PDBsum entry 1in8

DNA binding protein

PDB id

1in8

Contents

			Protein chain

					298 a.a. *

			Ligands

					ADP

			Waters ×155

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Structure and mechanism of the ruvb holliday junction branch migration motor.

Authors

C.D.Putnam, S.B.Clancy, H.Tsuruta, S.Gonzalez, J.G.Wetmur, J.A.Tainer.

Ref.

J Mol Biol, 2001, 311, 297-310. [DOI no: 10.1006/jmbi.2001.4852]

PubMed id

11478862

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.



	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]

PROCHECK

	Headers