Models of bacteriophage DNA packaging motors
Introduction
Purposeful biological activities depend on ATP-driven motors in several cases. Examples include the following (among others): (1) transportation of vesicles along microtubules that occurs via either kinesin- or dynein-motor molecules, (2) contraction of muscle that occurs via sliding along actin filaments of myosin-motor molecules (reviewed in Howard, 2001; Ishijima and Yanagida, 2001), and (3) DNA packaging in a bacteriophage capsid that occurs via sliding of a DNA molecule through a ring-shaped motor complex. Advantages of experimentation favor the use of bacteriophage DNA packaging motors as models for motors in general (reviewed in Fujisawa and Morita, 1997; Hendrix, 1998; Catalano, 2000).
The advantages of experimentation with bacteriophages include a well-developed platform of genetics and biochemistry. In the past, this platform has been used to reveal a variety of unexpected bacteriophage factors necessary for either DNA packaging or other events. These factors include scaffolding proteins for capsid assembly (reviewed in Dokland, 1999), chaperonins for protein folding (reviewed in King et al., 1996; Ang et al., 2000), transcription factors (reviewed in Hughes and Mathee, 1998), and accessory proteins for DNA packaging (reviewed in Fujisawa and Morita, 1997; Catalano, 2000). Genetic analysis is also effective in probing for new functions of proteins already known (recent examples for bacteriophage DNA packaging are in Cue and Feiss, 2001; Rao and Mitchell, 2001; Duffy and Feiss, 2002).
Kinesin-, dynein-, and myosin-based motors are potentially feedback regulated. However, studies of eukaryotic motors apparently do not include any data that would probe the presence of feedback regulation. From the perspective of biology, the most reasonable assumption is that eukaryotic motors really are feedback-regulated. For example, RNA transcription and DNA replication were once thought to be the product of single enzymes, but are now known to be the product of multimolecular complexes much more complicated than originally thought. The primary reason is regulation (reviewed in Nogales, 2000; Frick and Richardson, 2001; Katayama, 2001).
Bacteriophage DNA packaging occurs when a preassembled, DNA-free capsid (procapsid) binds and, then, packages a double-stranded DNA genome. The procapsid consists of a polyhedral protein shell that usually has a scaffolding protein associated with its internal surface. The scaffolding protein assists the assembly of the outer shell and departs from the capsid during subsequent DNA packaging. A ring of 12 subunits (called either the connector or the portal) interrupts the icosahedral lattice of the outer shell of the procapsid. The connector has a central channel that is traversed by the DNA genome during DNA packaging (reviewed in Valpuesta and Carrascosa, 1994; Fujisawa and Morita, 1997; Catalano, 2000). Both the connector and additional, accessory proteins constitute the DNA packaging motor. A schematic cross section of the procapsid of bacteriophage φ29 with DNA packaging motor is in Fig. 1a; a magnified view of the DNA packaging motor is in Fig. 1b (the author’s interpretation of data cited in the legend to Fig. 1). Further details are described in a subsequent section.
The hypothesis has been proposed that a bacteriophage DNA packaging motor works via an ATP-driven spinning connector that threads the grooves of the DNA double helix (to be called the rotating thread hypothesis) (Hendrix, 1978). Macroscopically, an analogy is the action of a turning worm gear. The primary rationale for the rotating thread hypothesis is a symmetry mismatch between the connector (sixfold rotational symmetry) and the outer shell at the point of attachment of the connector (fivefold rotational symmetry). The reasoning is that the symmetry mismatch reduces potential energy barriers during rotation. This hypothesis assumes a power stroke that rotates the connector. Power stroke-based models do not always use the power stroke to rotate the connector. The power stroke can work directly on the DNA molecule (Serwer, 1989; Smith et al., 2001; Guasch et al., 2002).
An alternative previous hypothesis (Serwer, 1988) is that the DNA packaging motor is an osmotic pressure-assisted, ATP-driven device for rectifying thermal motion. A device for rectifying thermal motion is sometimes called a thermal (Brownian) ratchet or motor (reviewed in Reimann and Hanggi, 2002; Parrondo and De Cisneros, 2002; Dill and Bromberg, 2003). If so, the DNA packaging motor does not resemble a macroscopic machine. Discrimination between a miniaturized version of a macroscopic machine-like motor and a thermal motion-dependent motor is an unsolved problem in the case of eukaryotic motors (reviewed in Kinosita, 1998; Astumian and Derenyi, 1999; Brokaw, 2001; Houdusse and Sweeney, 2001), as well as prokaryotic DNA packaging motors. Recent evidence does, however, favor a thermal ratchet in the case of myosin-based motors (reviewed in Ishii et al., 2002). Wang and Oster (2002) make the point that power stroke-driven motors may not be cleanly distinguishable from thermal ratchets when the details of the power stroke are (eventually) considered.
