Lessons learned from UvrD helicase: mechanism for directional movement

Abstract

How do molecular motors convert chemical energy to mechanical work? Helicases and nucleic acids offer simple motor systems for extensive biochemical and biophysical analyses. Atomic resolution structures of UvrD-like helicases complexed with DNA in the presence of AMPPNP, ADP.Pi, and Pi reveal several salient points that aid our understanding of mechanochemical coupling. Each ATPase cycle causes two motor domains to rotationally close and open. At a minimum, two motor-track contact points of alternating tight and loose attachment convert domain rotations to unidirectional movement. A motor is poised for action only when fully in contact with its track and, if applicable, working against a load. The orientation of domain rotation relative to the track determines whether the movement is linear, spiral, or circular. Motors powered by ATPases likely deliver each power stroke in two parts, before and after ATP hydrolysis. Implications of these findings for analyzing hexameric helicase, F(1)F(0) ATPase, and kinesin are discussed.

Figure 1

(a) A schematic diagram of F1F0 ATP synthase using ion gradients to generate torque, which in turn powers ATP synthesis. (b) A diagram of using energy released from ATP hydrolysis to transport ions against its concentration gradients.

Figure 2

(a) Structure of a kinesin dimer (PDB: 2NCD) shown in ribbon diagrams. One subunit is shown in cyan and the other in a blue-to-red gradient from the N- to C-terminus. The bound ADPs are shown as purple sticks. (b) A ribbon diagram of the two RecA-like domains, 1A and 2A of UvrD (PDB: 2IS6). Domain 1A contains the Walker A and B motifs and is shown in blue-to-red gradient. (c) The hand-over-hand mechanism. (d) The inchworm mechanism. The same ATPase-driven domain rotation and the same two motor-track contacts may enable movement in opposite directions by switching the tight contact to loose and loose to tight with regard to the ATPase cycle. Figures 2a–b and 4 were made using PyMol (www.pymol.org).

Figure 3

Diagram of four domains (1A, 1B, 2A, 2B) in UvrD. The ds and ss DNA are roughly orthogonal to each other when bound to UvrD. The 1A, 1B and 2B domain form a rigid body when UvrD is in full contact with ds- and ss-DNA. There are four critical contact points between UvrD and DNA, labeled as 1, 2, 3 and 4. (a) The 20° domain closing induced by ATP binding, and (b) the reverse of domain opening by ADP/Pi release around the grey double-arrowed axis. The tight contacts are indicated in deep red and loose contacts are indicated in pink.

Figure 4

Different ways of forming tight and loose contact with ssDNA by homologous UvrD and PcrA. (a) In UvrD-DNA complexes, each nucleotide from +1 to +5 position counting from the ds-ss junction is color-coded and highlighted by the electron densities. The Arg (R257) (indicated by the black arrow head) stacks with DNA bases (+4 and +5) in the ATP-free state and holds ssDNA in place during ATP binding. R257 together with F62 change their rotamer conformations when UvrD is bound to an ATP analogue and allows ssDNA translocation during ADP and Pi release. (b) In PcrA-DNA complexes, the last three nucleotides corresponding to +3, +4 and +5 in UvrD are colored accordingly. The trap-hole that stops ssDNA translocation during ATP binding is shown in a black box. Interestingly, PcrA does contain the equivalent of R257 (indicated by the grey arrow).

Figure 5

Linear, spiral and circular movement. (a) Linear movement by monomeric motor in an inchworm fashion. (b) Same monomeric motor can move along a helix in the spiral form. (c) Cylindrical motors often conduct circular (rotary) movement. (d) They can also move in a spiral form, e.g. around the helical backbone of nucleic acid, which may appear to be linear translocation.