Experimental Test of Connector Rotation during DNA Packaging into Bacteriophage ϕ29 Capsids

The bacteriophage ϕ29 generates large forces to compact its double-stranded DNA genome into a protein capsid by means of a portal motor complex. Several mechanical models for the generation of these high forces by the motor complex predict coupling of DNA translocation to rotation of the head-tail connector dodecamer. Putative connector rotation is investigated here by combining the methods of single-molecule force spectroscopy with polarization-sensitive single-molecule fluorescence. In our experiment, we observe motor function in several packaging complexes in parallel using video microscopy of bead position in a magnetic trap. At the same time, we follow the orientation of single fluorophores attached to the portal motor connector. From our data, we can exclude connector rotation with greater than 99% probability and therefore answer a long-standing mechanistic question.     

Experimental test of connector rotation during DNA packaging into bacteriophage phi29 capsids

The bacteriophage phi29 generates large forces to compact its double-stranded DNA genome into a protein capsid by means of a portal motor complex. Several mechanical models for the generation of these high forces by the motor complex predict coupling of DNA translocation to rotation of the head-tail connector dodecamer. Putative connector rotation is investigated here by combining the methods of single-molecule force spectroscopy with polarization-sensitive single-molecule fluorescence. In our experiment, we observe motor function in several packaging complexes in parallel using video microscopy of bead position in a magnetic trap. At the same time, we follow the orientation of single fluorophores attached to the portal motor connector. From our data, we can exclude connector rotation with greater than 99% probability and therefore answer a long-standing mechanistic question.     

Binding of pRNA to the N-terminal 14 amino acids of connector protein of bacteriophage phi29

During assembly, bacterial virus phi29 utilizes a motor to insert genomic DNA into a preformed protein shell called the procapsid. The motor contains one twelve-subunit connector with a 3.6 nm central channel for DNA transportation, six viral-encoded RNA (packaging RNA or pRNA) and a protein, gp16, with unknown stoichiometry. Recent DNA-packaging models proposed that the 5-fold procapsid vertexes and 12-fold connector (or the hexameric pRNA ring) represented a symmetry mismatch enabling production of a force to drive a rotation motor to translocate and compress DNA. There was a discrepancy regarding the location of the foothold for the pRNA. One model [C. Chen and P. Guo (1997) J. Virol., 71, 3864–3871] suggested that the foothold for pRNA was the connector and that the pRNA–connector complex was part of the rotor. However, one other model suggested that the foothold for pRNA was the 5-fold vertex of the capsid protein and that pRNA was the stator. To elucidate the mechanism of phi29 DNA packaging, it is critical to confirm whether pRNA binds to the 5-fold vertex of the capsid protein or to the 12-fold symmetrical connector. Here, we used both purified connector and purified procapsid for binding studies with in vitro transcribed pRNA. Specific binding of pRNA to the connector in the procapsid was found by photoaffinity crosslinking. Removal of the N-terminal 14 amino acids of the gp10 protein by proteolytic cleavage resulted in undetectable binding of pRNA to either the connector or the procapsid, as investigated by agarose gel electrophoresis, SDS–PAGE, sucrose gradient sedimentation and N-terminal peptide sequencing. It is therefore concluded that pRNA bound to the 12-fold symmetrical connector to form a pRNA–connector complex and that the foothold for pRNA is the connector but not the capsid protein.     

Crystal structure of 3WJ core revealing divalent ion-promoted thermostability and assembly of the Phi29 hexameric motor pRNA

