Structure and Mechanism of the Atpase That Powers Viral Genome

Structure and mechanism of the ATPase that powers PNAS PLUS viral genome packaging

Brendan J. Hilberta,1, Janelle A. Hayesa,1, Nicholas P. Stonea, Caroline M. Duffya, Banumathi Sankaranb, and Brian A. Kelcha,2

aDepartment of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605; and bBerkeley Center for Structural Biology, Lawrence Berkeley National Laboratory, Berkeley, CA 94720

Edited by Wei Yang, National Institutes of Health, Bethesda, MD, and approved June 5, 2015 (received for review April 9, 2015) Many viruses package their genomes into procapsids using an with portal; (ii) consequently, it was proposed that the active site ATPase machine that is among the most powerful known biological residues that catalyze ATP hydrolysis are derived from one sub- motors. However, how this motor couples ATP hydrolysis to DNA unit exclusively; (iii) the pore of the ring has a net negative charge translocation is still unknown. Here, we introduce a model system and is largely unconserved; and (iv) DNA was proposed to be with unique properties for studying motor structure and mecha- bound primarily by the nuclease domain. This model is sub- nism. We describe crystal structures of the packaging motor ATPase stantially different from models observed for other nucleic acid domain that exhibit nucleotide-dependent conformational changes translocases of the ASCE family, in which the pore is largely involving a large rotation of an entire subdomain. We also identify conserved and positively charged, and ATP hydrolysis is cata- the arginine finger residue that catalyzes ATP hydrolysis in a neigh- lyzed in trans (11–15). Resolving these disparities between the boring motor subunit, illustrating that previous models for motor proposed assembly and conserved features of the ASCE family is structure need revision. Our findings allow us to derive a structural necessary to understand the molecular mechanisms of motor action. model for the motor ring, which we validate using small-angle Several issues have prevented elucidation of the terminase X-ray scattering and comparisons with previously published data. mechanism. First, the TerL proteins that have been studied in We illustrate the model’s predictive power by identifying the mo- vitro thus far fail to form rings in isolation (16–19). Second, crystal tor’s DNA-binding and assembly motifs. Finally, we integrate our

structures of the TerL proteins from bacteriophages Sf6 and T4 BIOCHEMISTRY results to propose a mechanistic model for DNA translocation by this molecular machine. (20, 21) (gp2 and gp17, respectively; hereafter known as Sf6-TerL and T4-TerL) do not show ATP-dependent conformational ASCE ATPase | thermophile | bacteriophage | translocase | motor protein changes sufficient to power DNA translocation. Consequently, the structural mechanism coupling ATP hydrolysis to DNA translocation remains nebulous. Structural analysis of the WT ouble-stranded DNA (dsDNA) viruses ranging from bacte- TerL ATPase domain in different nucleotide states will be nec- riophages to the human pathogens of the herpesvirus family D essary to address this issue. form infectious virions by packaging their genomes into pre- To answer these critical questions, we used a novel model formed procapsids using a powerful ATPase machine (1). The system for understanding motor function. Because packaging viral genome packaging motor is a multicomponent molecular – machine that must complete several tasks in sequential order, motor ATPases are often insoluble (22 24), we first sought a the foremost of which is the ATP-dependent pumping of viral packaging motor system that is highly soluble and forms rings in DNA into the procapsid (Fig. 1A). Because the DNA progresses from a flexible state to a semicrystalline state as it fills the capsid Significance interior, the motor pumps against a tremendous force. The pressures inside the filled capsid are estimated to reach 60–70 Many viruses use a molecular motor to pump DNA into a pre- atm (2, 3), equivalent to 10-fold higher than a bottle of cham- formed protein shell called the capsid, a process that is essen- pagne. Thus, the viral packaging motor represents one of the tial for the formation of infectious virus particles. The ATPase most powerful biological motors known (2, 4). machine powering this process is the strongest known bi- The central component of the packaging motor is the ATPase ological motor. However, the structure and mechanism of this subunit, which drives DNA translocation. The ATPase subunit is motor are unknown. Here, we derive a structural model of the a member of the additional strand, conserved glutamate (ASCE) ATPase assembly using a combination of X-ray crystallography, superfamily of ATPases (5). In herpesviruses, as well as many small-angle X-ray scattering, molecular modeling, and bio- bacteriophages, this ATPase is from a specific clade of the ASCE chemical data. We identify residues critical for ATP hydrolysis family called the terminase family (1, 6). In viruses that use a and DNA binding, and derive a mechanistic model for the terminase-type motor for genome packaging, the motor consists translocation of DNA into the viral capsid. Our studies introduce of several proteins that assemble into homomeric rings (7) (Fig. a model for ATPase assembly and illustrate how DNA is pumped 1A). The large terminase (TerL) protein harbors the motor’s two with high force. enzymatic activities (7): the ATPase activity that pumps DNA into the capsid and an endonuclease domain that cleaves pack- Author contributions: B.J.H., J.A.H., and B.A.K. designed research; B.J.H., J.A.H., N.P.S., C.M.D., B.S., and B.A.K. performed research; B.A.K. contributed new reagents/analytic aged DNA from the remaining concatemeric DNA when the tools; B.J.H., J.A.H., N.P.S., C.M.D., B.S., and B.A.K. analyzed data; and B.J.H., J.A.H., and capsid is full. Cryoelectron microscopy (cryo-EM) studies in- B.A.K. wrote the paper. dicate that a pentamer of TerL subunits attaches to the capsid by The authors declare no conflict of interest. binding to a dodecameric assembly called portal (8). However, This article is a PNAS Direct Submission. there are conflicting reports as to the orientation of TerL relative Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, to portal during packaging (8–10). www.pdb.org (PDB ID codes 4ZNI, 4ZNJ, 4ZNK, and 4ZNL). A structural model for the bacteriophage T4 TerL ring has 1B.J.H. and J.A.H. contributed equally to this work. been previously proposed (8) with these salient features: (i) the 2To whom correspondence should be addressed. Email: [email protected]. nuclease domains assemble to constitute the ring distal to the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. capsid, with the ATPase domains protruding radially to interact 1073/pnas.1506951112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1506951112 PNAS Early Edition | 1of8 Downloaded by guest on October 1, 2021 F A trifluoride (BeF3) (26, 27) (Fig. S1 ). Thus, because the isolated TerLP74-26 protein can assume a pentamer consistent with its functional form, we argue that TerLP74-26 is an excellent minimal model for understanding packaging motor structure.

