PDBsum entry 1j7k

DNA binding protein

PDB id: 1j7k

Contents

Protein chain

299 a.a. *

Ligands

ACT ×2

ATP

HEZ

Metals

_CO

Waters ×263

* Residue conservation analysis

PDB id:

1j7k

Name:

DNA binding protein

Title:

Thermotoga maritima ruvb p216g mutant

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.

Resolution:

1.80Å R-factor: 0.212 R-free: 0.238

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

Date:

16-May-01 Release date: 08-Aug-01

Protein chain

Q56313  (RUVB_THEMA) -  Holliday junction branch migration complex subunit RuvB from Thermotoga maritima (strain ATCC 43589 / DSM 3109 / JCM 10099 / NBRC 100826 / MSB8)

Seq: 334 a.a.

Struc: 299 a.a.*

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

Enzyme reactions

• Enzyme class: E.C.3.6.4.- - ?????

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.

ABSTRACT

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.