Homomeric Ring Research Articles

Structural basis of DNA packaging by a ring-type ATPase from an archetypal viral system.

Many essential cellular processes rely on substrate rotation or translocation by a multi-subunit, ring-type NTPase. A large number of double-stranded DNA viruses, including tailed bacteriophages and herpes viruses, use a homomeric ring ATPase to processively translocate viral genomic DNA into procapsids during assembly. Our current understanding of viral DNA packaging comes from three archetypal bacteriophage systems: cos, pac and phi29. Detailed mechanistic understanding exists for pac and phi29, but not for cos. Here, we reconstituted in vitro a cos packaging system based on bacteriophage HK97 and provided a detailed biochemical and structural description. We used a photobleaching-based, single-molecule assay to determine the stoichiometry of the DNA-translocating ATPase large terminase. Crystal structures of the large terminase and DNA-recruiting small terminase, a first for a biochemically defined cos system, reveal mechanistic similarities between cos and pac systems. At the same time, mutational and biochemical analyses indicate a new regulatory mechanism for ATPase multimerization and coordination in the HK97 system. This work therefore establishes a framework for studying the evolutionary relationships between ATP-dependent DNA translocation machineries in double-stranded DNA viruses.

Biochemical and Biophysical Characterization of the dsDNA Packaging Motor from the Lactococcus lactis Bacteriophage Asccphi28.

Double-stranded DNA viruses package their genomes into pre-assembled protein procapsids. This process is driven by macromolecular motors that transiently assemble at a unique vertex of the procapsid and utilize homomeric ring ATPases to couple genome encapsidation to ATP hydrolysis. Here, we describe the biochemical and biophysical characterization of the packaging ATPase from Lactococcus lactis phage asccφ28. Size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), small angle X-ray scattering (SAXS), and negative stain transmission electron microscopy (TEM) indicate that the ~45 kDa protein formed a 443 kDa cylindrical assembly with a maximum dimension of ~155 Å and radius of gyration of ~54 Å. Together with the dimensions of the crystallographic asymmetric unit from preliminary X-ray diffraction experiments, these results indicate that gp11 forms a decameric D5-symmetric complex consisting of two pentameric rings related by 2-fold symmetry. Additional kinetic analysis shows that recombinantly expressed gp11 has ATPase activity comparable to that of functional ATPase rings assembled on procapsids in other genome packaging systems. Hence, gp11 forms rings in solution that likely reflect the fully assembled ATPases in active virus-bound motor complexes. Whereas ATPase functionality in other double-stranded DNA (dsDNA) phage packaging systems requires assembly on viral capsids, the ability to form functional rings in solution imparts gp11 with significant advantages for high-resolution structural studies and rigorous biophysical/biochemical analysis.

Insights into the Structure and Assembly of the Bacteriophage ϕ29 Double-Stranded DNA Packaging Motor

The tailed double-stranded DNA (dsDNA) bacteriophage 29 packages its 19.3-kbp genome into a preassembled procapsid structure by using a transiently assembled phage-encoded molecular motor. This process is remarkable considering that compaction of DNA to near-crystalline densities within the confined space of the capsid requires that the packaging motor work against significant entropic, enthalpic, and DNA-bending energies. The motor consists of three phage-encoded components: the dodecameric connector protein gp10, an oligomeric RNA molecule known as the prohead RNA (pRNA), and the homomeric ring ATPase gp16. Although atomic resolution structures of the connector and different pRNA subdomains have been determined, the mechanism of self-assembly and the resulting stoichiometry of the various motor components on the phage capsid have been the subject of considerable controversy. Here a subnanometer asymmetric cryoelectron microscopy (cryo-EM) reconstruction of a connector-pRNA complex at a unique vertex of the procapsid conclusively demonstrates the pentameric symmetry of the pRNA and illuminates the relative arrangement of the connector and the pRNA. Additionally, a combination of biochemical and cryo-EM analyses of motor assembly intermediates suggests a sequence of molecular events that constitute the pathway by which the motor assembles on the head, thereby reconciling conflicting data regarding pRNA assembly and stoichiometry. Taken together, these data provide new insight into the assembly, structure, and mechanism of a complex molecular machine. Viruses consist of a protein shell, or capsid, that protects and surrounds their genetic material. Thus, genome encapsidation is a fundamental and essential step in the life cycle of any virus. In dsDNA viruses, powerful molecular motors essentially pump the viral DNA into a preformed protein shell. This article describes how a viral dsDNA packaging motor self-assembles on the viral capsid and provides insight into its mechanism of action.

