DNA Packaging Machine Research Articles

Single-molecule packaging initiation in real time by a viral DNA packaging machine from bacteriophage T4.

Viral DNA packaging motors are among the most powerful molecular motors known. A variety of structural, biochemical, and single-molecule biophysical approaches have been used to understand their mechanochemistry. However, packaging initiation has been difficult to analyze because of its transient and highly dynamic nature. Here, we developed a single-molecule fluorescence assay that allowed visualization of packaging initiation and reinitiation in real time and quantification of motor assembly and initiation kinetics. We observed that a single bacteriophage T4 packaging machine can package multiple DNA molecules in bursts of activity separated by long pauses, suggesting that it switches between active and quiescent states. Multiple initiation pathways were discovered including, unexpectedly, direct DNA binding to the capsid portal followed by recruitment of motor subunits. Rapid succession of ATP hydrolysis was essential for efficient initiation. These observations have implications for the evolution of icosahedral viruses and regulation of virus assembly.

Delivery of vaccine genes and proteins into dendritic cells using the bacteriophage T4 DNA packaging machine (P3273)

Abstract Traditionally, vaccines are produced by empirical methods and current platforms generally do not allow complex designs to modify the quality and quantity of immune responses. We have developed a bacteriophage T4 platform that allows construction and delivery of multivalent vaccines containing both DNA and protein. Using this system, the composition of vaccines can be customized to address mechanistic questions as well as develop efficacious vaccines. The phage T4 DNA packaging machine consists of a molecular motor assembled at the portal vertex of an icosahedral head. The ATP-powered motor packages the 56µm-long 170-kb viral genome into 120nm x 86nm head to near crystalline density. We engineered this machine to deliver genes and proteins into mammalian cells. DNA molecules were translocated into emptied phage head and its outer surface was decorated with proteins fused to outer capsid proteins, Hoc and Soc. T4 nanoparticles carrying reporter genes, vaccine candidates, functional enzymes, and targeting ligands were efficiently delivered into cells, or targeted to antigen-presenting dendritic cells, and the delivered genes were abundantly expressed in vitro and in vivo. Mice delivered with a single dose of F1-V plague vaccine containing both gene and protein elicited robust antibody and cellular immune responses. This “progene delivery” approach might lead to new types of genetic therapies and prime-boost vaccines against complex infectious diseases such as HIV and malaria.

In vitro and in vivo delivery of genes and proteins using the bacteriophage T4 DNA packaging machine

The bacteriophage T4 DNA packaging machine consists of a molecular motor assembled at the portal vertex of an icosahedral head. The ATP-powered motor packages the 56-µm-long, 170-kb viral genome into 120 nm × 86 nm head to near crystalline density. We engineered this machine to deliver genes and proteins into mammalian cells. DNA molecules were translocated into emptied phage head and its outer surface was decorated with proteins fused to outer capsid proteins, highly antigenic outer capsid protein (Hoc) and small outer capsid protein (Soc). T4 nanoparticles carrying reporter genes, vaccine candidates, functional enzymes, and targeting ligands were efficiently delivered into cells or targeted to antigen-presenting dendritic cells, and the delivered genes were abundantly expressed in vitro and in vivo. Mice delivered with a single dose of F1-V plague vaccine containing both gene and protein in the T4 head elicited robust antibody and cellular immune responses. This “progene delivery” approach might lead to new types of vaccines and genetic therapies.

The emptied bacteriophage T4 head: a novel nanoparticle vehicle to simultaneously deliver HIV-1 antigens and genes into dendritic cells

Background Simultaneous delivery of HIV-1 antigens and genes by a single nanoparticle will greatly aid in the development of effective HIV-1 vaccines. We have created a highly stable, empty bacteriophage T4 capsid shell which can be engineered both inside and out. The empty head can be filled with foreign DNA using a powerful DNA packaging machine. Its surface can be arrayed with proteins as fusions of the outer capsid proteins, Hoc (highly antigenic outer capsid protein) and Soc (small outer capsid protein). The 120 x 86 nm head has a capacity to accommodate 170-kb foreign DNA and 1025 molecules of proteins. Methods

Portal-Large Terminase Interactions of the Bacteriophage T4 DNA Packaging Machine Implicate a Molecular Lever Mechanism for Coupling ATPase to DNA Translocation