The bacteriophage phi29 DNA packaging motor, one of the strongest biological motors characterized to date, is geared by a packaging RNA (pRNA) ring. When assembled from three RNA fragments, its three-way junction (3WJ) motif is highly thermostable, is resistant to 8 M urea, and remains associated at extremely low concentrations in vitro and in vivo. To elucidate the structural basis for its unusual stability, we solved the crystal structure of this pRNA 3WJ motif at 3.05 Å. The structure revealed two divalent metal ions that coordinate 4 nt of the RNA fragments. Single-molecule fluorescence resonance energy transfer (smFRET) analysis confirmed a structural change of 3WJ upon addition of Mg²⁺. The reported pRNA 3WJ conformation is different from a previously published construct that lacks the metal coordination sites. The phi29 DNA packaging motor contains a dodecameric connector at the vertex of the procapsid, with a central pore for DNA translocation. This portal connector serves as the foothold for pRNA binding to procapsid. Subsequent modeling of a connector/pRNA complex suggests that the pRNA of the phi29 DNA packaging motor exists as a hexameric complex serving as a sheath over the connector. The model of hexameric pRNA on the connector agrees with AFM images of the phi29 pRNA hexamer acquired in air and matches all distance parameters obtained from cross-linking, complementary modification, and chemical modification interference.     

Wi-Fi Live-Streaming Centrifuge Force Microscope for Benchtop Single-Molecule Experiments

The ability to apply controlled forces to individual molecules has been revolutionary in shaping our understanding of biophysics in areas as diverse as dynamic bond strength, biological motor operation, and DNA replication. However, the methodology to perform single-molecule experiments remains relatively inaccessible because of cost and complexity. In 2010, we introduced the centrifuge force microscope (CFM) as a platform for accessible and high-throughput single-molecule experimentation. The CFM consists of a rotating microscope with which prescribed centrifugal forces can be applied to microsphere-tethered biomolecules. In this work, we develop and demonstrate a next-generation Wi-Fi CFM that offers unprecedented ease of use and flexibility in design. The modular CFM unit fits within a standard benchtop centrifuge and connects by Wi-Fi to an external computer for live control and streaming at near gigabit speeds. The use of commercial wireless hardware allows for flexibility in programming and provides a streamlined upgrade path as Wi-Fi technology advances. To facilitate ease of use, detailed build and setup instructions, as well as LabVIEW-based control software and MATLAB-based analysis software, are provided. We demonstrate the instrument’s performance by analysis of force-dependent dissociation of short DNA duplexes of 7, 8, and 9 bp. We showcase the sensitivity of the approach by resolving distinct dissociation kinetic rates for a 7 bp duplex in which one G-C basepair is mutated to an A-T basepair.     

Large Terminase Conformational Change Induced by Connector Binding in Bacteriophage T7

During bacteriophage morphogenesis DNA is translocated into a preformed prohead by the complex formed by the portal protein, or connector, plus the terminase, which are located at an especial prohead vertex. The terminase is a powerful motor that converts ATP hydrolysis into mechanical movement of the DNA. Here, we have determined the structure of the T7 large terminase by electron microscopy. The five terminase subunits assemble in a toroid that encloses a channel wide enough to accommodate dsDNA. The structure of the complete connector-terminase complex is also reported, revealing the coupling between the terminase and the connector forming a continuous channel. The structure of the terminase assembled into the complex showed a different conformation when compared with the isolated terminase pentamer. To understand in molecular terms the terminase morphological change, we generated the terminase atomic model based on the crystallographic structure of its phage T4 counterpart. The docking of the threaded model in both terminase conformations showed that the transition between the two states can be achieved by rigid body subunit rotation in the pentameric assembly. The existence of two terminase conformations and its possible relation to the sequential DNA translocation may shed light into the molecular bases of the packaging mechanism of bacteriophage T7.     

Condensin extrudes DNA loops in steps up to hundreds of base pairs that are generated by ATP binding events

The condensin SMC protein complex organizes chromosomal structure by extruding loops of DNA. Its ATP-dependent motor mechanism remains unclear but likely involves steps associated with large conformational changes within the ∼50 nm protein complex. Here, using high-resolution magnetic tweezers, we resolve single steps in the loop extrusion process by individual yeast condensins. The measured median step sizes range between 20–40 nm at forces of 1.0–0.2 pN, respectively, comparable with the holocomplex size. These large steps show that, strikingly, condensin typically reels in DNA in very sizeable amounts with ∼200 bp on average per single extrusion step at low force, and occasionally even much larger, exceeding 500 bp per step. Using Molecular Dynamics simulations, we demonstrate that this is due to the structural flexibility of the DNA polymer at these low forces. Using ATP-binding-impaired and ATP-hydrolysis-deficient mutants, we find that ATP binding is the primary step-generating stage underlying DNA loop extrusion. We discuss our findings in terms of a scrunching model where a stepwise DNA loop extrusion is generated by an ATP-binding-induced engagement of the hinge and the globular domain of the SMC complex.     