The following terminology is used here. A molecular ratchet is a nanometer-scale device that rectifies the effects of either thermal motion (thermal ratchet) or an oscillating force. Some molecular ratchets are motors. Motor-like, thermal motion-dependent ratchets include both thermal ratchets and ratchets that rectify oscillating forces such as the forces generated by pulsed electrical fields. Some nonthermal ratchets are thermal motion-dependent (reviewed in Doering et al., 1995; Griess and Serwer, 2002; Reimann and Hanggi, 2002). The hypothesis that the DNA packaging motor is a ratchet will be called the motor-ratchet hypothesis. One possible motor-ratchet for DNA packaging is described in Fig. 2.
Recent studies of bacteriophages φ29, SPP1, and T3 confirm in detail the symmetry mismatch of the procapsid’s outer shell and the procapsid’s connector. Additional aspects of the connector structure are shown in these studies, including the association of RNA molecules in the case of φ29 (Morais et al., 2001; Simpson et al., 2000, Simpson et al., 2001; Guasch et al., 2002; T3: Valle et al., 1999; Valpuesta et al., 2000). In several studies of φ29 (Valpuesta et al., 1999; Simpson et al., 2000; Guasch et al., 2002), the assumption is made that power strokes drive DNA into the capsid. In contrast to the rotating thread hypothesis, these more recent hypotheses propose that rotation of the connector aligns an advancing DNA molecule with the site of the next power stroke. Each power stroke drives a two-nucleotide-pair length of the DNA molecule into the capsid (to be called rotating power stroke hypotheses). One rotating power stroke hypothesis proposes that the motor’s cycle involves storage of the energy in the connector (Simpson et al., 2000); another (Guasch et al., 2002) deliberately avoids the storage of energy by the connector. However, the data are not sufficient to conclude that either connector rotation or a power stroke drives DNA packaging.
On the other hand, the following question remains in the case of the motor-ratchet hypothesis: Can the motor-ratchet hypothesis explain the current data? These data include (1) genetic/biochemical analysis of the DNA packaging motor, (2) the energetics of packaging, (3) the detailed structure of the DNA packaging motor, and (4) the kinetics of packaging, as described below. The present article answers this question. The present article also compares the various models. The (unproven) assumption is made that the different double-stranded DNA bacteriophages all use the same mechanism for DNA packaging, as has been previously proposed (reviewed in Valpuesta and Carrascosa, 1994; Fujisawa and Morita, 1997; Catalano, 2000). Some results obtained here are applicable to biological motors in general.
Section snippets
Genetics and biochemistry
Early studies revealed that accessory proteins were necessary to package the genomes of all double-stranded DNA bacteriophages. Genetic removal of an accessory protein was found to block DNA packaging, but not to block either assembly of procapsids or anything else (reviewed in Casjens, 1985; Serwer, 1989). More recently, more detailed analysis of the biochemistry of mutants has been used to subdivide an accessory protein by the biochemical activity of the various parts of the protein. In the
A specific DNA packaging motor-ratchet
The term motor-ratchet defines a class of motors, not a specific motor. Among previously proposed specific motors in the motor-ratchet class, most have a cycle that does not include internal feedback. Fig. 2 illustrates the cycle of a specific motor that does include internal feedback as part of its ratchet-like cycle. In Fig. 2a, the left-hand image is an enlarged version of the motor of the right-hand image. The motor of Fig. 2 is a version of the motor of Serwer (1988), revised to include
Energetics
Nanometry of the packaging of latex sphere-bound φ29 DNA reveals that the φ29 DNA packaging motor is capable of a force as high as 40–70 pN. The data for this conclusion are obtained by stalling the motor with an external opposing force on the latex sphere (Smith et al., 2001). The terminal phase of φ29 DNA packaging is the time of the motor’s maximum load, 52 pN, when an external opposing force was not applied (Fig. 3d in Smith et al., 2001). Thus, the DNA packaging motor will not tolerate
The structure and activity of the connector
The φ29 connector at low resolution is a tapered, symmetric cylinder of 12 subunits. The wider end of the connector is held by the outer shell-scaffold of the procapsid; the narrower end projects outside of the procapsid (Simpson et al., 2000, Simpson et al., 2001; Ibarra et al., 2000; Guasch et al., 2002) (summarized in Fig. 1). Atomic force microscopy of isolated connectors reveals that the narrower end is flexible (Muller et al., 1997). X-Ray crystallography reveals two disordered
Dynamics of packaging
Nanometry reveals pauses that interrupt the entry of DNA into a φ29 capsid during the packaging of φ29 DNA in vitro (Smith et al., 2001). These pauses start at the beginning of DNA entry when resistance to DNA entry is too small to detect. As more DNA enters, the following two changes occur: The speed of entry decreases by a factor of about 10 (Fig. 1c in Smith et al., 2001) and the pause frequency increases by a factor of about 10 (Supplemental Fig. IIIa in Smith et al., 2001). So, the number
Feedback
From the perspective of physical chemistry, the rate of DNA packaging should decrease as more DNA is packaged and the resistance to packaging increases (Odijk, 1998; Kindt et al., 2001). From the perspective of biology, the opposite should be the case in vivo. The reason for the latter conclusion is that reducing the rate of DNA packaging increases the chance that the unpackaged DNA will be damaged by intracellular nucleases. Biologically, the capsid’s optimal course is to finish packaging as
Conclusions
Recently, research in the following areas has added constraints for models of DNA packaging motors: genetics/biochemistry of accessory proteins, high resolution structure of connectors, and single-molecule dynamics of DNA packaging. However, these constraints are not yet sufficient to determine the fundamentals of how this motor works. Previous hypotheses based primarily on structure propose models based on power strokes, in analogy with macroscopic motors. The analysis performed here includes
Acknowledgments
The author thanks Dwight L. Anderson for critical comments. Support was received from the National Institutes of Health (GM24365) and the Welch Foundation (AQ- 764).