DNA packaging by double-stranded DNA bacteriophages and herpesviruses is driven by a powerful molecular machine assembled at the portal vertex of the empty prohead. The phage T4 packaging machine consists of three components: dodecameric portal (gp20), pentameric large terminase motor (gp17), and 11- or 12-meric small terminase (gp16). These components dynamically interact and orchestrate a complex series of reactions to produce a DNA-filled head containing one viral genome per head. Here, we analyzed the interactions between the portal and motor proteins using a direct binding assay, mutagenesis, and structural analyses. Our results show that a portal binding site is located in the ATP hydrolysis-controlling subdomain II of gp17. Mutations at key residues of this site lead to temperature-sensitive or null phenotypes. A conserved helix-turn-helix (HLH) that is part of this site interacts with the portal. A recombinant HLH peptide competes with gp17 for portal binding and blocks DNA translocation. The helices apparently provide specificity to capture the cognate prohead, whereas the loop residues communicate the portal interaction to the ATPase center. These observations lead to a hypothesis in which a unique HLH-portal interaction in the symmetrically mismatched complex acts as a lever to position the arginine finger and trigger ATP hydrolysis. Transiently connecting the critical parts of the motor; subdomain I (ATP binding), subdomain II (controlling ATP hydrolysis), and C-domain (DNA movement), the portal-motor interactions might ensure tight coupling between ATP hydrolysis and DNA translocation.

Structure and function of the small terminase component of the DNA packaging machine in T4-like bacteriophages

Tailed DNA bacteriophages assemble empty procapsids that are subsequently filled with the viral genome by means of a DNA packaging machine situated at a special fivefold vertex. The packaging machine consists of a "small terminase" and a "large terminase" component. One of the functions of the small terminase is to initiate packaging of the viral genome, whereas the large terminase is responsible for the ATP-powered translocation of DNA. The small terminase subunit has three domains, an N-terminal DNA-binding domain, a central oligomerization domain, and a C-terminal domain for interacting with the large terminase. Here we report structures of the central domain in two different oligomerization states for a small terminase from the T4 family of phages. In addition, we report biochemical studies that establish the function for each of the small terminase domains. On the basis of the structural and biochemical information, we propose a model for DNA packaging initiation.

A Promiscuous DNA Packaging Machine from Bacteriophage T4

Author SummaryPhage T4 is a prototype for tailed bacteriophages, the most abundant organisms on the planet, as well as for large eukaryotic viruses such as herpes viruses. These viruses encode powerful machines to package their genomes tightly inside an icosahedral-shaped capsid “head.” Packaging into the capsid occurs via a dodecameric portal, localized in one of the vertexes of the capsid. Packaging requires precise orchestration of a series of steps: assembly of an empty prohead, concatemer cutting and attachment of the motor-DNA complex to the portal vertex, ATP-fueled DNA translocation until the head is full, DNA cutting to terminate packaging, detachment of the motor, and sealing of the packaged head by “neck” assembly. Sequential conformational changes, particularly in the portal, are thought to drive these transitions such that assembly proceeds directionally and irreversibly. Here, we found that the phage T4 packaging machine on various capsids is highly promiscuous, translocating DNA into proheads but also, unexpectedly, into previously filled virus heads. Other studies have shown that in filled viral capsids the structure of the portal is fundamentally altered, and it was thought that the packaging mechanism on full heads would be irreversible. We show that full heads, or heads that are emptied of most of their packaged DNA, can reassemble the packaging machine and use it to re-fill the capsid with any DNA molecules. These results challenge the classic sequential virus assembly models, suggest an explanation for the evolution of viral genomes that fit capsid volume, and point the way to a novel nanocapsid delivery system in which the viral packaging machine (portal and motor) could be used to translocate DNA and other therapeutic molecules into synthetic capsids.