Elastic Properties and Heterogeneous Stiffness of the Phi29 Motor Connector Channel

The DNA packaging motor of the bacteriophage ϕ29, comprising head-tail connector, ATPase, and pRNA, transports the viral DNA inside the procapsid against pressure differences of up to ∼60 atm during replication. Several models for the DNA packaging mechanism have been proposed, which attribute different roles to the connector, and require specific mechanical properties of the connector. To characterize these properties at the atomic level, and to understand how the connector withstands this large pressure, we have carried out molecular dynamics simulations of the whole connector both in equilibrium and under mechanical stress. The simulations revealed a quite heterogeneous distribution of stiff and soft regions, resembling that of typical composite materials that are also optimized to resist mechanical stress. In particular, the conserved middle α-helical region is found to be remarkably stiff, similar only to structural proteins forming viral shell, silk, or collagen. In contrast, large parts of the peripheral interface to the ϕ29 procapsid turned out to be rather soft. Force probe and umbrella sampling simulations showed that large connector deformations are remarkably reversible, and served to calculate the free energies required for these deformations. In particular, for an untwisting deformation by 12°, as postulated by the untwist-twist model, more than four times’ larger energy is required than is available from hydrolysis of one ATP molecule. Combined with previous experiments, this result is incompatible with the untwist-twist model. In contrast, our simulations support the recently proposed one-way revolution model and suggest in structural terms how the connector blocks DNA leakage. In particular, conserved loops at the rim of the central channel, which are in direct contact with the DNA, are found to be rather flexible and tightly anchored to the rigid central region. These findings suggest a check-valve mechanism, with the flexible loops obstructing the channel by interacting with the viral DNA.     

Structure of a viral DNA gatekeeper at 10 A resolution by cryo-electron microscopy

In tailed bacteriophages and herpes viruses, the viral DNA is packaged through the portal protein channel. Channel closure is essential to prevent DNA release after packaging. Here we present the connector structure from bacteriophage SPP1 using cryo-electron microscopy and single particle analysis. The multiprotein complex comprises the portal protein gp6 and the head completion proteins gp15 and gp16. Although we show that gp6 in the connector has a fold similar to that of the isolated portal protein, we observe conformational changes in the region of gp6 exposed to the DNA-packaging ATPase and to gp15. This reorganization does not cause closure of the channel. The connector channel traverses the full height of gp6 and gp15, but it is closed by gp16 at the bottom of the complex. Gp16 acts as a valve whose closure prevents DNA leakage, while its opening is required for DNA release upon interaction of the virus with its host.     

A Docking Model Based on Mass Spectrometric and Biochemical Data Describes Phage Packaging Motor Incorporation

