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.     

The nuclease domain of the SPP1 packaging motor coordinates DNA cleavage and encapsidation

The large terminase subunit is a central component of the genome packaging motor from tailed bacteriophages and herpes viruses. This two-domain enzyme has an N-terminal ATPase activity that fuels DNA translocation during packaging and a C-terminal nuclease activity required for initiation and termination of the packaging cycle. Here, we report that bacteriophage SPP1 large terminase (gp2) is a metal-dependent nuclease whose stability and activity are strongly and preferentially enhanced by Mn²⁺ ions. Mutation of conserved residues that coordinate Mn²⁺ ions in the nuclease catalytic site affect the metal-induced gp2 stabilization and impair both gp2-specific cleavage at the packaging initiation site pac and unspecific nuclease activity. Several of these mutations block also DNA encapsidation without affecting ATP hydrolysis or gp2 C-terminus binding to the procapsid portal vertex. The data are consistent with a mechanism in which the nuclease domain bound to the portal switches between nuclease activity and a coordinated action with the ATPase domain for DNA translocation. This switch of activities of the nuclease domain is critical to achieve the viral chromosome packaging cycle.     

Direct Interaction of the Bacteriophage SPP1 Packaging ATPase with the Portal Protein

DNA packaging in tailed bacteriophages and other viruses requires assembly of a complex molecular machine at a specific vertex of the procapsid. This machine is composed of the portal protein that provides a tunnel for DNA entry, an ATPase that fuels DNA translocation (large terminase subunit), and most frequently, a small terminase subunit. Here we characterized the interaction between the terminase ATPase subunit of bacteriophage SPP1 (gp2) and the procapsid portal vertex. We found, by affinity pulldown assays with purified proteins, that gp2 interacts with the portal protein, gp6, independently of the terminase small subunit gp1, DNA, or ATP. The gp2-procapsid interaction via the portal protein depends on gp2 concentration and requires the presence of divalent cations. Competition experiments showed that isolated gp6 can only inhibit gp2-procapsid interactions and DNA packaging at gp6:procapsid molar ratios above 10-fold. Assays with gp6 carrying mutations in distinct regions of its structure that affect the portal-induced stimulation of ATPase and DNA packaging revealed that none of these mutations impedes gp2-gp6 binding. Our results demonstrate that the SPP1 packaging ATPase binds directly to the portal and that the interaction is stronger with the portal embedded in procapsids. Identification of mutations in gp6 that allow for assembly of the ATPase-portal complex but impair DNA packaging support an intricate cross-talk between the two proteins for activity of the DNA translocation motor.     

The Structure of the Herpes Simplex Virus DNA-Packaging Terminase pUL15 Nuclease Domain Suggests an Evolutionary Lineage among Eukaryotic and Prokaryotic Viruses

Herpes simplex virus 1 (HSV-1), the prototypic member of herpesviruses, employs a virally encoded molecular machine called terminase to package the viral double-stranded DNA (dsDNA) genome into a preformed protein shell. The terminase contains a large subunit that is thought to cleave concatemeric viral DNA during the packaging initiation and completion of each packaging cycle and supply energy to the packaging process via ATP hydrolysis. We have determined the X-ray structure of the C-terminal domain of the terminase large-subunit pUL15 (pUL15C) from HSV-1. The structure shows a fold resembling those of bacteriophage terminases, RNase H, integrases, DNA polymerases, and topoisomerases, with an active site clustered with acidic residues. Docking analysis reveals a DNA-binding surface surrounded by flexible loops, indicating considerable conformational changes upon DNA binding. In vitro assay shows that pUL15C possesses non-sequence-specific, Mg²⁺-dependent nuclease activity. These results suggest that pUL15 uses an RNase H-like, metal ion-mediated catalysis mechanism for cleavage of viral concatemeric DNA. The structure reveals extra structural elements in addition to the RNase H-like fold core and variations in local architecture of the nuclease active site, which are conserved in herpesvirus terminases and bear great similarity to the phage T4 gp17 but are distinct from podovirus and siphovirus orthologs and cellular RNase H, delineating a new evolutionary lineage among a large family of eukaryotic viruses and simple and complex prokaryotic viruses.     