Structure of the TerL ATPase Domain. We next sought to elucidate the structural mechanism of genome packaging by TerLP74-26. Because DNA translocation is ATP-dependent, considerable insight can be obtained from the structure of the isolated ATPase domain. Therefore, we expressed, purified, and crystallized a construct that corresponds to the N-terminal ATPase domain of TerLP74-26 (residues 1–256; Fig. 1B). The structure in the absence of nucleotide was determined by single-wavelength B anomalous dispersion (SAD) (28) using selenomethionine (SeMet)- labeled protein, and phases were extended to 2.1 Å using diffraction data from native protein (Table S1). A second crystal form yielded another structure of the apo TerLP74-26 ATPasedomainto1.9Å. The structures from each crystal form are very similar (Cα rmsd of 0.8Å; Fig. S2A and Table S2). Unlike the full-length TerLP74-26,the Fig. 1. Genome packaging in bacteriophage and TerL constructs. (A) Mat- isolated ATPase domain does not form a pentameric arrangement uration of dsDNA viruses is a five-step process: (1) the small terminase (TerS) in either crystal form. subunit of the motor recognizes the concatemeric viral genome; (2) the We note three prominent features of the TerLP74-26 ATPase motor binds to the portal complex on the capsid; (3) TerL hydrolyzes ATP to domain (Fig. 2). First, TerLP74-26, as in other terminase ATPase translocate DNA into the capsid; (4) after the genome is translocated into domains (20, 21), contains a C-terminal subdomain (residues the capsid, TerL switches its enzymatic activity from translocation to DNA 221–251 in TerLP74-26) that sits above the ATPase active site. We cleavage; and (5) the motor is finally released for maturation of another “ ” virus while portal binds to the tail proteins to complete a mature, infectious call this region the lid subdomain with reference to the

virion. (B) TerL constructs used in this study. Full-length TerLP74-26 is produced structurally unrelated but analogous lid subdomains found in with an N-terminal T7-gp10 expression tag that is removed by prescission other ASCE ATPases (29). Lid subdomains are often used in protease (PP) cleavage. The C-terminal His10 tag is noncleavable. contacts between adjacent subunits in ASCE oligomers (11, 30), and their conformation can be modulated by ATP hydrolysis (11, 31). Second, we observe a large patch of six basic residues (R100, isolation to enable mechanistic analyses in a simplified system. R101, R102, R104, R128, and K130) in the turns and strands of We anticipated that TerL from a thermophilic phage would have the antiparallel section of the beta-sheet, a region that is specific higher stability, solubility, and propensity to form a pentamer. to the terminase ASCE subfamily (6). A basic residue is conserved Moreover, we expected that the features necessary for DNA at the Arg101 position in the other large terminase structures translocation would be more exaggerated in a thermophilic [Lys223 and Arg82 in T4 (8) and Sf6 (20), respectively] (Fig. S2C). motor because packaging is expected to be more difficult at high We hypothesize that this region binds to the DNA backbone temperature due to the increased entropic penalty of ordering DNA within the capsid (3). Here, we describe the TerL protein from the phage P74-26 (hereafter known as TerLP74-26), a thermophilic siphovirus that infects Thermus thermophilus (25). We show that the TerLP74-26 assembles into a pentamer that has ATPase and DNA-binding activity. We report the structure of the TerLP74-26 ATPase domain in both the apo and ADP•BeF3-bound states, and observe a large conformational change in a subdomain in response to ATP hydrolysis and release. We also show that ATP hydrolysis is catalyzed by a conserved arginine finger residue that is provided in trans by a neighboring TerL subunit. Finally, a combination of biochemical, biophysical, structural, and computational data is used to build a structural model for the TerL ring and a mechanistic model for how ATP hydrolysis results in translocation of DNA. Results

Characterization of TerLP74-26. We recombinantly expressed and purified TerLP74-26 in Escherichia coli (Fig. S1A). TerLP74-26 predominantly elutes in size exclusion chromatography (SEC) at a volume consistent with a pentamer [molecular mass by SEC (MMSEC)of∼270 kD, MMpentamer of 285 kD] with a small Fig. 2. Structure of the TerLP74-26 ATPase domain. Characteristics of the TerL ATPase domain are color-coded. The Walker A and Walker B motifs form the monomer peak (MMSEC of ∼57.3 kD, MMmonomer of 57.1 kD) (Fig. S1B). This observation is notable, because other TerL active site. The lid subdomain (residues 221–251) is adjacent to the ATP- proteins are predominantly monomeric in isolation (16–19), only binding site and contains large hydrophobic residues that are positioned forming a pentameric ring when bound to the procapsid (8). away from the hydrophobic core of the Rossmann fold. The hydrophobic residues Trp231 and Tyr238 are solvent-exposed, suggesting that they could TerLP74-26 is an active ATPase that exhibits apparent coopera- participate in protein–protein interactions. Arg100, Arg101, Arg102, Arg104, tivity in ATP hydrolysis, suggesting that the active sites are Arg128, and Lys130 form a continuous, solvent-exposed basic patch. Arg101 coupled (Fig. S1 C–E). TerLP74-26 tightly binds DNA when locked is identified as critical for DNA binding. Arg139, positioned ∼25 Å from the into an ATP-bound state by the ATP mimic ADP•beryllium active site, is established as necessary for catalysis in trans.