The molecular mechanism of scaffolding protein-mediated incorporation of one and only one DNA packaging motor/connector dodecamer at a unique vertex during lambdoid phage assembly has remained elusive because of the lack of structural information on how the connector and scaffolding proteins interact. We assembled and characterized a φ29 connector-scaffolding complex, which can be incorporated into procapsids during in vitro assembly. Native mass spectrometry revealed that the connector binds at most 12 scaffolding molecules, likely organized as six dimers. A data-driven docking model, using input from chemical cross-linking and mutagenesis data, suggested an interaction between the scaffolding protein and the exterior of the wide domain of the connector dodecamer. The connector binding region of the scaffolding protein lies upstream of the capsid binding region located at the C terminus. This arrangement allows the C terminus of scaffolding protein within the complex to both recruit capsid subunits and mediate the incorporation of the single connector vertex.The DNA packaging motor of double-stranded DNA bacteriophages translocates genomic DNA into a preformed procapsid to near crystalline density and is the strongest motor characterized to date. The packaging motor of the Bacillus subtilis phage φ29 can work against 57 piconewtons of internal force and translocate 2 bp of DNA per ATP hydrolyzed at a maximum velocity of 103 bp/s (1, 2). The motor complex is assembled on a dodecamer of the connector protein, which replaces a pentameric vertex in the procapsid and serves both as a portal for DNA passage and the docking site for the other packaging components (3).To successfully package a full-length genome, incorporation of one and only one connector vertex is essential (4). In vivo, nearly every assembled procapsid has one and only one connector vertex and is able to package DNA and mature into an infectious phage (5). This narrow distribution in which 95% of particles have a single connector vertex cannot be explained by random statistical incorporation. The control mechanism is coupled to the procapsid assembly process. Procapsid assembly requires the copolymerization of hundreds of copies each of the capsid and scaffolding proteins as well as a dodecamer of the portal or connector protein. The scaffolding protein acts to both activate the coat protein for assembly and ensure proper form determination. In the absence of scaffolding protein, uncontrolled polymerization results in the assembly of aberrant structures. In a properly assembled procapsid, the portal protein is located at one vertex, whereas scaffolding protein occupies the bulk of the interior space and is subsequently removed during DNA packaging by either proteolysis or simple release. Mutational studies have indicated that scaffolding protein is involved either directly or indirectly in the incorporation of the connector vertex during procapsid assembly in a variety of phages (6–8).In φ29, the connector vertex is specifically incorporated at one of the two 5-fold vertices lying on the long axis of a prolate procapsid composed of 235 copies of capsid protein and containing ∼180 copies of scaffolding protein (9, 10). The structure of the 33-kDa connector protein subunit consists of three long central α-helices bridging wide and narrow domains that are rich in β-sheets and extended polypeptides (Fig. 1A) (10–12). The 12 subunits are arranged to form a 75-Å-long tapered grommet-shaped structure with an external diameter of 69 Å at the wide end and 33 Å at the narrow end. By fitting the crystal structure of the connector dodecamer into the cryo-EM¹ density of the procapsid, the orientation of connector at the unique vertex of the procapsid was revealed. The wide domain of connector protein lies inside the procapsid, and the narrow domain is exposed to the exterior and makes contacts with the other parts of the motor complex (11). The 11-kDa scaffolding protein subunits form nanomolar affinity homodimers resembling arrows in solution. Each subunit contributes one side of the arrowhead and one-half of the long coiled coil shaft (Fig. 1B) (13). The subunit structure consists of three helical segments. A three-turn N-terminal helix (α1) followed by a five-residue loop, and an antiparallel five-turn helix (α2) makes up the arrowhead and part of the proximal part of the shaft. A three-residue loop and a seven-turn helix (α3) complete the shaft. The C-terminal 15 residues, which interact with capsid protein as determined in the in vitro assembly assay, are disordered in the crystal structure (14).Open in a separate windowFig. 1.The x-ray crystal structures of connector protein (Protein Data Bank code 1FOU, chains A and B) (A) and scaffolding protein (Protein Data Bank code 1NO4, chains A and B) (B).We have recently reported the development of an in vitro assembly system for phage φ29 in which purified connector protein complex can be successfully incorporated (15). The addition of connector protein dodecamers to coat and scaffolding subunits accelerated the rate of assembly and lowered the critical concentration, suggesting involvement in nucleation of assembly (15). Here we used native mass spectrometry, chemical cross-linking, and mutational analysis to characterize the interactions between the connector and the scaffolding proteins and develop a model of the scaffolding-connector complex, which provides a molecular model of how scaffolding protein might mediate stringent incorporation of one and only one connector dodecamer.     

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e. optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (>120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.     