Specificity of Interactions among the DNA-packaging Machine Components of T4-related Bacteriophages

Tailed bacteriophages use powerful molecular motors to package the viral genome into a preformed capsid. Packaging at a rate of up to ∼2000 bp/s and generating a power density twice that of an automobile engine, the phage T4 motor is the fastest and most powerful reported to date. Central to DNA packaging are dynamic interactions among the packaging components, capsid (gp23), portal (gp20), motor (gp17, large "terminase"), and regulator (gp16, small terminase), leading to precise orchestration of the packaging process, but the mechanisms are poorly understood. Here we analyzed the interactions between small and large terminases of T4-related phages. Our results show that the gp17 packaging ATPase is maximally stimulated by homologous, but not heterologous, gp16. Multiple interaction sites are identified in both gp16 and gp17. The specificity determinants in gp16 are clustered in the diverged N- and C-terminal domains (regions I-III). Swapping of diverged region(s), such as replacing C-terminal RB49 region III with that of T4, switched ATPase stimulation specificity. Two specificity regions, amino acids 37-52 and 290-315, are identified in or near the gp17-ATPase "transmission" subdomain II. gp16 binding at these sites might cause a conformational change positioning the ATPase-coupling residues into the catalytic pocket, triggering ATP hydrolysis. These results lead to a model in which multiple weak interactions between motor and regulator allow dynamic assembly and disassembly of various packaging complexes, depending on the functional state of the packaging machine. This might be a general mechanism for regulation of the phage packaging machine and other complex molecular machines.

‘Let the phage do the work’: Using the phage P22 coat protein structures as a framework to understand its folding and assembly mutants

The amino acid sequence of viral capsid proteins contains information about their folding, structure and self-assembly processes. While some viruses assemble from small preformed oligomers of coat proteins, other viruses such as phage P22 and herpesvirus assemble from monomeric proteins (Fuller and King, 1980; Newcomb et al., 1999). The subunit assembly process is strictly controlled through protein:protein interactions such that icosahedral structures are formed with specific symmetries, rather than aberrant structures. dsDNA viruses commonly assemble by first forming a precursor capsid that serves as a DNA packaging machine (Earnshaw, Hendrix, and King, 1980; Heymann et al., 2003). DNA packaging is accompanied by a conformational transition of the small precursor procapsid into a larger capsid for isometric viruses. Here we highlight the pseudo-atomic structures of phage P22 coat protein and rationalize several decades of data about P22 coat protein folding, assembly and maturation generated from a combination of genetics and biochemistry.

Kinetic Analysis of the Genome Packaging Reaction in Bacteriophage λ

Bacteriophage lambda is a double-stranded DNA virus that infects the Escherichia coli bacterium. lambda genomic DNA is replicated via rolling circle replication, resulting in multiple genomes linked head to tail at the cos site. To insert a single lambda genome into the viral capsid, the lambda terminase enzyme introduces symmetric nicks, 12 bp apart, at the cos site, and then promotes a strand separation reaction, releasing the tail end of the previous genome and leaving a binary complex consisting of lambda terminase bound to the head end of the adjacent genome. Next, the genome is translocated into the interior of the capsid particle, in a process that requires ATP hydrolysis by lambda terminase. Even though DNA packaging has been studied extensively, currently no bulk assays are available that have been optimized to report directly on DNA translocation. Rather, these assays are sensitive to assembly steps reflecting formation of the active, DNA packaging machine. In this work, we have modified the DNase protection assay commonly used to study DNA packaging in several bacteriophage systems, such that it reports directly on the kinetics of the DNA packaging reaction. We have analyzed our DNA packaging data according to an N-step sequential minimal kinetic model and have estimated an overall packaging rate of 119 +/- 8 bp/s, at 4 degrees C and 1 mM ATP. Furthermore, we have measured an apparent step size for the this reaction (m(obs)) of 410 +/- 150 bp. The magnitude of this value indicates that our assay is most likely sensitive to both mechanical steps associated with DNA insertion as well as occasional slow steps that are repeated every >410 bp. These slow steps may be reflective of the pausing events observed in recent single-molecule studies of DNA packaging in bacteriophage lambda [Fuller, D. N., et al. (2007) J. Mol. Biol. 373, 1113-1122]. Finally, we show that either ATP or ADP is required for terminase cutting at cos, to generate the active, DNA packaging complex.

The Structure of the ATPase that Powers DNA Packaging into Bacteriophage T4 Procapsids

Packaging the viral genome into empty procapsids, an essential event in the life cycle of tailed bacteriophages and some eukaryotic viruses, is a process that shares features with chromosome assembly. Most viral procapsids possess a special vertex containing a dodecameric portal protein that is used for entry and exit of the viral genome. The portal and an ATPase are parts of the genome-packaging machine. The ATPase is required to provide energy for translocation and compaction of the negative charges on the genomic DNA. Here we report the atomic structure of the ATPase component in a phage DNA-packaging machine. The bacteriophage T4 ATPase has the greatest similarity to monomeric helicases, suggesting that the genome is translocated by an inchworm mechanism. The similarity of the packaging machines in the double-stranded DNA (dsDNA) bacteriophage T4 and dsRNA bacteriophage varphi12 is consistent with the evolution of many virions from a common ancestor.