Nucleotide-induced asymmetry within ATPase activator ring drives σ54–RNAP interaction and ATP hydrolysis

It is largely unknown how the typical homomeric ring geometry of ATPases associated with various cellular activities enables them to perform mechanical work. Small-angle solution X-ray scattering, crystallography, and electron microscopy (EM) reconstructions revealed that partial ATP occupancy caused the heptameric closed ring of the bacterial enhancer-binding protein (bEBP) NtrC1 to rearrange into a hexameric split ring of striking asymmetry. The highly conserved and functionally crucial GAFTGA loops responsible for interacting with σ54-RNA polymerase formed a spiral staircase. We propose that splitting of the ensemble directs ATP hydrolysis within the oligomer, and the ring's asymmetry guides interaction between ATPase and the complex of σ54 and promoter DNA. Similarity between the structure of the transcriptional activator NtrC1 and those of distantly related helicases Rho and E1 reveals a general mechanism in homomeric ATPases whereby complex allostery within the ring geometry forms asymmetric functional states that allow these biological motors to exert directional forces on their target macromolecules.

Feeling the Pressure: Dynamics of Viral DNA Packaging are Controlled by the Confined Genome within the Capsid

Double-stranded DNA viruses, including tailed bacteriophages and mammalian herpesviruses, package their genomes into pre-formed protein capsids. The packaging process is driven by a molecular complex known as the packaging motor. This motor is a ring-shaped oligomeric ATPase that utilizes the chemical energy from ATP binding and hydrolysis to perform the mechanical work. We have recently presented a detailed mechanochemical characterization for the bacteriophage phi29 motor, a homo-pentamer that translocates DNA in cycles composed of alternating dwells and bursts (1). We now aim to investigate the behavior of the motor at different stages of packaging, as the motor needs to overcome increasing amounts of internal pressure generated by the compressed dsDNA. We find that the effect of the internal pressure on the motor dynamics is more complex than simply exerting an opposing force to packaging. Through a detailed analysis using ultra-high-resolution DNA translocation data, we find that the internal pressure affects multiple kinetic transitions in the mechanochemical cycle, including events in both the dwell phase and the burst phase. This analysis allows us to make an accurate estimation of the internal force as a function of DNA filling, which is important for the understanding of the DNA organization inside the capsid and the ejection energetics. Remarkably, the motor changes its step size and displays a new class of long-lived pauses towards the completion of packaging. Finally, we determine the structural elements in the packaging complex that are responsible for the signal transduction from the capsid to the motor.(1) G. Chistol∗, S. Liu∗, C. L. Hetherington, J. R. Moffitt, S. Grimes, P. J. Jardine, C. Bustamante, “High Degree of Coordination and Division of Labor Among Subunits in a Homomeric Ring ATPase”, Cell, in press (2012).

High Degree of Coordination and Division of Labor among Subunits in a Homomeric Ring ATPase

Ring NTPases of the ASCE superfamily perform a variety of cellular functions. An important question about the operation of these molecular machines is how the ring subunits coordinate their chemical and mechanical transitions. Here, we present a comprehensive mechanochemical characterization of a homomeric ring ATPase-the bacteriophage φ29 packaging motor-a homopentamer that translocates double-stranded DNA in cycles composed of alternating dwells and bursts. We use high-resolution optical tweezers to determine the effect of nucleotide analogs on the cycle. We find that ATP hydrolysis occurs sequentially during the burst and that ADP release is interlaced with ATP binding during the dwell, revealing a high degree of coordination among ring subunits. Moreover, we show that the motor displays an unexpected division of labor: although all subunits of the homopentamer bind and hydrolyze ATP during each cycle, only four participate in translocation, whereas the remaining subunit plays an ATP-dependent regulatory role.

Complete Characterization of the Mechanochemical Cycle of a Homomeric Ring ATPase

Understanding how multimeric ring ATPases couple chemical transitions to mechanical motion and coordinate their subunits is essential for elucidating the functions of these important molecular machines. Here we used high-resolution optical tweezers to study one such ATPase - the bacteriophage phi29 DNA packaging motor. This pentameric motor is known to translocate DNA by cycling through a dwell phase, in which subunits load ATPs, and a burst phase, in which 10 base pairs of DNA are packaged in four 2.5-base-pair steps. By monitoring packaging in buffers containing various nucleotides and nucleotide analogs, we determined the exact timing of chemical transitions (nucleotide hydrolysis and product release) with respect to the mechanical phases of the motor's cycle. Our results reveal an intricate coordination mechanism with multiple levels of communication between neighboring subunits. ADP release and ATP binding occur in a concerted alternating fashion, with ATP binding to one subunit facilitating the release of ADP in the following subunit. ATP hydrolysis also appears to occur sequentially around the ring through an intersubunit allosteric effect. Moreover, our data suggest that all five subunits of the ring bind and hydrolyze ATP. Only four out of the five hydrolysis events are coupled to DNA translocation, while the fifth subunit consumes ATP to fulfill a regulatory role, resetting the motor for the next mechanochemical cycle. The phi29 motor is able to tolerate a single inactive subunit and still function, albeit at a much slower pace. These new findings allow us to present the most complete mechanochemical model of a homomeric ring ATPase to date, which should provide insight into the operating principles of other ring-shaped ATPases.