Helical inchworming: a novel translocation mechanism for a ring ATPase

Ring ATPases perform a variety of tasks in the cell. Their function involves complex communication and coordination among the often identical subunits. Translocases in this group are of particular interest as they involve both chemical and mechanical actions in their operation. We study the DNA packaging motor of bacteriophage φ29, and using single-molecule optical tweezers and single-particle cryo-electron microscopy, have discovered a novel translocation mechanism for a molecular motor.     

The PLB measurement for the connector in Phi29 bacteriophage reveals the function of its channel loop

The connector protein, also known as the portal protein, located at the portal vertex in the Phi29 bacteriophage has been found to play a key role in the genome DNA packaging motor. There is a disordered region, composed of 12 sets of 18-residue loops N229–N246, that has been assumed to serve as a “clamp” to retain the DNA within the pressurized capsid when DNA is fully packaged. However, the process remains undefined about how the clamping of DNA occurs and what signal is used to engage the channel loops to clamp the DNA near the end of DNA packaging. In this study, we use the planar lipid bilayer (PLB) membrane technique to study the connector with its loops cleaved. The channel properties are compared with those of the connector with corresponding wild-type loops at different membrane potentials. On the basis of the hypothesis of the Donnan effects in the flashing Brownian ratchet model, we associate the PLB experimental results with the outcomes from the relevant biochemical experiments on the proheads containing the connectors without the loops, which enables us to provide a clear picture about how the DNA clamping occurs. A mathematical relationship between the Donnan potential and the DNA packaging density is established, demonstrating that they are both in essence the same signal that is received and transmitted by the connector to dictate DNA clamping and the termination of DNA packaging. At the end of the study, the PLB technique is proposed as a viral research tool, and its potential use to study the functions of specific domains in a portal protein of the tailed bacteriophages is highlighted.     

A Minimal Optical Trapping and Imaging Microscopy System

We report the construction and testing of a simple and versatile optical trapping apparatus, suitable for visualizing individual microtubules (∼25 nm in diameter) and performing single-molecule studies, using a minimal set of components. This design is based on a conventional, inverted microscope, operating under plain bright field illumination. A single laser beam enables standard optical trapping and the measurement of molecular displacements and forces, whereas digital image processing affords real-time sample visualization with reduced noise and enhanced contrast. We have tested our trapping and imaging instrument by measuring the persistence length of individual double-stranded DNA molecules, and by following the stepping of single kinesin motor proteins along clearly imaged microtubules. The approach presented here provides a straightforward alternative for studies of biomaterials and individual biomolecules.     

The yeast dynein Dyn2-Pac11 complex is a dynein dimerization/processivity factor: structural and single-molecule characterization

Cytoplasmic dynein is the major microtubule minus end–directed motor. Although studies have probed the mechanism of the C-terminal motor domain, if and how dynein''s N-terminal tail and the accessory chains it binds regulate motor activity remain to be determined. Here, we investigate the structure and function of the Saccharomyces cerevisiae dynein light (Dyn2) and intermediate (Pac11) chains in dynein heavy chain (Dyn1) movement. We present the crystal structure of a Dyn2-Pac11 complex, showing Dyn2-mediated Pac11 dimerization. To determine the molecular effects of Dyn2 and Pac11 on Dyn1 function, we generated dyn2Δ and dyn2Δpac11Δ strains and analyzed Dyn1 single-molecule motor activity. We find that the Dyn2-Pac11 complex promotes Dyn1 homodimerization and potentiates processivity. The absence of Dyn2 and Pac11 yields motors with decreased velocity, dramatically reduced processivity, increased monomerization, aggregation, and immobility as determined by single-molecule measurements. Deleting dyn2 significantly reduces Pac11-Dyn1 complex formation, yielding Dyn1 motors with activity similar to Dyn1 from the dyn2Δpac11Δ strain. Of interest, motor phenotypes resulting from Dyn2-Pac11 complex depletion bear similarity to a point mutation in the mammalian dynein N-terminal tail (Loa), highlighting this region as a conserved, regulatory motor element.     