Assembly of Bacteriophage Lambda Terminase into a Viral DNA Maturation and Packaging Machine

Terminase enzymes are common to complex double-stranded DNA viruses and function to package viral DNA into the capsid. We recently demonstrated that the bacteriophage lambda terminase gpA and gpNu1 proteins assemble into a stable heterotrimer with a molar ratio gpA1/gpNu1(2). This terminase protomer possesses DNA maturation and packaging activities that are dependent on the E. coli integration host factor protein (IHF). Here, we show that the protomer further assembles into a homogeneous tetramer of protomers of composition (gpA1/gpNu1(2))4. Electron microscopy shows that the tetramer forms a ring structure large enough to encircle duplex DNA. In contrast to the heterotrimer, the ring tetramer can mature and package viral DNA in the absence of IHF. We propose that IHF induced bending of viral DNA facilitates the assembly of four terminase protomers into a ring tetramer that represents the catalytically competent DNA maturation and packaging complex in vivo. This work provides, for the first time, insight into the functional assembly state of a viral DNA packaging motor.

Structural changes of bacteriophage φ29 upon DNA packaging and release

Cryo-electron microscopy three-dimensional reconstructions have been made of mature and of emptied bacteriophage phi29 particles without making symmetry assumptions. Comparisons of these structures with each other and with the phi29 prohead indicate how conformational changes might initiate successive steps of assembly and infection. The 12 adsorption capable 'appendages' were found to have a structure homologous to the bacteriophage P22 tailspikes. Two of the appendages are extended radially outwards, away from the long axis of the virus, whereas the others are around and parallel to the phage axis. The appendage orientations are correlated with the symmetry-mismatched positions of the five-fold related head fibers, suggesting a mechanism for partial cell wall digestion upon rotation of the head about the tail when initiating infection. The narrow end of the head-tail connector is expanded in the mature virus. Gene product 3, bound to the 5' ends of the genome, appears to be positioned within the expanded connector, which may potentiate the release of DNA-packaging machine components, creating a binding site for attachment of the tail.

A Critical Coiled Coil Motif in the Small Terminase, gp16, from Bacteriophage T4: Insights into DNA Packaging Initiation and Assembly of Packaging Motor

Double-stranded DNA packaging in bacteriophages is driven by one of the most powerful force-generating molecular motors reported to date. The phage T4 motor is composed of the small terminase protein, gpl6 (18kDa), the large terminase protein, gp17 (70kDa), and the dodecameric portal protein gp20 (61kDa). gp16, which exists as an oligomer in solution, is involved in the recognition of the viral DNA substrate, the very first step in the DNA packaging pathway, and stimulates the ATPase and packaging activities associated with gp17. Sequence analyses using COILS2 revealed the presence of coiled coil motifs (CCMs) in gp16. Sixteen T4-family and numerous phage small terminases show CCMs in the corresponding region of the protein, suggesting a common structural and functional theme. Biochemical properties such as reversible thermal denaturation and analytical gel filtration data suggest that the central CCM-1 is critical for oligomerization of gp16. Mutations in CCM-1 that change the hydrophobicity of key residues, or pH 6.0, destabilized coiled coil interactions, resulting in a loss of gp16 oligomerization. The gp16 oligomers are in a dynamic equilibrium with lower M(r) intermediate species and monomer. Monomeric gp16 is unable to stimulate gp17-ATPase, an activity essential for DNA packaging, while conversion back into oligomeric form restored the activity. These data for the first time defined a CCM that is critical for structure and function of the small terminase. We postulate a packaging model in which the gp16 CCM is implicated in the regulation of packaging initiation and assembly of a supramolecular DNA packaging machine on the viral concatemer.

