DNA Packaging Motor Research Articles

High resolution structure of hexameric herpesvirus DNA-packaging motor elucidates revolving mechanism and ends 20-year fervent debate

High resolution structure of hexameric herpesvirus DNA-packaging motor elucidates revolving mechanism and ends 20-year fervent debate

A thermophilic phage uses a small terminase protein with a fixed helix–turn–helix geometry

Tailed bacteriophages use a DNA-packaging motor to encapsulate their genome during viral particle assembly. The small terminase (TerS) component of this DNA-packaging machinery acts as a molecular matchmaker that recognizes both the viral genome and the main motor component, the large terminase (TerL). However, how TerS binds DNA and the TerL protein remains unclear. Here we identified gp83 of the thermophilic bacteriophage P74-26 as the TerS protein. We found that TerSP76-26 oligomerizes into a nonamer that binds DNA, stimulates TerL ATPase activity, and inhibits TerL nuclease activity. A cryo-EM structure of TerSP76-26 revealed that it forms a ring with a wide central pore and radially arrayed helix-turn-helix domains. The structure further showed that these helix-turn-helix domains, which are thought to bind DNA by wrapping the double helix around the ring, are rigidly held in an orientation distinct from that seen in other TerS proteins. This rigid arrangement of the putative DNA-binding domain imposed strong constraints on how TerSP76-26 can bind DNA. Finally, the TerSP76-26 structure lacked the conserved C-terminal β-barrel domain used by other TerS proteins for binding TerL. This suggests that a well-ordered C-terminal β-barrel domain is not required for TerSP76-26 to carry out its matchmaking function. Our work highlights a thermophilic system for studying the role of small terminase proteins in viral maturation and presents the structure of TerSP76-26, revealing key differences between this thermophilic phage and its mesophilic counterparts.

Insertion of channel of phi29 DNA packaging motor into polymer membrane for high-throughput sensing

The connector channel of bacteriophage phi29 DNA packaging motor has been inserted into the lipid bilayer membrane and has shown potential for the sensing of DNA, RNA, chemicals, peptides, and antibodies. Properties such as high solubility and large channel size have made phi29 channel an advantageous system for those applications; however, previously studied lipid membranes have short lifetimes, and they are frangible and unstable under voltages higher than 200 mV. Thus, the application of this lipid membrane platform for clinical applications is challenging. Here we report the insertion of the connector into the stable polymer membrane in MinION flow cell that contains 2048 wells for high-throughput sensing by the liposome-polymer fusion process. The successful insertion of phi29 connector was confirmed by a unique gating phenomenon. Peptide translocation through the inserted phi29 connector was also observed, revealing the potential of applying phi29 connector for high-throughput peptide sensing.

Dynamic Interrogation of a Viral DNA Packaging Motor Complex

Dynamic Interrogation of a Viral DNA Packaging Motor Complex

Retained Stability of the RNA Structure in DNA Packaging Motor with a Single Mg2+ Ion Bound at the Double Mg-Clamp Structure.

It has been known that the binding of two Mg2+ ions to the phosphate groups of RNA is essential for enhancing the stability of a three-way junction oligomeric prohead RNA (3WJ-pRNA). Here, we investigate the coupling between binding of two Mg2+ ions and the stability of the complex structure by calculating the rupture force of 3WJ-pRNA using steered molecular dynamics simulations. Our simulations showed that the mechanical stability of the pRNA structure is largely retained with a single Mg2+ ion bound. Because of a strong electrostatic repulsion between the adjacent Mg2+ ions, the rupture force with two Mg2+ ions bound is smaller than the sum of the rupture forces with either one of Mg2+ ions bound. Our results suggest that binding of the second Mg2+ ion could be redundant for maintaining the rigidity of 3WJ-pRNA, which allows 3WJ-pRNA to have a flexible working range of Mg2+ concentration in a cell.

ATP/ADP modulates gp16-pRNA conformational change in the Phi29 DNA packaging motor.

