Mechanical operation and intersubunit coordination of ring-shaped molecular motors: insights from single-molecule studies

Abstract

Ring NTPases represent a large and diverse group of proteins that couple their nucleotide hydrolysis activity to a mechanical task involving force generation and some type of transport process in the cell. Because of their shape, these enzymes often operate as gates that separate distinct cellular compartments to control and regulate the passage of chemical species across them. In this manner, ions and small molecules are moved across membranes, biopolymer substrates are segregated between cells or moved into confined spaces, double-stranded nucleic acids are separated into single strands to provide access to the genetic information, and polypeptides are unfolded and processed for recycling. Here we review the recent advances in the characterization of these motors using single-molecule manipulation and detection approaches. We describe the various mechanisms by which ring motors convert chemical energy to mechanical force or torque and coordinate the activities of individual subunits that constitute the ring. We also examine how single-molecule studies have contributed to a better understanding of the structural elements involved in motor-substrate interaction, mechanochemical coupling, and intersubunit coordination. Finally, we discuss how these molecular motors tailor their operation-often through regulation by other cofactors-to suit their unique biological functions.

Figure 1

The architecture of ring-shaped motors from the ASCE superfamily of NTPases. (A) A crystal structure gallery of representative ASCE ring motors. The bovine papillomavirus replicative helicase E1 with a ssDNA substrate (PDB:2GXA) (7); The E. coli transcription termination factor Rho helicase with ssRNA (PDB: 3ICE) (8); The Bacillus stearothermophilus replicative helicase DnaB with ssDNA (PDB: 4ESV) (9); The motor domain of the Pseudomonas aeruginosa dsDNA translocase FtsK (PDB: 2IUU) (94); The E. coli protein unfoldase and polypeptide translocase ClpX (PDB: 3HWS) (110); The bovine mitochondrial F₁-ATPase with the three catalytic sites encircled (α-subunits shown in green, β in red, and γ in blue) (PDB: 1BMF) (6). In all structures, nucleotides and their analogs are bound at the interface between adjacent subunits (black). Note that with the exception of the F₁-ATPase, each ring consists of identical subunits that are colored differently for clarity. (B) Diagram of the core ASCE fold (modified from Lyubimov et al. (28)). WA, Walker A motif; WB, Walker B motif; CE, catalytic glutamate; RF, arginine finger. Note that the positions of CE and RF may vary in different motors and only the most common locations are shown.

Figure 2

Biological systems that contain ring-shaped molecular motors. (A) Packaging motor translocates the viral genome into a preformed capsid. (B) Eukaryotic replicative helicase unwinds duplex DNA at the replication fork. Note that the bacterial helicase DnaB has a 5′→3′ polarity and translocates along the lagging-strand template. (C) SpoIIIE translocates dsDNA at the bacterial division septum during sporulation. (D) F₀F₁-ATP synthase generates ATP by using the proton gradient across the membrane. (E) ClpXP unfolds and degrades proteins.

Figure 3

Models of mechanochemical coupling and intersubunit coordination for three ASCE ring ATPases. (A) F₁-ATPase model adapted from Adachi et al. (78). (Solid line) The γ-subunit rotation angle versus time. (Circles) The three catalytic sites. (Asterisk) Sites accessible to nucleotides from solution. (Highlighted arrow) The γ-subunit orientation. (B) φ29 gp16 model adapted from Chistol et al. (58). (Solid line) Motor position on DNA versus time. (Circles) The five catalytic sites. (Gray-shaded area) DNA. The special subunit (thick outline) contacts a pair of DNA backbone phosphates (red circles) during the dwell phase. (C) ClpX model adapted from Sen et al. (53) and Stinson et al. (112). (Solid line) Motor position on the unfolded polypeptide versus time. (Circles) The six catalytic sites: (solid outline) two high-affinity catalytic sites that bind nucleotides at any [ATP]; (dashed outline) two low-affinity catalytic sites that bind nucleotides only at high [ATP]; (crossed circles) the two ATPase sites that remain empty due to ring distortion.

Figure 4

The φ29 packaging motor is capable of disrupting the biotin-streptavidin linkage. (A) The packaging complex is assembled in a dual-trap optical tweezers instrument. Streptavidin is bound to a biotin on DNA. (B) Trajectories of individual motors exhibit clear pauses at the location of the roadblock (black arrows). For clarity, some traces are offset vertically. In ∼50% of the cases, the motor successfully overcomes the roadblock (blue). In the remaining cases, the motor slips (red). Because the inner diameter of packaging motor cannot accommodate both dsDNA and streptavidin, these data suggest that the motor can disrupt the biotin-streptavidin linkage.