Bacteriophage lambda terminase: alterations of the high-affinity ATPase affect viral DNA packaging

DNA packaging by large DNA viruses such as the tailed bacteriophages and the herpesviruses involves DNA translocation into a preformed protein shell, called the prohead. Translocation is driven by an ATP hydrolysis-powered DNA packaging motor. The bacteriophages encode a heterodimeric viral DNA packaging protein, called terminase. The terminases have an ATPase center located in the N terminus of the large subunit implicated in DNA translocation. In previous work with phage lambda, lethal mutations that changed ATP-reactive residues 46 and 84 of gpA, the large terminase subunit, were studied. These mutant enzymes retained the terminase endonuclease and helicase activities, but had severe defects in virion assembly, and lacked the terminase high-affinity ATPase activity. Surprisingly, in the work described here, we found that enzymes with the conservative gpA changes Y46F and Y46A had only mild packaging defects. These mild defects contrast with their profound virion assembly defects. Thus, these mutant enzymes have, in addition to the mild DNA packaging defects, a severe post-DNA packaging defect. In contrast, the gpA K84A enzyme had similar virion assembly and DNA packaging defects. The DNA packaging energy budget, i.e. DNA packaged/ATP hydrolyzed, was unchanged for the mutant enzymes, indicating that DNA translocation is tightly coupled to ATP hydrolysis. A model is proposed in which gpA residues 46 and 84 are important for terminase's high-affinity ATPase activity. Assembly of the translocation complex remodels this ATPase so that residues 46 and 84 are not crucial for the activated translocation ATPase. Changing gpA residues 46 and 84 primarily affects assembly, rather than the activity, of the translocation complex.     

Assembly architecture and DNA binding of the bacteriophage P22 terminase small subunit

Morphogenesis of bacteriophage P22 involves the packaging of double-stranded DNA into a preassembled procapsid. DNA is translocated by a powerful virally encoded molecular motor called terminase, which comprises large (gp2, 499 residues) and small (gp3, 162 residues) subunits. While gp2 contains the phosphohydrolase and endonuclease activities of terminase, the function of gp3 may be to regulate specific and nonspecific modes of DNA recognition as well as the enzymatic activities of gp2. Electron microscopy shows that wild-type gp3 self-assembles into a stable and monodisperse nonameric ring. A three-dimensional reconstruction at 18 Å resolution provides the first glimpse of P22 terminase architecture and implies two distinct modes of interaction with DNA—involving a central channel of 20 Å diameter and radial spikes separated by 34 Å. Electromobility shift assays indicate that the gp3 ring binds double-stranded DNA nonspecifically in vitro via electrostatic interactions between the positively charged C-terminus of gp3 (residues 143-152) and phosphates of the DNA backbone. Raman spectra show that nonameric rings formed by subunits truncated at residue 142 retain the subunit fold despite the loss of DNA-binding activity. Difference density maps between gp3 rings containing full-length and C-terminally truncated subunits are consistent with localization of residues 143-152 along the central channel of the nonameric ring. The results suggest a plausible molecular mechanism for gp3 function in DNA recognition and translocation.     

Subunit conformations and assembly states of a DNA-translocating motor: the terminase of bacteriophage P22

Bacteriophage P22, a podovirus infecting strains of Salmonella typhimurium, packages a 42-kbp genome using a headful mechanism. DNA translocation is accomplished by the phage terminase, a powerful molecular motor consisting of large and small subunits. Although many of the structural proteins of the P22 virion have been well characterized, little is known about the terminase subunits and their molecular mechanism of DNA translocation. We report here structural and assembly properties of ectopically expressed and highly purified terminase large and small subunits. The large subunit (gp2), which contains the nuclease and ATPase activities of terminase, exists as a stable monomer with an α/β fold. The small subunit (gp3), which recognizes DNA for packaging and may regulate gp2 activity, exhibits a highly α-helical secondary structure and self-associates to form a stable oligomeric ring in solution. For wild-type gp3, the ring contains nine subunits, as demonstrated by hydrodynamic measurements, electron microscopy, and native mass spectrometry. We have also characterized a gp3 mutant (Ala 112 → Thr) that forms a 10-subunit ring, despite a subunit fold indistinguishable from wild type. Both the nonameric and decameric gp3 rings exhibit nonspecific DNA-binding activity, and gp2 is able to bind strongly to the DNA/gp3 complex but not to DNA alone. We propose a scheme for the roles of P22 terminase large and small subunits in the recruitment and packaging of viral DNA and discuss the model in relation to proposals for terminase-driven DNA translocation in other phages.     

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.     

Analysis of capsid portal protein and terminase functional domains: interaction sites required for DNA packaging in bacteriophage T4.

