PDBsum entry 1in8

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DNA binding protein PDB id
Protein chain
298 a.a. *
Waters ×155
* Residue conservation analysis
PDB id:
Name: DNA binding protein
Title: Thermotoga maritima ruvb t158v
Structure: Holliday junction DNA helicase ruvb. Chain: a. Synonym: ruvb. Engineered: yes. Mutation: yes
Source: Thermotoga maritima. Organism_taxid: 2336. Gene: ruvb. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.
1.90Å     R-factor:   0.226     R-free:   0.249
Authors: C.D.Putnam,S.B.Clancy,H.Tsuruta,J.G.Wetmur,J.A.Tainer
Key ref:
C.D.Putnam et al. (2001). Structure and mechanism of the RuvB Holliday junction branch migration motor. J Mol Biol, 311, 297-310. PubMed id: 11478862 DOI: 10.1006/jmbi.2001.4852
12-May-01     Release date:   08-Aug-01    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
Q56313  (RUVB_THEMA) -  Holliday junction ATP-dependent DNA helicase RuvB
334 a.a.
298 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 helicase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: ATP + H2O = ADP + phosphate
+ H(2)O
Bound ligand (Het Group name = ADP)
corresponds exactly
+ phosphate
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     response to DNA damage stimulus   5 terms 
  Biochemical function     nucleotide binding     6 terms  


DOI no: 10.1006/jmbi.2001.4852 J Mol Biol 311:297-310 (2001)
PubMed id: 11478862  
Structure and mechanism of the RuvB Holliday junction branch migration motor.
C.D.Putnam, S.B.Clancy, H.Tsuruta, S.Gonzalez, J.G.Wetmur, J.A.Tainer.
The RuvB hexamer is the chemomechanical motor of the RuvAB complex that migrates Holliday junction branch-points in DNA recombination and the rescue of stalled DNA replication forks. The 1.6 A crystal structure of Thermotoga maritima RuvB together with five mutant structures reveal that RuvB is an ATPase-associated with diverse cellular activities (AAA+-class ATPase) with a winged-helix DNA-binding domain. The RuvB-ADP complex structure and mutagenesis suggest how AAA+-class ATPases couple nucleotide binding and hydrolysis to interdomain conformational changes and asymmetry within the RuvB hexamer implied by the crystallographic packing and small-angle X-ray scattering in solution. ATP-driven domain motion is positioned to move double-stranded DNA through the hexamer and drive conformational changes between subunits by altering the complementary hydrophilic protein- protein interfaces. Structural and biochemical analysis of five motifs in the protein suggest that ATP binding is a strained conformation recognized both by sensors and the Walker motifs and that intersubunit activation occurs by an arginine finger motif reminiscent of the GTPase-activating proteins. Taken together, these results provide insights into how RuvB functions as a motor for branch migration of Holliday junctions.
  Selected figure(s)  
Figure 2.
Figure 2. RuvB nucleotide recognition and an implied strained ATP-bound conformation. (a) Details of the nucleotide-binding site reveal that the phosphate groups are coordinated by the Walker A motif (including Lys64 and Thr65) with the ADP moiety contacted by residues of the sensor 2 motif (Pro216 and Arg217). Sensor 1 (Thr158) and Walker B (Asp109 and Glu110) motifs are located near the position of the g-phosphate group. The isosurface of the simulated annealing omit difference density is shown for ADP, contoured at 3s (green). (b) Structure-based mutational analysis reveals the importance of ATP hydrolysis in branch migration and the key roles played by sensor 1, sensor 2, and arginine finger in RuvB. Biochemical characterization of the DNA-dependent ATPase activity of RuvB mutants[21] and branch migration of an in vitro reconstituted RuvAB-Holliday junction complex.[51 and 52] Proteins scored as inactive, "-", in branch migration activity are either wholly or substantially compromised, as they showed less than 3 % of wild-type activity after an incubation of 60 minutes. (c) Overlay of the wild-type RuvB protein (blue) with structures of the sensor 1 mutations Ala156Ser (yellow), Thr158Val (light blue), and the Walker A mutation Lys64Arg (light brown). (d) Overlay of the sensor 2 mutation Pro216Gly (yellow) with wild-type RuvB, illustrating some of the structural rearrangements required to accommodate the misregistered ATP (Figure 2(c) in the nucleotide-binding site. (e) Details of ATP binding from the Pro216Gly structure (red) and ADP binding from the wild-type structure (blue) demonstrating the reorientation of the both adenine and ribose moieties and the phosphate misregistration, where the ATP g-phosphate group binds at the b position and the ATP b-phosphate group binds at the a position. This structure suggests that binding ATP in the appropriate conformation channels binding energy into a strained RuvB conformation. (f) Overlay of the arginine finger mutation Arg170Ala (yellow) with wild-type RuvB, suggesting the dramatic loss of ATPase and branch migration assay are due to loss of the guanidium functionality, as structural perturbations are small.
Figure 6.