Packaging of phage phi29 genome requires the ATPase gp16 and prohead RNA (pRNA). The highly conserved pRNA forms the interface between the connector complex and gp16. Understanding how pRNA interacts with gp16 under packaging conditions can shed light on the molecular mechanism of the packaging motor. Here, we present 3D models of the pRNA–gp16 complex and its conformation change in response to ATP or ADP binding. Using a combination of crystallography, small angle X-ray scattering and chemical probing, we find that the pRNA and gp16 forms a ‘Z’-shaped complex, with gp16 specifically binds to pRNA domain II. The whole complex closes in the presence of ATP, and pRNA domain II rotates open as ATP hydrolyzes, before resetting after ADP is released. Our results suggest that pRNA domain II actively participates in the packaging process.

Channel from bacterial virus T7 DNA packaging motor for the differentiation of peptides composed of a mixture of acidic and basic amino acids

Protein mutations can result in dysfunctional cell signaling pathways; therefore it is of significance to develop a robust platform for the detection of protein mutations. Here, we report that the channel of bacterial virus T7 DNA packaging motor is able to discriminate peptides containing a mixture of acidic (negatively charged) and basic (positively charged) amino acids. Peptides were differentiated based on their current signatures created by their unique charge compositions. In combination with protease digestion, peptides with the locational differences of single amino acid were also identified. The results suggest that the T7 motor channel has the potential for peptide differentiation, mutation verification, and analysis of protein sequence.

Evidence that a catalytic glutamate and an 'Arginine Toggle'act in concert to mediate ATP hydrolysis and mechanochemical coupling in a viral DNA packaging motor.

ASCE ATPases include ring-translocases such as cellular helicases and viral DNA packaging motors (terminases). These motors have conserved Walker A and B motifs that bind Mg2+-ATP and a catalytic carboxylate that activates water for hydrolysis. Here we demonstrate that Glu179 serves as the catalytic carboxylate in bacteriophage λ terminase and probe its mechanistic role. All changes of Glu179 are lethal: non-conservative changes abrogate ATP hydrolysis and DNA translocation, while the conservative E179D change attenuates ATP hydrolysis and alters single molecule translocation dynamics, consistent with a slowed chemical hydrolysis step. Molecular dynamics simulations of several homologous terminases suggest a novel mechanism, supported by experiments, wherein the conserved Walker A arginine ‘toggles’ between interacting with a glutamate residue in the ‘lid’ subdomain and the catalytic glutamate upon ATP binding; this switch helps mediate a transition from an ‘open’ state to a ‘closed’ state that tightly binds nucleotide and DNA, and also positions the catalytic glutamate next to the γ-phosphate to align the hydrolysis transition state. Concomitant reorientation of the lid subdomain may mediate mechanochemical coupling of ATP hydrolysis and DNA translocation. Given the strong conservation of these structural elements in terminase enzymes, this mechanism may be universal for viral packaging motors.

Nano-channel of viral DNA packaging motor as single pore to differentiate peptides with single amino acid difference

Detection, differentiation, mapping, and sequencing of proteins are important in proteomics for the assessment of cell development such as protein methylation or phosphorylation as well as the diagnosis of diseases including metabolic disorder, mental illness, immunological ailments, and malignant cancers. Nanopore technology has demonstrated the potential for the sequencing or sensing of DNA, RNA, chemicals, or other macromolecules. Due to the diversity of protein in shape, structure and charge and the composition versatility of 20 amino acids, the sequencing of proteins remains challenging. Herein, we report the application of the channel of bacteriophage T7 DNA packaging motor for the differentiation of an assortment of peptides of a single amino acid difference. Explicit fingerprints or signatures were obtained based on current blockage and dwell time of individual peptide. Data from the clear mapping of small proteins after protease digestion suggests the potential of using T7 motor channel for proteomics including protein sequencing.

Molecular switch-like regulation enables global subunit coordination in a viral ring ATPase

Subunits in multimeric ring-shaped motors must coordinate their activities to ensure correct and efficient performance of their mechanical tasks. Here, we study WT and arginine finger mutants of the pentameric bacteriophage φ29 DNA packaging motor. Our results reveal the molecular interactions necessary for the coordination of ADP-ATP exchange and ATP hydrolysis of the motor's biphasic mechanochemical cycle. We show that two distinct regulatory mechanisms determine this coordination. In the first mechanism, the DNA up-regulates a single subunit's catalytic activity, transforming it into a global regulator that initiates the nucleotide exchange phase and the hydrolysis phase. In the second, an arginine finger in each subunit promotes ADP-ATP exchange and ATP hydrolysis of its neighbor. Accordingly, we suggest that the subunits perform the roles described for GDP exchange factors and GTPase-activating proteins observed in small GTPases. We propose that these mechanisms are fundamental to intersubunit coordination and are likely present in other ring ATPases.