2of8 | www.pnas.org/cgi/doi/10.1073/pnas.1506951112 Hilbert et al. Downloaded by guest on October 1, 2021 PNAS PLUS during packaging. In support of this hypothesis, we observe a ADP•BeF3 states occurs in the lid subdomain. Upon binding the sulfate ion bound to Arg101 in all of our crystal forms. The third ATP analog, the lid rotates ∼13° in a rigid body motion (Fig. 3B region of note is a conserved arginine that is on the opposite face and Movie S1). Upon binding the ATP mimic, the P-loop resi- from the active site. We propose that this residue is used to dues Arg39 and Gln40 rotate to contact the gamma-phosphate ’ activate ATP hydrolysis in a neighboring subunit in the assembly mimic directly. The P-loop s interactions with several lid residues (Ser221, Trp225, Arg228, and Tyr232) result in a commensurate (discussed below). C To determine the conformational changes induced by ATP rotation of the lid toward the ATP adenine ring (Fig. 3 ). Thus, the motion appears to be induced by the P-loop, which bridges binding, we solved a 2.0-Å crystal structure of the TerL P74-26 the gamma-phosphate mimic and residues at the base of the lid. ATPase domain cocrystallized with the ATP mimic, ADP•BeF3. • The rotation of the lid upon ATP binding results in a reorientation The packing of the ADP BeF3 cocrystal is unrelated to the apo of the lid’s hydrophobic residues such that they are now less sol- • crystals, with three ADP BeF3-bound TerLP74-26 ATPase vent-exposed, potentially altering TerL’s interactions within the domains in the asymmetric unit (Fig. S2B). The residues of the motor (Fig. 3D). Walker A and B motifs are in the active conformation (Fig. 3A). The largest conformational change between the apo and Identification of the Trans-acting Arginine Finger. The structures of TerL ATPases reported here and elsewhere (20, 21) display incomplete active sites, presumably because all were crystallized without formation of an active ring. These monomeric TerL A B structures have only two positively charged residues within the P-loop that contact the gamma-phosphate of ATP to stabilize the transition state for hydrolysis. However, other ASCE family members have at least three positive residues that contact the gamma-phosphate (11–15, 31, 32). Indeed, our ATPase domain construct lacks ATPase activity (Fig. S3A), confirming that the active site in our structure is incomplete. In ASCE-type ATPa- ses, the third positive charge is provided in trans by a residue known as the “arginine finger,” which is most often arginine,

although it can also be lysine (29). The location of the arginine BIOCHEMISTRY C finger within the Rossmann fold varies widely across the ASCE family, which means that ASCE subfamilies often have different relative orientations of their ATPase domains within the oligo- mer (33). Thus, the identification of the trans-acting arginine finger in TerL is necessary both for elucidation of the ATPase mechanism and for determining the architecture of the TerL ring assembly. We used a two-step strategy to identify the arginine finger of TerLP74-26. First, we screened mutants of surface-exposed arginines in full-length TerLP74-26 for ATPase activity. For mutants that showed no ATPase activity in this initial scan, we tested D whether an inactive TerLP74-26 protein (mutated Walker B motif) could restore activity by donating its intact arginine finger. We measured steady-state ATPase activity for 11 different arginine mutants in full-length TerLP74-26 (Fig. 4A). The R39A, R139A, and R235A variants completely abrogate ATPase activity, making them candidates for the arginine finger. Of these candidate residues, Arg39 and Arg235 reside within or near the in cis Fig. 3. Structure of the TerL ATPase domain in complex with an ATP active site, suggesting that they may affect catalysis . Con- P74-26 versely, Arg139 is located ∼25 Å away from the gamma-phosphate analog. (A) ADP•BeF3 is bound in the TerL active site. An omit map density − 2 (blue mesh) contoured to 3σ (0.34 e /Å ) shows ADP•BeF3, a magnesium ion of ATP (Fig. 2). (green), and waters (red) bound in the TerL active site. Ser44, an ADP beta- To assess which of our candidates is the arginine finger, we phosphate oxygen, fluoride, and three waters coordinate the magnesium developed a biochemical complementation assay. We mixed ion. Glu150 is pointed toward the BeF3 moiety to catalyze ATP hydrolysis. each of our three candidate arginine finger mutants with the (B) Conformational changes upon ATP hydrolysis and release. The ATPase E150A variant whose active site is disabled in cis (Fig. S1C) but • • domain of the ADP BeF3-bound structure (light purple, with ADP BeF3 in can still donate an intact arginine finger to an adjacent subunit in green) was superposed onto the Rossmann fold of the apo structure (dark the assembly. Thus, E150A should restore activity to an arginine purple). The lid was not used for superposition. Vectors (orange arrows) finger mutant, but not to a mutant whose ATPase activity is show alpha-carbon position differences of 2.5 Å or greater between the two in cis structures. The teal bar marks the lid’s axis of rotation, which lies just off the disabled . Mixing E150A with R139A results in restoration ATP gamma-phosphate mimic. (C) P-loop interactions with the lid (green) of ATPase activity, whereas mixtures of E150A with R39A or appear to drive conformational changes. Hydrogen bonds are shown as R235A exhibit no significant ATP hydrolysis (Fig. 4B). ATPase yellow dashes. (Left) Backbone carbonyls of P-loop residues Arg39 and Gln40 activity is maximal at a 1:1 ratio of E150A to R139A (Fig. S3B), (dark purple) interact with side chains of lid residues Ser221 and Trp225. as expected for complementation of an arginine finger mutant. • (Right) Arg39 and Gln40 change position to bind ADP BeF3, pulling at the In contrast, a double mutant combining both the R139A and base of the lid. The new Gln40 position binds Tyr232, stabilizing the new lid E150A mutations in the same protein has no activity, and cannot conformation. The Trp225 indole rearranges to stack with the adenine base (purple disks), and Arg39 and Gln40 side chains engage the beryllium fluo- be complemented by either the E150A or R139A single mutant. ride (purple dashes). (D) Schematic illustration depicting the conformational Our complementation results mirror the results of similar exchange of the lid subdomain in response to nucleotide hydrolysis and periments from Cox et al. (34) definitively establishing that the release. The lid is held fixed such that the Rossmann fold rotates by ASCE family member RecA uses trans-interactions to catalyze ∼13° outward. ATP hydrolysis.