Bacteriophage phi29 Translocates DNA Along A Left-Handed Helical Path During Packaging

Bacteriophage phi29 employs a homomeric ring of RecA-like ATPases in order to package its dsDNA genome into the capsid at near-crystalline density. Previous single-molecule measurements of packaging have revealed the coordination of motor subunits, the step size of the motor, and the sensitivity of the motor to substrate modifications, thereby suggesting structural and kinetic models for the mechanism of translocation. However, traditional single-molecule experiments measure only the projection of the motor's motion onto the DNA longitudinal axis.We directly observe that phi29 translocates DNA along a left-handed helical path by monitoring rotation of a bead attached to the side of the substrate DNA in a laser tweezers. Simultaneously, the response to applied torque is measured. This novel experiment probes the details of force and torque generation by the packaging motor. Combining these measurements with angstrom-scale laser tweezers observations of motor stepping suggests specific geometric models for the interaction of the motor and DNA during translocation.The rotor bead technique introduced here allows tracking of the complete three-dimensional trajectory of a dsDNA translocase in action. It also permits the application of torque in a laser tweezers apparatus using commercially-available microspheres.

Bacteriophage Phi29 Negatively Twists DNA During Packaging

Bacteriophage phi29 employs a homomeric ring of RecA-like ATPases in order to package its dsDNA genome into the capsid at near-crystalline density. Previous single-molecule measurements of packaging have revealed the coordination of motor subunits, the step size of the motor, and the sensitivity of the motor to substrate modifications, thereby suggesting structural and kinetic models for the mechanism of translocation.We directly observe that phi29 negatively supercoils DNA during packaging against applied force by monitoring rotation of a bead attached to the side of the substrate DNA in a laser tweezers. Simultaneously, torque generation and response to applied torque are measured. In this way, we probe the details of force and torque generation by the packaging motor. Combining these measurements with angstrom-scale laser tweezers observations of motor stepping suggests specific geometric models for the interaction of the motor and DNA during translocation.The magnetic rotor bead technique introduced here allows simple application of torque and straightforward measurement of twist in a laser tweezers apparatus at arbitrary forces. We propose that this method can be applied with ease to a number of existing single-molecule experiments.

Intersubunit coordination in a homomeric ring ATPase.

Homomeric ring-ATPases perform many vital and varied tasks in the cell, ranging from chromosome segregation to protein degradation. Here we report the first direct observation of the inter-subunit coordination and the step size of such a ring-ATPase, the dsDNA packaging motor in the bacteriophage φ29. Using high-resolution optical tweezers, we find that packaging occurs in increments of 10 bp. Statistical analysis of the preceding dwell times reveals that multiple ATPs bind during each dwell, and application of high force reveals that these 10-bp increments are composed of four 2.5-bp steps. These results indicate that the hydrolysis cycles of the individual subunits are highly coordinated via a mechanism novel for ring-ATPases. In addition, a step size that is a non-integer number of base pairs demands new models for motor-DNA interactions.

Concealed enzyme coordination

Coordination between subunits is crucial for the proper functioning of multi-component molecular machines. A single-molecule study now allows glimpses into the mechanism used by subunits of one such machine. Homomeric ring ATPases are found in all forms of life and are involved in many processes such as chromosome segregation, protein unfolding and ATP synthesis. This remarkable functional diversity is generated by a single, highly conserved, structural core, responsible for the binding and hydrolysis of ATP. How these enzymes coordinate ATP hydrolysis and mechanistic function is largely unknown. One such ring ATPase from a bacteriophage, ϕ29, helps load the dsDNA genome into the viral shell. Moffitt et al. show that this five-membered motor packages the DNA in 10-base-pair bursts, each consisting of four individual 2.5-bp steps corresponding to the hydrolysis of a single ATP. Such a non-integral step size is unprecedented, and raises intriguing mechanistic questions about ATP hydrolysis within rings, and the interactions of the ring with DNA.

Homomeric Ring Assemblies of Eukaryotic Sm Proteins Have Affinity for Both RNA and DNA: CRYSTAL STRUCTURE OF AN OLIGOMERIC COMPLEX OF YEAST SmF

Sm and Sm-like proteins are key components of small ribonucleoproteins involved in many RNA and DNA processing pathways. In eukaryotes, these complexes contain seven unique Sm or Sm-like (Lsm) proteins assembled as hetero-heptameric rings, whereas in Archaea and bacteria six or seven-membered rings are made from only a single polypeptide chain. Here we show that single Sm and Lsm proteins from yeast also have the capacity to assemble into homo-oligomeric rings. Formation of homo-oligomers by the spliceosomal small nuclear ribonucleoprotein components SmE and SmF preclude hetero-interactions vital to formation of functional small nuclear RNP complexes in vivo. To better understand these unusual complexes, we have determined the crystal structure of the homomeric assembly of the spliceosomal protein SmF. Like its archaeal/bacterial homologs, the SmF complex forms a homomeric ring but in an entirely novel arrangement whereby two heptameric rings form a co-axially stacked dimer via interactions mediated by the variable loops of the individual SmF protein chains. Furthermore, we demonstrate that the homomeric assemblies of yeast Sm and Lsm proteins are capable of binding not only to oligo(U) RNA but, in the case of SmF, also to oligo(dT) single-stranded DNA.