lawson (2008).
Crystallization and preliminary X-ray analysis of the O-carbamoyltransferase NovN from the novobiocin-biosynthetic cluster of Streptomyces spheroides.
  Acta Crystallogr Sect F Struct Biol Cryst Commun, 64, 1000-1002.  
18093089 L.A.Plesniak, K.Botsch, M.Leibrand, M.Kelly, D.Sem, J.A.Adams, and P.Jennings (2008).
Transferred NOE and saturation transfer difference NMR studies of novobiocin binding to EnvZ suggest binding mode similar to DNA gyrase.
  Chem Biol Drug Des, 71, 28-35.  
17910061 M.Bello, G.Pérez-Hernández, D.A.Fernández-Velasco, R.Arreguín-Espinosa, and E.García-Hernández (2008).
Energetics of protein homodimerization: effects of water sequestering on the formation of beta-lactoglobulin dimer.
  Proteins, 70, 1475-1487.  
18086667 S.Chopra, R.M.Dooling, C.G.Horner, and E.E.Howell (2008).
A balancing act between net uptake of water during dihydrofolate binding and net release of water upon NADPH binding in R67 dihydrofolate reductase.
  J Biol Chem, 283, 4690-4698.  
18218621 S.L.Newstead, J.A.Potter, J.C.Wilson, G.Xu, C.H.Chien, A.G.Watts, S.G.Withers, and G.L.Taylor (2008).
The structure of Clostridium perfringens NanI sialidase and its catalytic intermediates.
  J Biol Chem, 283, 9080-9088.
PDB codes: 2bf6 2vk5 2vk6 2vk7
  17329822 C.E.Stevenson, C.L.Freel Meyers, C.T.Walsh, and D.M.Lawson (2007).
Crystallization and preliminary X-ray analysis of the O-methyltransferase NovP from the novobiocin-biosynthetic cluster of Streptomyces spheroides.
  Acta Crystallogr Sect F Struct Biol Cryst Commun, 63, 236-238.  
17237207 M.W.Freyer, R.Buscaglia, A.Hollingsworth, J.Ramos, M.Blynn, R.Pratt, W.D.Wilson, and E.A.Lewis (2007).
Break in the heat capacity change at 303 K for complex binding of netropsin to AATT containing hairpin DNA constructs.
  Biophys J, 92, 2516-2522.  
17242738 Z.Li, and T.Lazaridis (2007).
Water at biomolecular binding interfaces.
  Phys Chem Chem Phys, 9, 573-581.  
16705642 A.V.Gribenko, T.R.Keiffer, and G.I.Makhatadze (2006).
Amino acid substitutions affecting protein dynamics in eglin C do not affect heat capacity change upon unfolding.
  Proteins, 64, 295-300.  
16385472 B.S.Blagg, and T.D.Kerr (2006).
Hsp90 inhibitors: small molecules that transform the Hsp90 protein folding machinery into a catalyst for protein degradation.
  Med Res Rev, 26, 310-338.  
16784236 C.A.McElroy, A.Manfredo, P.Gollnick, and M.P.Foster (2006).
Thermodynamics of tryptophan-mediated activation of the trp RNA-binding attenuation protein.
  Biochemistry, 45, 7844-7853.  
16906619 N.Shimokhina, A.Bronowska, and S.W.Homans (2006).
Contribution of ligand desolvation to binding thermodynamics in a ligand-protein interaction.
  Angew Chem Int Ed Engl, 45, 6374-6376.  
16411240 P.Jakimowicz, M.Tello, C.L.Meyers, C.T.Walsh, M.J.Buttner, R.A.Field, and D.M.Lawson (2006).
The 1.6-A resolution crystal structure of NovW: a 4-keto-6-deoxy sugar epimerase from the novobiocin biosynthetic gene cluster of Streptomyces spheroides.
  Proteins, 63, 261-265.
PDB code: 2c0z
16569821 R.H.Flatman, A.Eustaquio, S.M.Li, L.Heide, and A.Maxwell (2006).
Structure-activity relationships of aminocoumarin-type gyrase and topoisomerase IV inhibitors obtained by combinatorial biosynthesis.
  Antimicrob Agents Chemother, 50, 1136-1142.  
17038336 Y.Y.Huang, J.Y.Deng, J.Gu, Z.P.Zhang, A.Maxwell, L.J.Bi, Y.Y.Chen, Y.F.Zhou, Z.N.Yu, and X.E.Zhang (2006).
The key DNA-binding residues in the C-terminal domain of Mycobacterium tuberculosis DNA gyrase A subunit (GyrA).
  Nucleic Acids Res, 34, 5650-5659.  
16257377 G.A.Holdgate, and W.H.Ward (2005).
Measurements of binding thermodynamics in drug discovery.
  Drug Discov Today, 10, 1543-1550.  