Hilbert et al. PNAS Early Edition | 3of8 Downloaded by guest on October 1, 2021 large terminase family (Fig. 4C and Fig. S3D). Hence, we expect A that the arginine finger is playing a similar role throughout the family of large terminases. Taken together, our results indicate that Arg139 is the arginine finger, which mediates ATP hydrolysis in TerL in trans. Our identification of the arginine finger illustrates that (i) ATP hydrolysis is catalyzed in trans and (ii) the previously proposed structural model (8) must be substantially refined to account for these trans-interactions within the TerL ring.

Modeling of the TerL Ring. Our two key results, the TerLP74-26 ATPase domain structure and identification of the arginine finger residue, allow us to create a model for the TerL ring assembly. The structure provides a basis for molecular docking, and the arginine finger provides a spatial restraint that must B be satisfied for the TerL assembly. We used the program M-ZDOCK (35, 36) to model the TerL ATPase ring. Fivefold symmetry was the only constraint applied. We used the trans- arginine finger interaction to validate the docking results in- dependently. Although many ATPase rings are asymmetrical during function (12, 37), we assume fivefold symmetry for sim- plicity. Regardless, most ringed ATPase structures were first modeled as symmetrical assemblies (32, 38), and these models were refined later to show asymmetry during function (9, 10, 12, 37). After docking of the ATPase ring, we used the full-length crystal structure of T4-TerL (8) to orient a homology model of the P74-26 nuclease domain, which results in minimal steric clashes between the two domains. The soft energetic potential used in M-ZDOCK results in slightly shorter interatomic dis- tances (35), and therefore a constricted ring; thus, we treat the molecular docking results in a qualitative fashion. Molecular docking produced a TerL assembly that is consistent with our identification of the arginine finger. Arg139 contacts the ATP gamma-phosphate in a neighboring subunit, despite C no restraint for this interaction imposed during the calculation (Fig. 5 A and B). Thus, these models satisfy a critical spatial interaction identified by our biochemical results. The lid subdomains interact with an adjacent subunit, with the nuclease domains arranged radially (Fig. 5C). We tested our model’s plausibility by comparing our TerL assembly with other TerL crystal structures and a cryo-EM re- Fig. 4. Identification of the TerL arginine finger residue. (A) ATP hydrolysis construction. First, orthologous TerL ATPase crystal structures

rates of TerLP74-26 arginine mutants. The only arginine mutants that abolish (8, 20) can be identically positioned without significant steric ATPase activity are R39A, R139A, and R235A (compare activity with activity clashes of the ATPase domains (Fig. S4 A and C). In fact, the of the inactive E150A mutant). The final TerL concentration is 0.5 μM. For all N-terminal extension that is unique to T4 is placed on the out- relevant panels in this figure, error bars are the SD from at least three side of the ring rather than lining the pore of the ring as in the replicates. R39 is a conserved P-loop residue proposed by Sun et al. (8) to be a previous proposal (8). Moreover, the pores of our Sf6-TerL and cis-acting arginine finger. (B) Arginine finger complementation assay. Dif- B μ T4-TerL ATPase models are positively charged (Fig. S4 and ferent TerL variants at 0.5 M each are mixed either with an alternate TerL D). Second, our structural model predicts that for other TerL mutant in a 1:1 ratio (as indicated by the “+” sign) or with buffer, and their steady-state ATPase activity is measured. Note that the R139A/E150A double proteins that are monomeric in isolation, ring assembly would be mutant is a single variant and not a mixture. Only a mixture of the R139A enhanced by ATP mediating cross-subunit interactions. Indeed, and E150A mutants significantly restores activity. Schematic illustrations il- this dependency has been observed for T4 (39), T3 (40), and lustrate that the Walker B mutant (E150A) has a debilitated active site but lambda (41). Third, our T4-TerL ring model is a bowl-like as- can donate its intact arginine finger (shown as an arrow), whereas the ar- sembly that reasonably fits the cryo-EM map (8) for an actively ginine finger mutant has an intact active site but lacks ATPase activity due to packaging T4 phage (Fig. S4 E and F). However, this fitting loss of the trans-acting residue. (C) Arginine finger residue is conserved in positions the ATPase domain ring distal to the capsid, whereas other TerL ortholog sequences. A logo diagram (46) was made from a se- the nuclease domains interact with portal (Fig. S4E), an inter- quence alignment of 70 TerL proteins. The residue numbering is shown for action that has been observed recently (10). Thus, our revised TerL . P74-26 TerL ring model is a different arrangement than that previously proposed. We examined whether our structural model is globally con- We verified that the R139A mutation does not perturb sistent with a molecular envelope, as calculated from small-angle ATPase domain structure by solving the 2.5-Å resolution struc- X-ray scattering (SAXS) data. Whereas WT TerLP74-26 exhibits ture of the isolated ATPase domain with the R139A mutation. slight aggregation, we obtained high-quality SAXS data by This structure is essentially identical to the WT structure (Cα exploiting the R104E-TerL mutant, a more soluble variant = C P74-26 rmsd 0.2 Å; Fig. S3 ), suggesting the observed deficit is not than the WT protein (Fig. S5 A and B). R104E-TerLP74-26 is well due to structural changes in cis. In support of this finding, we folded and has a radius of gyration of 48.0 ± 2.0 Å (Fig. S5 B and observe that arginine or lysine is conserved at this position in the C), consistent with a pentameric assembly. We performed an