Figure 6. Structurally implied mechanism for branch migration. Illustration of a mechanism for RuvB branch migration involving a rotation of the RuvB hexamer (green, cyan, and blue subunits) relative to the RuvA tetramer (yellow bar). Stepwise migration of the DNA is indicated by motion of the circled numbers through the center of the hexamer, although the fundamental translocation step size is unknown. The 2-fold symmetry of the loading of the nucleotide binding sites is based on pre-steady state kinetics of RuvB, which hydrolyzes two ATP molecules per hexamer.[38 and 45] The starting state (a) with two ATP and two ADP molecules is inferred from the optimal nucleotide ratio (2 ATPgS:1 ATP) for forming topologically underwound DNA, [21, 38 and 49] equivalent to step (b), and the productive arginine finger geometry observed in the AMP-PNP bound NSF-D2. [41] ATP hydrolysis in step (b) may drive rotation of the RuvB hexamer (c) by opening of the ADP-bound state along DNA as well as through interactions with RuvA. ATP serves as an allosteric effector for ADP release, [45] which may be driven by interface changes between subunits that may be released after rotation (d) or during rotation. Hydrolysis of ATP by RuvB is kinetically rapid and ADP release is slow. [45]
  The above figures are reprinted by permission from Elsevier: J Mol Biol (2001, 311, 297-310) copyright 2001.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
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PDB codes: 2oap 2oaq
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Structure-function analysis of the three domains of RuvB DNA motor protein.
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Three-dimensional structural views of branch migration and resolution in DNA homologous recombination.
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15292509 R.Amit, O.Gileadi, and J.Stavans (2004).
Direct observation of RuvAB-catalyzed branch migration of single Holliday junctions.
  Proc Natl Acad Sci U S A, 101, 11605-11610.  
15210950 T.Hishida, Y.W.Han, S.Fujimoto, H.Iwasaki, and H.Shinagawa (2004).
Direct evidence that a conserved arginine in RuvB AAA+ ATPase acts as an allosteric effector for the ATPase activity of the adjacent subunit in a hexamer.
  Proc Natl Acad Sci U S A, 101, 9573-9577.  
14747526 Y.M.Loo, and T.Melendy (2004).
Recruitment of replication protein A by the papillomavirus E1 protein and modulation by single-stranded DNA.
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Ordered ATP hydrolysis in the gamma complex clamp loader AAA+ machine.
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12774115 D.Li, R.Zhao, W.Lilyestrom, D.Gai, R.Zhang, J.A.DeCaprio, E.Fanning, A.Jochimiak, G.Szakonyi, and X.S.Chen (2003).
Structure of the replicative helicase of the oncoprotein SV40 large tumour antigen.
  Nature, 423, 512-518.
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Holliday junction binding activity of the human Rad51B protein.
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Crystal structure of the SF3 helicase from adeno-associated virus type 2.
  Structure, 11, 1025-1035.
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Reconstitution of the Mcm2-7p heterohexamer, subunit arrangement, and ATP site architecture.
  J Biol Chem, 278, 4491-4499.  
12586394 R.Giraldo (2003).
Common domains in the initiators of DNA replication in Bacteria, Archaea and Eukarya: combined structural, functional and phylogenetic perspectives.
  FEMS Microbiol Rev, 26, 533-554.  
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Uncoupling of the ATPase activity from the branch migration activity of RuvAB protein complexes containing both wild-type and ATPase-defective RuvB proteins.
  Genes Cells, 8, 721-730.  
14566326 T.Pape, H.Meka, S.Chen, G.Vicentini, M.van Heel, and S.Onesti (2003).
Hexameric ring structure of the full-length archaeal MCM protein complex.
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Nucleotide-dependent conformational changes in the sigma54-dependent activator DctD.
  J Bacteriol, 185, 6215-6219.  
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Cooperative kinetics of both Hsp104 ATPase domains and interdomain communication revealed by AAA sensor-1 mutants.
  EMBO J, 21, 12-21.  
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Analysis of the AAA sensor-2 motif in the C-terminal ATPase domain of Hsp104 with a site-specific fluorescent probe of nucleotide binding.
  Proc Natl Acad Sci U S A, 99, 2732-2737.  
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Crystal structure of ClpA, an Hsp100 chaperone and regulator of ClpAP protease.
  J Biol Chem, 277, 46743-46752.
PDB codes: 1k6k 1ksf
12434150 I.Rouiller, B.DeLaBarre, A.P.May, W.I.Weis, A.T.Brunger, R.A.Milligan, and E.M.Wilson-Kubalek (2002).
Conformational changes of the multifunction p97 AAA ATPase during its ATPase cycle.
  Nat Struct Biol, 9, 950-957.  
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Helicase structure and mechanism.
  Curr Opin Struct Biol, 12, 123-133.  
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The structure of bacterial DnaA: implications for general mechanisms underlying DNA replication initiation.
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PDB code: 1l8q
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Motors and switches: AAA+ machines within the replisome.
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ATPases as drug targets: learning from their structure.
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Structural analysis of DNA replication fork reversal by RecG.
  Cell, 107, 79-89.
PDB code: 1gm5
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.