Bacteriophage DNA packaging results from an ATP-driven translocation of concatemeric DNA into the prohead by the phage terminase complexed with the portal vertex dodecamer of the prohead. Functional domains of the bacteriophage T4 terminase and portal gene 20 product (gp20) were determined by mutant analysis and sequence localization within the structural genes. Interaction regions of the portal vertex and large terminase subunit (gp17) were determined by genetic (terminase-portal intergenic suppressor mutations), biochemical (column retention of gp17 and inhibition of in vitro DNA packaging by gp20 peptides), and immunological (co-immunoprecipitation of polymerized gp20 peptide and gp17) studies. The specificity of the interaction was tested by means of a phage T4 HOC (highly antigenicoutercapsid protein) display system in which wild-type, cs20, and scrambled portal peptide sequences were displayed on the HOC protein of phage T4. Binding affinities of these recombinant phages as determined by the retention of these phages by a His-tag immobilized gp17 column, and by co-immunoprecipitation with purified terminase supported the specific nature of the portal protein and terminase interaction sites. In further support of specificity, a gp20 peptide corresponding to a portion of the identified site inhibited packaging whereas the scrambled sequence peptide did not block DNA packaging in vitro.The portal interaction site is localized to 28 residues in the central portion of the linear sequence of gp20 (524 residues). As judged by two pairs of intergenic portal-terminase suppressor mutations, two separate regions of the terminase large subunit gp17 (central and COOH-terminal) interact through hydrophobic contacts at the portal site. Although the terminase apparently interacts with this gp20 portal peptide, polyclonal antibody against the portal peptide appears unable to access it in the native structure, suggesting intimate association of gp20 and gp17 possibly internalizes terminase regions within the portal in the packasome complex. Both similarities and differences are seen in comparison to analogous sites which have been identified in phages T3 and lambda.     

Energy-independent helicase activity of a viral genome packaging motor

The assembly of complex double-stranded DNA viruses includes a genome packaging step where viral DNA is translocated into the confines of a preformed procapsid shell. In most cases, the preferred packaging substrate is a linear concatemer of viral genomes linked head-to-tail. Viral terminase enzymes are responsible for both excision of an individual genome from the concatemer (DNA maturation) and translocation of the duplex into the capsid (DNA packaging). Bacteriophage λ terminase site-specifically nicks viral DNA at the cos site in a concatemer and then physically separates the nicked, annealed strands to mature the genome in preparation for packaging. Here we present biochemical studies on the so-called helicase activity of λ terminase. Previous studies reported that ATP is required for strand separation, and it has been presumed that ATP hydrolysis is required to drive the reaction. We show that ADP and nonhydrolyzable ATP analogues also support strand separation at low (micromolar) concentrations. In addition, the Escherichia coli integration host factor protein (IHF) strongly stimulates the reaction in a nucleotide-independent manner. Finally, we show that elevated concentrations of nucleotide inhibit both ATP- and IHF-stimulated strand separation by λ terminase. We present a model where nucleotide and IHF interact with the large terminase subunit and viral DNA, respectively, to engender a site-specifically bound, catalytically competent genome maturation complex. In contrast, binding of nucleotide to the low-affinity ATP binding site in the small terminase subunit mediates a conformational switch that down-regulates maturation activities and activates the DNA packaging activity of the enzyme. This affords a motor complex that binds tightly, but nonspecifically, to DNA as it translocates the duplex into the capsid shell. These studies have yielded mechanistic insight into the assembly of the maturation complex on viral DNA and its transition to a mobile packaging motor that may be common to all of the complex double-stranded DNA viruses.     

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. lambda, 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 beta-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.     

Regulation by interdomain communication of a headful packaging nuclease from bacteriophage T4

In genome packaging by tailed bacteriophages and herpesviruses, a concatemeric DNA is cut and inserted into an empty procapsid. A series of cuts follow the encapsidation of each unit-length 'headful' genome, but the mechanisms by which cutting is coupled to packaging are not understood. Here we report the first biochemical characterization of a headful nuclease from bacteriophage T4. Our results show that the T4 nuclease, which resides in the C-terminal domain of large 'terminase' gp17, is a weak endonuclease and regulated by a variety of factors; Mg, NaCl, ATP, small terminase gp16 and N-terminal ATPase domain. The small terminase, which stimulates gp17-ATPase, also stimulates nuclease in the presence of ATP but inhibits in the absence of ATP suggesting interdomain crosstalk. Comparison of the 'relaxed' and 'tensed' states of the motor show that a number of basic residues lining the nuclease groove are positioned to interact with DNA in the tensed state but change their positions in the relaxed state. These results suggest that conformational changes in the ATPase center remodel the nuclease center via an interdomain 'communication track'. This might be a common regulatory mechanism for coupling DNA cutting to DNA packaging among the headful packaging nucleases from dsDNA viruses.     

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.     