4of8 | www.pnas.org/cgi/doi/10.1073/pnas.1506951112 Hilbert et al. Downloaded by guest on October 1, 2021 PNAS PLUS several surface arginines in full-length TerLP74-26 and measured ABthe ability of these mutants to interact with DNA in the presence of ADP•BeF3 (Fig. 6E). Mutation of Arg101 causes loss of DNA binding, whereas mutation of residues distal to the pore (Arg39 and Arg58) has no measurable effect (Fig. 6F). Although a defect in binding ATP could cause loss of DNA binding, the R101E mutant retains WT levels of ATPase activity (Fig. S5D), indicating that the DNA-binding defect is not due to loss of ATP

Fig. 5. Derivation of the TerLP74-26 ring assembly structure. (A) Model of the TerLP74-26 ATPase ring derived from symmetry-constrained docking. A bot- tom-up view shows each of the five subunits in different colors, with the ATP ligand in green. The arginine finger and the conserved basic site (Arg101)

are shown as colored spheres. (B) Side view of the TerLP74-26 ATPase ring derived from symmetry-constrained docking. Colors and the image are BIOCHEMISTRY produced as in A.(C) Schematic depiction of the TerL ring. The TerL ring is shown from the perspective of the pore, with the ring artificially “broken” and flattened on the page so that all subunits can be viewed simultaneously. Each subunit is colored as shown in other panels in this figure. The nuclease domains are depicted as translucent squares to represent the ambiguity of the placement of the nuclease domain within the TerL ring. The loops protruding from the sides of each subunit represent the conserved basic patch

(Arg101 in TerLP74-26). The gamma-phosphate of ATP is contacted by the arginine finger from a neighboring subunit.

ab initio calculation of the TerLP74-26 molecular envelope using our SAXS data and imposing fivefold symmetry. The calculated molecular envelope fits the scattering data well (χ2 = 0.79) and is roughly shaped like a bowl, with a pore in the center and a V-shaped cross-section. The overall bowl-like shape of the TerLP74-26 ring model fits the SAXS reconstruction well; the

ATPase domains form the V-shaped base, and the nuclease Fig. 6. Validation of the TerL ring assembly model. (A) Model of TerLP74-26 domains form the bowl’s lip (Fig. 6A). We note that extra density fits the ab initio SAXS envelope. The modeled TerLP74-26 ring structure (col- is present between the nuclease domains, possibly indicating ored cartoon) is superposed onto the SAXS envelope calculated using GAS- conformational heterogeneity in the bowl’s lip or a poor fit of BOR (47) (imposing fivefold symmetry). Dummy atoms for the SAXS our nuclease domain homology model. Regardless, our model envelope are shown as gray spheres. (B) Hydrophobic patch on the lid subdomain mediates critical intersubunit interactions. The structural model is broadly consistent with reconstructions from both SAXS suggests that Trp231 and Tyr238 of the lid hydrophobic patch (ball-and-stick and cryo-EM. representation) mediate critical interactions with a neighboring subunit for

We tested various aspects of our structural model to de- positioning the arginine finger. (C)TerLP74-26 lid hydrophobic patch is critical termine whether it has predictive power. Our model predicts that for TerL function. Steady-state ATPase activity is shown for 0.5 μM TerL. the lid’s hydrophobic patch (Trp231 and Tyr238) contacts an Mutation of either W231 or Y238 results in a large decrease in ATPase ac- adjacent subunit for arginine finger positioning (Fig. 6B). To test tivity. Error bars are the SD from at least three replicates. (D) Electrostatic map the hypothesis that these residues are important for ATPase of the docked TerLP74-26 ATPase ring, with positive and negative surface po- tentials shown in blue and red, respectively. Note the positive charge lining the activity, we individually mutated Trp231 and Tyr238 to ala- pore of the TerLP74-26 ATPase ring. The electrostatic surface was calculated nine. Mutation at each of these residues results in severe loss using the APBS plug-in for PyMOL (DeLano Scientific) (48). Figure is colored C ’ of ATPase activity (Fig. 6 ). Thus, the lid s hydrophobic patch is by electrostatic potential (kbT/ec) as indicated. (E) DNA binding for multiple critical for TerL activity. arginine mutants. EMSA was carried out as in Fig. S1F, including several As an additional test of our model’s predictive power, we used arginine mutants. Arg101 is required for DNA binding, whereas Arg39 and our model to identify the DNA-binding motif. Basic residues line Arg58 are dispensable. (F) Mutation of the conserved pore arginine abro- the pore of the TerL ring, with a central pore residue (Arg101) gates DNA binding. The ability of three different arginine mutants to bind D C DNA tightly in the EMSA assay was mapped onto the model of the TerLP74-26 that is conserved (Fig. 6 and Fig. S2 ). A positive electrostatic ring. As predicted by the structural model, Arg101 is necessary for DNA environment in the pore is similar to other ASCE nucleic acid binding and mutations distal to the pore (R39A and R58A) have no mea- translocases (12, 13, 15). We hypothesize that Arg101 binds surable effect on DNA binding. A 100-bp DNA ladder (New England BioLabs) DNA during packaging. To test this hypothesis, we mutated is used as a standard.