Continuous Allosteric Regulation of a Viral Packaging Motor by a Sensor that Detects the Density and Conformation of Packaged DNA

We report evidence for an unconventional type of allosteric regulation of a biomotor. We show that the genome-packaging motor of phage ϕ29 is regulated by a sensor that detects the density and conformation of the DNA packaged inside the viral capsid, and slows the motor by a mechanism distinct from the effect of a direct load force on the motor. Specifically, we show that motor-ATP interactions are regulated by a signal that is propagated allosterically from inside the viral shell to the motor mounted on the outside. This signal continuously regulates the motor speed and pausing in response to changes in either density or conformation of the packaged DNA, and slows the motor before the buildup of large forces resisting DNA confinement. Analysis of motor slipping reveals that the force resisting packaging remains low (<1 pN) until ∼70% and then rises sharply to ∼23 pN at high filling, which is a several-fold lower value than was previously estimated under the assumption that force alone slows the motor. These findings are consistent with recent studies of the stepping kinetics of the motor. The allosteric regulatory mechanism we report allows double-stranded DNA viruses to achieve rapid, high-density packing of their genomes by limiting the buildup of nonequilibrium load forces on the motor.     

Mechanism of force generation of a viral DNA packaging motor

A large family of multimeric ATPases are involved in such diverse tasks as cell division, chromosome segregation, DNA recombination, strand separation, conjugation, and viral genome packaging. One such system is the Bacillus subtilis phage phi 29 DNA packaging motor, which generates large forces to compact its genome into a small protein capsid. Here we use optical tweezers to study, at the single-molecule level, the mechanism of force generation in this motor. We determine the kinetic parameters of the packaging motor and their dependence on external load to show that DNA translocation does not occur during ATP binding but is likely triggered by phosphate release. We also show that the motor subunits act in a coordinated, successive fashion with high processivity. Finally, we propose a minimal mechanochemical cycle of this DNA-translocating ATPase that rationalizes all of our findings.     

DnaB helicase dynamics in bacterial DNA replication resolved by single-molecule studies

In Escherichia coli, the DnaB helicase forms the basis for the assembly of the DNA replication complex. The stability of DnaB at the replication fork is likely important for successful replication initiation and progression. Single-molecule experiments have significantly changed the classical model of highly stable replication machines by showing that components exchange with free molecules from the environment. However, due to technical limitations, accurate assessments of DnaB stability in the context of replication are lacking. Using in vitro fluorescence single-molecule imaging, we visualise DnaB loaded on forked DNA templates. That these helicases are highly stable at replication forks, indicated by their observed dwell time of ∼30 min. Addition of the remaining replication factors results in a single DnaB helicase integrated as part of an active replisome. In contrast to the dynamic behaviour of other replisome components, DnaB is maintained within the replisome for the entirety of the replication process. Interestingly, we observe a transient interaction of additional helicases with the replication fork. This interaction is dependent on the τ subunit of the clamp-loader complex. Collectively, our single-molecule observations solidify the role of the DnaB helicase as the stable anchor of the replisome, but also reveal its capacity for dynamic interactions.     

The Interaction of DNA with Bacteriophage φ29 Connector: A Study by AFM and TEM

The connector of bacteriophage φ29 is involved in DNA packaging during viral morphogenesis and we have studied itsin vitrobinding to DNA using either linear or circular DNA. The protein–DNA complexes have been analyzed by transmission electron microscopy (TEM) and by atomic force microscopy (AFM) of samples directly deposited on mica. TEM showed the presence of a specific binding due to the interaction of the protein with the free ends of the DNA. The study of these samples by AFM showed two major types of morphologies: The interaction of the connector with circular DNA revealed that the strands of DNA that enter and exit the protein complex form an angle with a mean value of 132°. Nevertheless, when the connector was incubated with linear DNA (and later circularized), there was an additional bend angle of about 168°. Further morphological analysis of the latter samples by AFM revealed a structure of the protein–DNA complex consistent with the DNA traversing the connector, probably through the inner channel. On the other hand, images from the samples obtained by incubation of the connector with circular DNA were consistent with an interaction of the DNA with the outer side of the connector.