Allostery and molecular machines.

Machines undergo repeated energy (fuel)-driven cycling between various functional and structural states. Such cycling involves motions of the machine parts, which take place in a highly coordinated manner in time and space. This description applies not only to man-made machines but also to the molecular machines that mediate many of the key processes in all forms of life [1]. Examples include protein synthesis by the ribosome, DNA unwinding by helicases during replication, protein degradation by the proteasome and protein folding by chaperones. The coordinated movements of most biomolecular machines are achieved by allosteric regulation of ATP binding and hydrolysis. Consequently, a molecular level understanding of how the essential machines of life work requires invoking and further developing allosteric theory. Hence, the motivation for the Royal Society Discussion Meeting entitled ‘Allostery and molecular machines' that took place in June 2017. The meeting brought together scientists interested in allostery, in general, with others who are studying specific biomolecular machines of interest. The meeting led to many interesting discussions and resulted in this special issue of the Philosophical Transactions B . In 1965, Monod, Wyman & Changeux published a seminal paper [2] that described a so-called ‘concerted model' (referred to here and generally as the MWC model) for cooperative ligand binding by oligomeric proteins. This issue contains a paper by one of the co-authors of this landmark paper in which the MWC model is explained in brief and its application to the nicotinic acetylcholine receptor is then described [3]. The MWC model remains important but its elegance has come at the price of several restrictive assumptions. One key assumption is that cooperativity is due to a shift in equilibrium between different quaternary …

Methods for construction and characterization of simple or special multifunctional RNA nanoparticles based on the 3WJ of phi29 DNA packaging motor.

The field of RNA nanotechnology has developed rapidly over the last decade, as more elaborate RNA nanoarchitectures and therapeutic RNA nanoparticles have been constructed, and their applications have been extensively explored. Now it is time to offer different levels of RNA construction methods for both the beginners and the experienced researchers or enterprisers. The first and second parts of this article will provide instructions on basic and simple methods for the assembly and characterization of RNA nanoparticles, mainly based on the pRNA three-way junction (pRNA-3WJ) of phi29 DNA packaging motor. The third part of this article will focus on specific methods for the construction of more sophisticated multivalent RNA nanoparticles for therapeutic applications. In these parts, some simple protocols are provided to facilitate the initiation of the RNA nanoparticle construction in labs new to the field of RNA nanotechnology. This article is intended to serve as a general reference aimed at both apprentices and senior scientists for their future design, construction and characterization of RNA nanoparticles based on the pRNA-3WJ of phi29 DNA packaging motor.

Challenging a DNA Packaging Motor with a Modified Substrate

The DNA packaging motor of the bacteriophage phi29 is a powerful molecular machine that couples the free energy of ATP hydrolysis with DNA translocation in order to complete the production of new viral particles. The active part of this motor is a pentameric ring ATPase which mechanochemical cycle has been described with exquisite detail using hi-resolution optical tweezers. It was described that in each turn of the cycle, the motor packages DNA taking discrete steps of 10 base pairs (bp) each, in what is called a burst of translocation. At the same time it was shown that this 10 bp burst is composed of four 2.5 bp sub-steps, presumably reflecting the power stroke of the individual ATPases. Several models can explain what is the origin of the burst size: the helical pitch of B-form DNA is 10.5 bp/turn of the double helix, suggesting that the structure of the substrate is what determines the burst size; however, the non-integer nature of the sub-steps within the burst allows to hypothesize that is the ATPase’s conformational change what sets the burst size. Yet another possibility is that the DNA packaging motor switches the local conformation of the DNA substrate from B-form to A-form during packaging (DNA scrunching). To test the above hypotheses we challenged the phi29 DNA packaging motor with a double-stranded RNA substrate (that adopts the A-form of nucleic acids) and measured the packaging activity using optical tweezers.