Hilbert et al. PNAS Early Edition | 5of8 Downloaded by guest on October 1, 2021 binding. Thus, we have identified the critical DNA-binding motif relative to the docked model because the docked model exhibits in TerL, supporting our structural model for the TerL ring. contacts that are clearly too close with several overlapping atoms, which would artificially constrict the ring. Furthermore, Discussion our model positions the nuclease domain based on the full- Our results suggest a substantially different organization of the TerL length T4-TerL structure, which crystallized as a monomer (8). ring than the initial model that was proposed previously in a The nuclease domain position may be ortholog-dependent or ground-breaking study of the T4 terminase. The previous model, may be altered upon ring formation and/or upon portal binding. which is based on fitting the T4-TerL crystal structure to a cryo-EM Nuclease position is important because the nuclease domain reconstruction with a resolution of 34 Å, proposed that the TerL may be playing a key role in pentamer assembly, considering that ATPase domains form a ring that contacts portal through the lid the isolated TerLP74-26 ATPase domain crystallizes as a monomer. subdomains (8). The pore of the ATPase ring has a net negative Although ongoing and future investigations will refine the details of charge and is lined by a portion of TerL that is unique to T4. The our structural model, we have established that it captures essential nuclease domain was proposed to form a ring distal to portal that grips aspects of the TerL ring through its consistency with prior data, as and translocates DNA through the pore. Because the ATPase active well as its predictive power. Our structural model, combined with sites do not contact a neighboring subunit, Sun et al. (8) proposed our nucleotide-dependent structural changes, allows us to derive a that an invariant arginine in the P-loop (Arg162 in T4 TerL, Arg39 preliminary mechanistic model for DNA translocation in this family. in TerLP74-26) acts as the arginine finger to catalyze ATP hydrolysis. Based on our TerL ring model and the observed conforma- trans Our identification of the conserved -acting arginine finger tional changes upon ATP hydrolysis and release, we propose a indicates that the previous model requires significant modifica- mechanistic model for DNA translocation. During genome cis tion. Although the P-loop arginine previously proposed as the - packaging, DNA is gripped in the center of the TerL ring. In this acting arginine finger is necessary for packaging (42) and ATPase model, it is assumed that the TerL ring hydrolyzes ATP one A activity (8) (Fig. 4 ), our data do not support the hypothesis that it subunit at a time, and not in a concerted all-or-none mechanism. is the arginine finger. Instead, we propose that this residue, Arg39 in TerLP74-26, is conceptually analogous to the sensor II arginine found in the family of ATPases known as the ATPases associated with diverse cellular activities (AAA+), which aids ATP hydrolysis A in cis and confers movement of the AAA+ lid subdomain upon ATP hydrolysis (11, 31). In order for the previous model to ac- commodate the trans-acting arginine finger, a rotation of the ATPase domain of ∼110° is necessary. Although formally possible, this rotation of the ATPase domain does not fit the cryo-EM density for the actively translocating motor (8) and would disrupt portal interactions. Therefore, we disfavor this model. Our docked model for the TerL ring satisfies the distance constraint imposed by the arginine finger and revises the orientation of TerL relative to portal. We propose that the nuclease domains form a radially arranged ring that is proximal to portal, whereas the ATPase ring is distal. Our model positions the lid subdomains at the interface between adjacent ATPase subunits, where they assist in positioning the arginine finger, as supported by our mutagenesis data (Fig. 6C). In the previously proposed model (8), the lid residues interact with portal (Fig. S5 E and F). However, because our sample lacks portal, the observed ATPase defects in lid mutants are not due to a disruption of portal interactions, but are consistent with our proposed role in TerL B assembly. Our updated model accounts for functional conservation across the TerL family. First, our model contains a largely basic patch lining the pore. Within this patch, we identify Arg101 as a critical component of the DNA-binding motif. With conservation of a basic residue at this position, our model is congruent with the terminase family. Notably, in the previously proposed structural model, the equivalent residue in T4-TerL is positioned distal to the pore and the nuclease domain was hypothesized to bind E F DNA (8) (Fig. S5 and ), both of which are inconsistent with Fig. 7. Proposed model for the mechanism of DNA translocation by TerL. our observations. Second, our identification of the arginine finger (A) TerL ring is shown as in Fig. 6F. The nuclease domains interact with the in TerL brings the mechanism of ATP hydrolysis into accord not portal complex (translucent gray rectangle) and possibly the procapsid (black only with the terminase family but also with the rest of the ASCE curve). DNA is not shown but interacts with TerL through the DNA in- family. Conservation of the DNA-binding and catalytic mecha- teraction motif (Arg101). Each subunit’s lid is bound tightly to the Rossmann nism therefore supports our updated structural model. fold of the adjacent subunit. (B) Upon ATP hydrolysis and release by the Although our model allows accurate predictions for regions of magenta subunit, the lid stays bound to the blue subunit and the Rossmann function, the overall model is qualitative in nature. As previously fold rotates 13° upward. To allow for this movement, the adjacent red mentioned, the ring dimensions are slightly constricted due to subunit must also move in concert with the magenta subunit. To represent that the second site of symmetry breaking is unknown, the other three the docking algorithm, with the pore’s smallest inner diameter ∼ ATPase domains are faded. After hydrolyzing ATP, the magenta subunit measuring 16 Å, as calculated from the Cβ positions of surface- releases DNA to the red subunit to translocate DNA upward through the exposed arginines lining the pore (we use the Cβ position because pore; into the pore of the portal complex; and, ultimately, inside the pro- it is rigidly fixed). Thus, the modeled pore is too small to accom- capsid. The release of DNA at each cycle by the ATP-hydrolyzing subunit modate dsDNA. We propose that the TerL ring is expanded allows for unidirectional DNA translocation.