Functional Analysis of the Bacteriophage T4 DNA-packaging ATPase Motor

Packaging of double-stranded DNA into bacteriophage capsids is driven by one of the most powerful force-generating motors reported to date. The phage T4 motor is constituted by gene product 16 (gp16) (18 kDa; small terminase), gp17 (70 kDa; large terminase), and gp20 (61 kDa; dodecameric portal). Extensive sequence alignments revealed that numerous phage and viral large terminases encode a common Walker-B motif in the N-terminal ATPase domain. The gp17 motif consists of a highly conserved aspartate (Asp255) preceded by four hydrophobic residues (251MIYI254), which are predicted to form a beta-strand. Combinatorial mutagenesis demonstrated that mutations that compromised hydrophobicity, or integrity of the beta-strand, resulted in a null phenotype, whereas certain changes in hydrophobicity resulted in cs/ts phenotypes. No substitutions, including a highly conservative glutamate, are tolerated at the conserved aspartate. Biochemical analyses revealed that the Asp255 mutants showed no detectable in vitro DNA packaging activity. The purified D255E, D255N, D255T, D255V, and D255E/E256D mutant proteins exhibited defective ATP binding and very low or no gp16-stimulated ATPase activity. The nuclease activity of gp17 is, however, retained, albeit at a greatly reduced level. These data define the N-terminal ATPase center in terminases and show for the first time that subtle defects in the ATP-Mg complex formation at this center lead to a profound loss of phage DNA packaging.

The Functional Domains of Bacteriophage T4 Terminase

The packaging of double-stranded genomic DNA into some viral and all bacteriophage capsids is driven by powerful molecular motors. In bacteriophage T4, the motor consists of the portal protein assembly composed of twelve copies of gene product 20 (gp20, 61 kDa) and an oligomeric terminase complex composed of gp16 (18 kDa) and gp17 (70 kDa). The packaging motor drives the 171-kbp T4 DNA into the capsid utilizing the free energy of ATP hydrolysis. Evidence suggests that gp17 is the key component of the motor; it exhibits ATPase, nuclease, and in vitro DNA-packaging activities. The N- and C-terminal halves of gp17 were expressed and purified to homogeneity and found to have ATPase and nuclease activities, respectively. The N-terminal domain exhibited 2-3-fold higher Kcat values for gp16-stimulated ATPase than the full-length gp17. Neither of the domains, individually or together, exhibited in vitro DNA-packaging activity, suggesting that communication between the domains is essential for DNA packaging. The domains, in particular the C-terminal domain or a mixture of both the N- and C-terminal domains, inhibited in vitro DNA packaging that is catalyzed by full-length gp17. In conjunction with genetic evidence, these data suggest that the domains compete with the full-length gp17 for binding sites on the portal protein. A model for the assembly of the T4 DNA-packaging machine is presented.

The portal protein plays essential roles at different steps of the SPP1 DNA packaging process

A large number of viruses use a specialized portal for entry of DNA to the viral capsid and for its polarized exit at the beginning of infection. These families of viruses assemble an icosahedral procapsid containing a portal protein oligomer in one of its 12 vertices. The viral ATPase (terminase) interacts with the portal vertex to form a powerful molecular motor that translocates DNA to the procapsid interior against a steep concentration gradient. The portal protein is an essential component of this DNA packaging machine. Characterization of single amino acid substitutions in the portal protein gp6 of bacteriophage SPP1 that block DNA packaging identified sequential steps in the packaging mechanism that require its action. Gp6 is essential at early steps of DNA packaging and for DNA translocation to the capsid interior, it affects the efficiency of DNA packaging, it is a central component of the headful sensor that determines the size of the packaged DNA molecule, and is essential for closure of the portal pore by the head completion proteins to prevent exit of the DNA encapsidated. Functional regions of gp6 necessary at each step are identified within its primary structure. The similarity between the architecture of portal oligomers and between the DNA packaging strategies of viruses using portals strongly suggests that the portal protein plays the same roles in a large number of viruses.

A minimal kinetic model for a viral DNA packaging machine.

Terminase enzymes are common to both eukaryotic and prokaryotic double-stranded DNA viruses. These enzymes possess ATPase and nuclease activities that work in concert to "package" a viral genome into an empty procapsid, and it is likely that terminase enzymes from disparate viruses utilize a common packaging mechanism. Bacteriophage lambda terminase possesses a site-specific nuclease activity, a so-called helicase activity, a DNA translocase activity, and multiple ATPase catalytic sites that function to package viral DNA. Allosteric interactions between the multiple catalytic sites have been reported. This study probes these catalytic interactions using enzyme kinetic, photoaffinity labeling, and vanadate inhibition studies. The ensemble of data forms the basis for a minimal kinetic model for lambda terminase. The model incorporates an ADP-driven conformational reorganization of the terminase subunits assembled on viral DNA, which is central to the activation of a catalytically competent packaging machine. The proposed model provides a unifying mechanism for allosteric interaction between the multiple catalytic sites of the holoenzyme and explains much of the kinetic data in the literature. Given that similar packaging mechanisms have been proposed for viruses as dissimilar as lambda and the herpes viruses, the model may find general utility in our global understanding of the enzymology of virus assembly.