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The PLB measurement for the connector in Phi29 bacteriophage reveals the function of its channel loop
2021, Biophysical JournalCitation Excerpt :It has been confirmed that the motor protein GP16 plays a central role in the entire packaging process, mediating the ATP binding and hydrolysis that provide the input energy needed to drive the motor (6–12). However, the roles that the connector GP10 plays in force generation in the DNA packaging motor are still intensely debated (13–21). The connector in bacteriophage Phi29 had been deemed as a passive conduit for the DNA translocation until Hendrix proposed a model (13) in which the connector may actively package the DNA into the prohead by its self-rotation in the prohead because of the symmetry mismatch between the fivefold symmetric prohead shell and 12-fold symmetric connector.
Discovery of a new motion mechanism of biomotors similar to the earth revolving around the sun without rotation
2013, VirologyCitation Excerpt :Recently, the bacteriophage phi29 DNA packaging motor has been described as a handicapped pentamer with one non-functional subunit and four rotating subunits (Chistol et al., 2012; Moffitt et al., 2009; Yu et al., 2010). Many other models have also been proposed for the motor action of dsDNA viruses (Guo et al., 1998; Chen and Guo, 1997; Black and Silverman, 1978; Maluf et al., 2006; Moffitt et al., 2009; Aathavan et al., 2009) (for review, see (Guo, 1994; Guo and Lee, 2007; Rao and Feiss, 2008; Zhang et al., 2012; Serwer, 2003)). However, the latest thorough investigation has unexpectedly revealed that the phi29 DNA packaging motor is a hexameric revolution motor that does not rotate (Guo et al., 1998; Shu et al., 2007; Zhang et al., 2012; Schwartz et al., 2013b; Schwartz et al., 2013a; Zhao et al., in press), a discovery disparate to previous models.
Revolution rather than rotation of AAA+ hexameric phi29 nanomotor for viral dsDNA packaging without coiling
2013, VirologyCitation Excerpt :It was suggested that viral DNA packaging motors operate by a rotation mechanism involving a five-/six-fold mismatch structure (Hendrix, 1978). Many subsequent models have been proposed describing the packaging motor of dsDNA (Khan et al., 1995; Serwer, 2003; Astumian, 1997; Guo et al., 1998; Hendrix, 1978;Grimes and Anderson, 1997; Chen and Guo, 1997; Guasch et al., 2002; Hou et al., 2010; Morita et al., 1995b; Sabanayagam et al., 2007; Oram et al., 2008; Serwer, 2010; Shu et al., 2007; Maluf et al., 2006; Yu et al., 2010; Aathavan et al., 2009). The most well-studied bacteriophage phi29 DNA packaging motor was also the first constructed with purified components (Guo et al., 1986) and has been shown to consist of three major components that interact with each other in unison (Fig. 1) (Guo et al., 1987a, 1987c; Lee and Guo, 2006; Ibarra et al., 2001).
The ATPase of the phi29 DNA packaging motor is a member of the hexameric AAA+ superfamily
2013, VirologyCitation Excerpt :The phi29 DNA packaging motor is composed of a dodecameric connector at the vertex of the procapsid, geared by a pRNA ring (Guo et al., 1987a) which encircles the N-terminus of the connector (Xiao et al., 2005, 2008; Atz et al., 2007), and a ring of gp16 which functions as an ATPase to drive the motor (Guo et al., 1987c; Ibarra et al., 2001). The connector was recently revealed to only allow for unidirectional movement of dsDNA (Jing et al., 2010), and a model using a “push through a one-way valve” mechanism was described (Schwartz et al., 2012; Fang et al., 2012) which agrees nicely with the previously proposed ratchet (Serwer, 2003) and compression (Ray et al., 2010a, 2010b) models. This mechanism describes dsDNA as being pushed through the connector channel by the ATPase gp16 while the connector functions like a valve to prevent DNA from slipping out of the capsid during the packaging process.
Stochastic mechano-chemical kinetics of molecular motors: A multidisciplinary enterprise from a physicist's perspective
2013, Physics ReportsCitation Excerpt :Most of the models envisage execution of a power stroke by the genome packaging motor. However, alternative scenarios that envisage a Brownian ratchet mechanism for these motors have also been proposed [1420–1422]. An ATP-binding cassette (ABC) transporter is a membrane-bound machine.