6of8 | www.pnas.org/cgi/doi/10.1073/pnas.1506951112 Hilbert et al. Downloaded by guest on October 1, 2021 This assumption is consistent with the mechanism of ATP hy- ring structure. Alternatively, the TerL assembly may form an PNAS PLUS drolysis in other ringed ASCE ATPases (12, 13, 15, 43). When open lock washer shape that dynamically alternates which sub- one subunit hydrolyzes ATP, it undergoes a conformational units cap the ends of the lock washer, as has been proposed for rearrangement such that the lid pivots 13° around the Rossmann the ASCE helicase DnaB (12). Although our current model is B fold (Fig. 3 ). If we further assume that the lid remains bound to fivefold symmetrical, recent studies of ASCE family members the neighboring subunit throughout packaging, as seen in ho- illustrate that significant asymmetry exists during motor function mologs (44), the result would be the Rossmann fold pivoting (12, 13, 37). Future refinement of our structural model will outward and upward toward the capsid (Fig. 7 and Movie S2). identify how TerL asymmetry drives DNA translocation. We propose that the conformational change from one ATP hydrolysis event propagates to an adjacent subunit, sterically Materials and Methods exerting force on the adjacent subunit such that the two subunits Cloning, Expression, and Purification of TerLP74-26. The TerLP74-26 gene was move in concert. Because TerL only binds DNA tightly in the synthesized by Genscript Corporation and subcloned into a modified pET24a ATP-bound form (Fig. S1F), ATP hydrolysis at one subunit will vector using standard procedures. TerLP74-26 was recombinantly expressed in lead to that individual subunit releasing DNA. During a hydro- BLR-DE3 E. coli cells and purified by nickel affinity, ion exchange, and SEC lysis event, the ATP-hydrolyzing subunit loses its grip on DNA (details are provided in SI Materials and Methods). but initiates motion that is propagated to the adjacent ATP- bound subunit, which is still gripping DNA through interaction ATPase and DNA-Binding Assays. ATPase activity was measured using a with Arg101. The conformational change of the ATP-hydrolyz- standard coupled enzyme assay. DNA binding was measured by EMSA (details ing subunit results in an upward translocation motion of DNA at are provided in SI Materials and Methods). the adjacent subunit and resets the motor for unidirectional translocation. We estimate that this motion would translocate Crystallization, Diffraction Data Collection, SAXS, and Docking. SeMet-labeled,

DNA perpendicular to the plane of the ring by ∼8 Å per hy- apo, and ADP•BeF3-bound TerLP74-26 ATPase domain were crystallized with drolysis event, or about 2.4 bp. In addition, we predict that DNA the hanging drop vapor diffusion method in solutions containing buffered would rotate in the plane of the ring by ∼2.3° for each step. ammonium sulfate. Crystals were frozen in a cryogenic buffer containing Because our model is qualitative, our estimated step rotation and elevated respective precipitant concentrations supplemented with 30% (vol/vol) size should be viewed with reservation. However, these values ethylene glycol. Diffraction data were collected at Advanced Light Source (ALS) compare favorably with the 2.5-bp translocation (43) and ∼3.5° beamline 5.0.1, at Advanced Photon Source beamline 23-ID-B, at Brookhaven

rotation per step (45) measured for the phi29 motor at low National Laboratory beamline X25, or using a home source. SAXS data were BIOCHEMISTRY packaging force. Our proposed “lever-like” mechanism for obtained on filtered R104E-TerLP74-26 at the Structurally Integrated Biology force generation is in contrast to the previous model, wherein a for Life Sciences (SIBYLS) beamline at the ALS. The TerLP74-26 ATPase ring “spring-like” motion of DNA-bound nuclease domains trans- was modeled using M-ZDOCK (35) (details are provided in SI Materials locates DNA through the pore (8). Because several other and Methods). studies have evaluated DNA packaging in the context of the ACKNOWLEDGMENTS. We thank the Rhind, Rando, Schiffer, Royer, Ryder, previous TerL structural and mechanistic models, our work il- and Bolon laboratories for discussions and use of instrumentation. We thank lustrates that these studies should be reinterpreted within the beamline scientists at ALS sector 5 (Lawrence Berkeley National Laboratory), context of the updated TerL model. APS 23-ID-B (Argonne National Laboratory), and NSLS X25 (Brookhaven It is unclear how this conformational change will affect sub- National Laboratory) for technical support with X-ray diffraction data units further downstream than the two moving subunits. The collection, and Z. Maben and Dr. J. Birtley for assistance with data collection of the ADP•BeF3 cocrystals. This work was initiated by a grant from the breakage of symmetry at the ATP hydrolysis site necessitates at Worcester Foundation. B.A.K. is a Pew Scholar in the Biomedical Sciences, least one other site of symmetry breaking to maintain a closed supported by the Pew Charitable Trusts.