Defining the Bacteriophage T4 DNA Packaging Machine: Evidence for a C-terminal DNA Cleavage Domain in the Large Terminase/Packaging Protein gp17

Double-stranded DNA packaging in bacteriophage T4 and other viruses occurs by translocation of DNA into an empty prohead by a packaging machine assembled at the portal vertex. Coordinated with this complex process is the cutting of concatemeric DNA to initiate and terminate DNA packaging and encapsidate one genome-length viral DNA. The catalytic site responsible for cutting, and the mechanisms by which cutting is precisely coordinated with DNA translocation remained as interesting open questions. Phage T4, unlike the phages with defined ends (e.g. λ, T3, T7), packages DNA in a strictly headful manner, and exhibits no strict sequence specificity to initiate or terminate DNA packaging. Previous evidence suggests that the large terminase protein gp17, a key component of the T4 packaging machine, possesses a non-specific DNA cutting activity. A histidine-rich metal-binding motif, H382-X 2-H385-X 16-C402-X 8-H411-X 2-H414-X 15-H430-X 5-H436, in the C-terminal half of gp17 is thought to be involved in the terminase cleavage. Here, exhaustive site-directed mutagenesis revealed that none of the cysteine and histidine residues other than the H436 residue is critical for function. On the other hand, a cluster of conserved residues within this region, D401, E404, G405, and D409, are found to be critical for function. Biochemical analyses showed that the D401 mutants exhibited a novel phenotype, showing a loss of in vivo DNA cutting activity but not the DNA packaging activity. The functional nature of the critical residues and their disposition in the conserved loop region between two predicted β-strands suggest that these residues are part of a metal-coordinated catalytic site that cleaves the phosphodiester bond of DNA substrate. The data suggest that the T4 terminase consists of at least two functional domains, an N-terminal DNA-translocating ATPase domain and a C-terminal DNA-cutting domain. Although the DNA recognition mechanisms may be distinct, it appears that T4 and other phage terminases employ a common catalytic paradigm for phosphodiester bond cleavage that is used by numerous nucleases.

Defining the ATPase Center of Bacteriophage T4 DNA Packaging Machine: Requirement for a Catalytic Glutamate Residue in the Large Terminase Protein gp17

Double-stranded DNA packaging in icosahedral bacteriophages is driven by an ATPase-coupled packaging machine constituted by the portal protein and two non-structural packaging/terminase proteins assembled at the unique portal vertex of the empty viral capsid. Recent studies show that the N-terminal ATPase site of bacteriophage T4 large terminase protein gp17 is critically required for DNA packaging. It is likely that this is the DNA translocating ATPase that powers directional translocation of DNA into the viral capsid. Defining this ATPase center is therefore fundamentally important to understand the mechanism of ATP-driven DNA translocation in viruses. Using combinatorial mutagenesis and biochemical approaches, we have defined the catalytic carboxylate residue that is required for ATP hydrolysis. Although the original catalytic carboxylate hypothesis suggested the presence of a catalytic glutamate between the Walker A (SRQLGKT 161–167) and Walker B (MIYID 251–255) motifs, none of the four candidate glutamic acid residues, E198, E208, E220 and E227, is required for function. However, the E256 residue that is immediately adjacent to the putative Walker B aspartic acid residue (D255) exhibited a phenotypic pattern that is consistent with the catalytic carboxylate function. None of the amino acid substitutions, including the highly conservative D and Q, was tolerated. Biochemical analyses showed that the purified E256V, D, and Q mutant gp17s exhibited a complete loss of gp16-stimulated ATPase activity and in vitro DNA packaging activity, whereas their ATP binding and DNA cleavage functions remained intact. The data suggest that the E256 mutants are trapped in an ATP-bound conformation and are unable to catalyze the ATP hydrolysis-transduction cycle that powers DNA translocation. Thus, this study for the first time identified and characterized a catalytic glutamate residue that is involved in the energy transduction mechanism of a viral DNA packaging machine.