Insights on Viral DNA Packaging Motor Mechanisms from the Effects of Motor Residue Changes on Single-Molecule Packaging Dynamics

Many dsDNA viruses utilize a molecular motor to translocate DNA into preassembled viral prohead shells. We use optical tweezers to measure the dynamics of packaging of single DNA molecules by the bacteriophage lambda motor. We used site-directed mutagenesis to investigate the roles of various motor protein residues in translocation function. We found that residue changes in the proposed Walker A and Walker B ATP binding motif and catalytic glutamate cause varying impairments, ranging from partial impairment to total abrogation of DNA translocation activity. Altogether, we studied 23 mutants and identified 11 with no detectable translocation and 12 having varying impairments. The results support the proposed motif assignments. Observed changes in motor velocity and pausing and slipping for the partly impaired mutants implicates residues involved in ATP binding and positioning, coupling between ATP binding and DNA gripping, and catalysis of hydrolysis.

RNA nanoparticle distribution and clearance in the eye after subconjunctival injection with and without thermosensitive hydrogels

Thermodynamically and chemically stable RNA nanoparticles derived from the three-way junction (3WJ) of the pRNA from bacteriophage phi29 DNA packaging motor were examined previously for ocular delivery. It was reported that, after subconjunctival injection, RNA nanoparticles with tri-way shape entered the corneal cells but not the retinal cells, whereas particle with four-way shape entered both corneal and retinal cells. The present study evaluated ocular delivery of RNA nanoparticles with various shapes and sizes, and assessed the effect of thermosensitive hydrogels (poly(lactic-co-glycolic acid)-b-poly(ethylene glycol)-b-poly(lactic-co-glycolic acid); PLGA-PEG-PLGA) for increasing the retention of RNA nanoparticles in the eye. Fluorescence imaging of mouse eyes and fluorescence microscopy of dissected eye tissues from the conjunctiva, cornea, retina, and sclera were performed to determine the distribution and clearance of the nanoparticles in the eyes after subconjunctival injection in vivo. RNA nanoparticles entered the cells of the conjunctiva, cornea, retina, and sclera after subconjunctival delivery. The clearance of RNA pentagon was slower than both RNA square and triangle of the same designed edge length (10nm) in the eye, and the clearance of RNA squares of the longer edge lengths (10 and 20nm) was slower than RNA square of the shorter edge length (5nm), thus indicating that the size could affect ocular pharmacokinetics of the nanoparticles. At 24h after the injection, approximately 6–10% of the fluorescence signal from the larger nanoparticles in the study (RNA square of 20nm edge length and RNA pentagon of 10nm edge length) remained in the eye, and up to 70% of the retinal cells contained the nanoparticles. The results suggest that the larger nanoparticles were “gulped” in conjunctival, corneal, retinal, and scleral cells, similar to the behavior observed in macrophages. Additionally, the combination of RNA nanoparticles with the thermosensitive polymers increased the retention of the nanoparticles in the eye.

Altering the speed of a DNA packaging motor from bacteriophage T4.

The speed at which a molecular motor operates is critically important for the survival of a virus or an organism but very little is known about the underlying mechanisms. Tailed bacteriophage T4 employs one of the fastest and most powerful packaging motors, a pentamer of gp17 that translocates DNA at a rate of up to ∼2000-bp/s. We hypothesize, guided by structural and genetic analyses, that a unique hydrophobic environment in the catalytic space of gp17-adenosine triphosphatase (ATPase) determines the rate at which the ‘lytic water’ molecule is activated and OH− nucleophile is generated, in turn determining the speed of the motor. We tested this hypothesis by identifying two hydrophobic amino acids, M195 and F259, in the catalytic space of gp17-ATPase that are in a position to modulate motor speed. Combinatorial mutagenesis demonstrated that hydrophobic substitutions were tolerated but polar or charged substitutions resulted in null or cold-sensitive/small-plaque phenotypes. Quantitative biochemical and single-molecule analyses showed that the mutant motors exhibited 1.8- to 2.5-fold lower rate of ATP hydrolysis, 2.5- to 4.5-fold lower DNA packaging velocity, and required an activator protein, gp16 for rapid firing of ATPases. These studies uncover a speed control mechanism that might allow selection of motors with optimal performance for organisms’ survival.