15728908 R.H.Flatman, A.J.Howells, L.Heide, H.P.Fiedler, and A.Maxwell (2005).
Simocyclinone D8, an inhibitor of DNA gyrase with a novel mode of action.
  Antimicrob Agents Chemother, 49, 1093-1100.  
15853944 S.S.Garrido, A.C.Scatigno, E.Trovatti, D.C.Carvalho, and R.Marchetto (2005).
Probing the binding of the coumarin drugs using peptide fragments of DNA gyrase B protein.
  J Pept Res, 65, 502-511.  
14988501 A.Cauerhff, F.A.Goldbaum, and B.C.Braden (2004).
Structural mechanism for affinity maturation of an anti-lysozyme antibody.
  Proc Natl Acad Sci U S A, 101, 3539-3544.
PDB code: 1p2c
15489226 M.Katragadda, D.Morikis, and J.D.Lambris (2004).
Thermodynamic studies on the interaction of the third complement component and its inhibitor, compstatin.
  J Biol Chem, 279, 54987-54995.  
12604539 C.H.Gross, J.D.Parsons, T.H.Grossman, P.S.Charifson, S.Bellon, J.Jernee, M.Dwyer, S.P.Chambers, W.Markland, M.Botfield, and S.A.Raybuck (2003).
Active-site residues of Escherichia coli DNA gyrase required in coupling ATP hydrolysis to DNA supercoiling and amino acid substitutions leading to novobiocin resistance.
  Antimicrob Agents Chemother, 47, 1037-1046.  
14527324 G.L.Verdine, and D.P.Norman (2003).
Covalent trapping of protein-DNA complexes.
  Annu Rev Biochem, 72, 337-366.  
12044152 D.Lafitte, V.Lamour, P.O.Tsvetkov, A.A.Makarov, M.Klich, P.Deprez, D.Moras, C.Briand, and R.Gilli (2002).
DNA gyrase interaction with coumarin-based inhibitors: the role of the hydroxybenzoate isopentenyl moiety and the 5'-methyl group of the noviose.
  Biochemistry, 41, 7217-7223.
PDB code: 1kzn
12163065 J.Mendes, R.Guerois, and L.Serrano (2002).
Energy estimation in protein design.
  Curr Opin Struct Biol, 12, 441-446.  
11151007 J.C.Burnett, P.Botti, D.J.Abraham, and G.E.Kellogg (2001).
Computationally accessible method for estimating free energy changes resulting from site-specific mutations of biomolecules: systematic model building and structural/hydropathic analysis of deoxy and oxy hemoglobins.
  Proteins, 42, 355-377.  
11514673 K.M.Peterson, K.V.Gopalan, A.Nandy, and D.K.Srivastava (2001).
Influence of Glu-376 --> Gln mutation on enthalpy and heat capacity changes for the binding of slightly altered ligands to medium chain acyl-CoA dehydrogenase.
  Protein Sci, 10, 1822-1834.  
10677211 J.C.Burnett, G.E.Kellogg, and D.J.Abraham (2000).
Computational methodology for estimating changes in free energies of biomolecular association upon mutation. The importance of bound water in dimer-tetramer assembly for beta 37 mutant hemoglobins.
  Biochemistry, 39, 1622-1633.  
10933791 M.J.Snider, S.Gaunitz, C.Ridgway, S.A.Short, and R.Wolfenden (2000).
Temperature effects on the catalytic efficiency, rate enhancement, and transition state affinity of cytidine deaminase, and the thermodynamic consequences for catalysis of removing a substrate "anchor".
  Biochemistry, 39, 9746-9753.  
9989504 A.M.Bilwes, L.A.Alex, B.R.Crane, and M.I.Simon (1999).
Structure of CheA, a signal-transducing histidine kinase.
  Cell, 96, 131-141.
PDB code: 1b3q
10488102 H.Kondo, M.Shiroishi, M.Matsushima, K.Tsumoto, and I.Kumagai (1999).
Crystal structure of anti-Hen egg white lysozyme antibody (HyHEL-10) Fv-antigen complex. Local structural changes in the protein antigen and water-mediated interactions of Fv-antigen and light chain-heavy chain interfaces.
  J Biol Chem, 274, 27623-27631.
PDB code: 1c08
10398392 I.Jelesarov, and H.R.Bosshard (1999).
Isothermal titration calorimetry and differential scanning calorimetry as complementary tools to investigate the energetics of biomolecular recognition.
  J Mol Recognit, 12, 3.  
9730839 K.L.Peterson, K.M.Peterson, and D.K.Srivastava (1998).
Thermodynamics of ligand binding and catalysis in human liver medium-chain acyl-CoA dehydrogenase: comparative studies involving normal and 3'-dephosphorylated C8-CoAs and wild-type and Asn191 --> Ala (N191A) mutant enzymes.
  Biochemistry, 37, 12659-12671.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB code is shown on the right.