Biochemical characterization of an ATPase activity associated with the large packaging subunit gp17 from bacteriophage T4

Double-stranded DNA-packaging in icosahedral bacteriophages is believed to be driven by a packaging "machine" constituted by the portal protein and the two packaging/terminase proteins assembled at the unique portal vertex of the empty prohead shell. Although ATP hydrolysis is evidently the principal driving force, which component of the packaging machinery functions as the translocating ATPase has not been elucidated. Evidence suggests that the large packaging subunit is a strong candidate for the translocating ATPase. We have constructed new phage T4 terminase recombinants under the control of phage T7 promoter and overexpressed the packaging/terminase proteins gp16 and gp17 in various configurations. The hexahistidine-tagged-packaging proteins were purified to near homogeneity by Ni(2+)-agarose chromatography and were shown to be highly active for packaging DNA in vitro. The large packaging subunit gp17 but not the small subunit gp16 exhibited an ATPase activity. Although gp16 lacked ATPase activity, it enhanced the gp17-associated ATPase activity by >50-fold. The gp16 enhancement was specific and was due to an increased catalytic rate for ATP hydrolysis. A phosphorylated gp17 was demonstrated under conditions of low catalytic rates but not under high catalytic rates in the presence of gp16. The data are consistent with the hypothesis that a weak ATPase is transformed into a translocating ATPase of high catalytic capacity after assembly of the packaging machine.     

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.     

Isolation and characterization of T4 bacteriophage gp17 terminase,a large subunit multimer with enhanced ATPase activity

Phage T4 terminase is a two-subunit enzyme that binds to the prohead portal protein and cuts and packages a headful of concatameric DNA. To characterize the T4 terminase large subunit, gp17 (70 kDa), gene 17 was cloned and expressed as a chitin-binding fusion protein. Following cleavage and release of gp17 from chitin, two additional column steps completed purification. The purification yielded (i) homogeneous soluble gp17 highly active in in vitro DNA packaging ( approximately 10% efficiency, >10(8) phage/ml of extract); (ii) gp17 lacking endonuclease and contaminating protease activities; and (iii) a DNA-independent ATPase activity stimulated >100-fold by the terminase small subunit, gp16 (18 kDa), and modestly by portal gp20 and single-stranded binding protein gp32 multimers. Analyses revealed a preparation of highly active and slightly active gp17 forms, and the latter could be removed by immunoprecipitation using antiserum raised against a denatured form of the gp17 protein, leaving a terminase with the increased specific activity (approximately 400 ATPs/gp17 monomer/min) required for DNA packaging. Analysis of gp17 complexes separated from gp16 on glycerol gradients showed that a prolonged enhanced ATPase activity persisted after exposure to gp16, suggesting that constant interaction of the two proteins may not be required during packaging.     

Structural basis for the nuclease activity of a bacteriophage large terminase

The DNA‐packaging motor in tailed bacteriophages requires nuclease activity to ensure that the genome is packaged correctly. This nuclease activity is tightly regulated as the enzyme is inactive for the duration of DNA translocation. Here, we report the X‐ray structure of the large terminase nuclease domain from bacteriophage SPP1. Similarity with the RNase H family endonucleases allowed interactions with the DNA to be predicted. A structure‐based alignment with the distantly related T4 gp17 terminase shows the conservation of an extended β‐sheet and an auxiliary β‐hairpin that are not found in other RNase H family proteins. The model with DNA suggests that the β‐hairpin partly blocks the active site, and in vivo activity assays show that the nuclease domain is not functional in the absence of the ATPase domain. Here, we propose that the nuclease activity is regulated by movement of the β‐hairpin, altering active site access and the orientation of catalytically essential residues.     

Nucleotides regulate the conformational state of the small terminase subunit from bacteriophage lambda: implications for the assembly of a viral genome-packaging motor

Terminase enzymes are responsible for "packaging" of viral DNA into a preformed procapsid. Bacteriophage lambda terminase is composed of two subunits, gpA and gpNu1, in a gpA(1).gpNu1(2) holoenzyme complex. The larger gpA subunit is responsible for preparation of viral DNA for packaging, and is central to the packaging motor complex. The smaller gpNu1 subunit is required for site-specific assembly of the packaging motor on viral DNA. Terminase assembly at the packaging initiation site is regulated by ATP binding and hydrolysis at the gpNu1 subunit. Characterization of the catalytic and structural interactions between the DNA and nucleotide binding sites of gpNu1 is thus central to our understanding of the packaging motor at the molecular level. The high-resolution structure of the DNA binding domain of gpNu1 (gpNu1-DBD) was recently determined in our lab [de Beer, T., et al. (2002) Mol. Cell 9, 981-991]. The structure reveals the presence of a winged-helix-turn-helix DNA binding motif, but the location of the ATPase catalytic site in gpNu1 remains unknown. In this work, nucleotide binding to the gpNu1-DBD was probed using acrylamide fluorescence quenching and fluorescence-monitored ligand binding studies. The data indicate that the minimal DBD dimer binds both ATP and ADP at two equivalent but highly cooperative binding sites. The data further suggest that ATP and ADP induce distinct conformations of the dimer but do not affect DNA binding affinity. The implications of these results with respect to the assembly and function of a terminase DNA-packaging motor are discussed.