Mg2+‐Dependent High Mechanical Anisotropy of Three‐Way‐Junction pRNA as Revealed by Single‐Molecule Force Spectroscopy

AbstractMechanical anisotropy is ubiquitous in biological tissues but is hard to reproduce in synthetic biomaterials. Developing molecular building blocks with anisotropic mechanical response is the key towards engineering anisotropic biomaterials. The three‐way‐junction (3WJ) pRNA, derived from ϕ29 DNA packaging motor, shows strong mechanical anisotropy upon Mg2+ binding. In the absence of Mg2+, 3WJ‐pRNA is mechanically weak without noticeable mechanical anisotropy. In the presence of Mg2+, the unfolding forces can differ by more than 4‐fold along different pulling directions, ranging from about 47 pN to about 219 pN. Mechanical anisotropy of 3WJ‐pRNA stems from pulling direction dependent cooperativity for the rupture of two Mg2+ binding sites, which is a novel mechanism for the mechanical anisotropy of biomacromolecules. It is anticipated that 3WJ‐pRNA can be used as a key element for the construction of biomaterials with controllable mechanical anisotropy.

Mg2+ -Dependent High Mechanical Anisotropy of Three-Way-Junction pRNA as Revealed by Single-Molecule Force Spectroscopy.

Mechanical anisotropy is ubiquitous in biological tissues but is hard to reproduce in synthetic biomaterials. Developing molecular building blocks with anisotropic mechanical response is the key towards engineering anisotropic biomaterials. The three-way-junction (3WJ) pRNA, derived from ϕ29 DNA packaging motor, shows strong mechanical anisotropy upon Mg2+ binding. In the absence of Mg2+ , 3WJ-pRNA is mechanically weak without noticeable mechanical anisotropy. In the presence of Mg2+ , the unfolding forces can differ by more than 4-fold along different pulling directions, ranging from about 47 pN to about 219 pN. Mechanical anisotropy of 3WJ-pRNA stems from pulling direction dependent cooperativity for the rupture of two Mg2+ binding sites, which is a novel mechanism for the mechanical anisotropy of biomacromolecules. It is anticipated that 3WJ-pRNA can be used as a key element for the construction of biomaterials with controllable mechanical anisotropy.

Dual Tumor-Targeting Nanocarrier System for siRNA Delivery Based on pRNA and Modified Chitosan.

Highly specific and efficient delivery of siRNA is still unsatisfactory. Herein, a dual tumor-targeting siRNA delivery system combining pRNA dimers with chitosan nanoparticles (CNPPs) was designed to improve the specificity and efficiency of siRNA delivery. In this dual delivery system, folate-conjugated and PEGylated chitosan nanoparticles encapsulating pRNA dimers were used as the first class of delivery system and would selectively deliver intact pRNA dimers near or into target cells. pRNA dimers simultaneously carrying siRNA and targeting aptamer, the second class of delivery system, would specifically deliver siRNA into the target cells via aptamer-mediated endocytosis or proper particle size. To certify the delivering efficiency of this dual system, CNPPs, pRNA dimers alone, chitosan nanoparticles containing siRNA with folate conjugation and PEGylation (CNPS), and chitosan nanoparticles containing pRNA dimers alone (CN) were first prepared. Then, we observed that treatment with CNPPs resulted in increased cellular uptake, higher cell apoptosis, stronger cell cytotoxicity, and more efficacious gene silencing compared to the other three formulations. Higher accumulation of siRNA in the tumor site, stronger tumor inhibition, and longer circulating time were also observed with CNPPs compared to other formulations. In conclusion, this dual nanocarrier system showed high targeting and favorable therapeutic efficacy both in vitro and in vivo. Thereby, a new approach is provided in this study for specific and efficient delivery of siRNA, which lays a foundation for the development of pRNA hexamers, which can simultaneously carry six different substances.

Fingerprinting and Differentiation of Small Proteins with a Large Channel of Bacteriophage PHI29 DNA Packaging Motor

Fingerprinting and Differentiation of Small Proteins with a Large Channel of Bacteriophage PHI29 DNA Packaging Motor