1. Rao VB, Feiss M (2008) The bacteriophage DNA packaging motor. Annu Rev Genet 14. Chen Z, Yang H, Pavletich NP (2008) Mechanism of homologous recombination from 42:647–681. the RecA-ssDNA/dsDNA structures. Nature 453(7194):489–494. 2. Smith DE, et al. (2001) The bacteriophage straight phi29 portal motor can package 15. Enemark EJ, Joshua-Tor L (2006) Mechanism of DNA translocation in a replicative DNA against a large internal force. Nature 413(6857):748–752. hexameric helicase. Nature 442(7100):270–275. 3. Evilevitch A, Castelnovo M, Knobler CM, Gelbart WM (2004) Measuring the force 16. Leffers G, Rao VB (2000) Biochemical characterization of an ATPase activity associated ejecting DNA from phage †. J Phys Chem B 108(21):6838–6843. with the large packaging subunit gp17 from bacteriophage T4. J Biol Chem 275(47): 4. Fuller DN, Raymer DM, Kottadiel VI, Rao VB, Smith DE (2007) Single phage T4 DNA 37127–37136. packaging motors exhibit large force generation, high velocity, and dynamic vari- 17. Gual A, Camacho AG, Alonso JC (2000) Functional analysis of the terminase large subunit, – ability. Proc Natl Acad Sci USA 104(43):16868–16873. G2P, of Bacillus subtilis bacteriophage SPP1. JBiolChem275(45):35311 35319. 5. Iyer LM, Makarova KS, Koonin EV, Aravind L (2004) Comparative genomics of the 18. Baumann RG, Black LW (2003) Isolation and characterization of T4 bacteriophage FtsK-HerA superfamily of pumping ATPases: Implications for the origins of chromo- gp17 terminase, a large subunit multimer with enhanced ATPase activity. J Biol Chem – some segregation, cell division and viral capsid packaging. Nucleic Acids Res 32(17): 278(7):4618 4627. 19. White JH, Richardson CC (1988) Gene 19 of bacteriophage T7. Overexpression, puri- 5260–5279. fication, and characterization of its product. J Biol Chem 263(5):2469–2476. 6. Burroughs AM, Iyer LM, Aravind L (2007) Comparative genomics and evolutionary 20. Zhao H, Christensen TE, Kamau YN, Tang L (2013) Structures of the phage Sf6 large trajectories of viral ATP dependent DNA-packaging systems. Genome Dyn 3:48–65. terminase provide new insights into DNA translocation and cleavage. Proc Natl Acad 7. Feiss M, Rao VB (2012) The bacteriophage DNA packaging machine. Adv Exp Med Biol Sci USA 110(20):8075–8080. 726:489–509. 21. Sun S, Kondabagil K, Gentz PM, Rossmann MG, Rao VB (2007) The structure of the 8. Sun S, et al. (2008) The structure of the phage T4 DNA packaging motor suggests a ATPase that powers DNA packaging into bacteriophage T4 procapsids. Mol Cell 25(6): mechanism dependent on electrostatic forces. Cell 135(7):1251–1262. 943–949. 9. Hegde S, Padilla-Sanchez V, Draper B, Rao VB (2012) Portal-large terminase in- 22. Guo P, Grimes S, Anderson D (1986) A defined system for in vitro packaging of DNA- teractions of the bacteriophage T4 DNA packaging machine implicate a molecular gp3 of the Bacillus subtilis bacteriophage phi 29. Proc Natl Acad Sci USA 83(10): – lever mechanism for coupling ATPase to DNA translocation. J Virol 86(8):4046 4057. 3505–3509. 10. Dixit AB, Ray K, Thomas JA, Black LW (2013) The C-terminal domain of the bacte- 23. Ibarra B, Valpuesta JM, Carrascosa JL (2001) Purification and functional character- riophage T4 terminase docks on the prohead portal clip region during DNA pack- ization of p16, the ATPase of the bacteriophage Phi29 packaging machinery. Nucleic aging. Virology 446(1-2):293–302. Acids Res 29(21):4264–4273. 11. Erzberger JP, Mott ML, Berger JM (2006) Structural basis for ATP-dependent DnaA 24. Nadal M, et al. (2010) Structure and inhibition of herpesvirus DNA packaging ter- assembly and replication-origin remodeling. Nat Struct Mol Biol 13(8):676–683. minase nuclease domain. Proc Natl Acad Sci USA 107(37):16078–16083. 12. Itsathitphaisarn O, Wing RA, Eliason WK, Wang J, Steitz TA (2012) The hexameric 25. Yu MX, Slater MR, Ackermann H-W (2006) Isolation and characterization of Thermus helicase DnaB adopts a nonplanar conformation during translocation. Cell 151(2): bacteriophages. Arch Virol 151(4):663–679. 267–277. 26. Bigay J, Deterre P, Pfister C, Chabre M (1987) Fluoride complexes of aluminium or 13. Thomsen ND, Berger JM (2009) Running in reverse: The structural basis for trans- beryllium act on G-proteins as reversibly bound analogues of the gamma phosphate location polarity in hexameric helicases. Cell 139(3):523–534. of GTP. EMBO J 6(10):2907–2913.

Hilbert et al. PNAS Early Edition | 7of8 Downloaded by guest on October 1, 2021 27. Kagawa R, Montgomery MG, Braig K, Leslie AG, Walker JE (2004) The structure of 44. Stinson BM, et al. (2013) Nucleotide binding and conformational switching in the bovine F1-ATPase inhibited by ADP and beryllium fluoride. EMBO J 23(14):2734–2744. hexameric ring of a AAA+ machine. Cell 153(3):628–639. 28. Hendrickson WA, Teeter MM (1981) Structure of the hydrophobic protein crambin de- 45. Liu S, et al. (2014) A viral packaging motor varies its DNA rotation and step size to termined directly from the anomalous scattering of sulphur. Nature 290(5802):107–113. preserve subunit coordination as the capsid fills. Cell 157(3):702–713. 29. Erzberger JP, Berger JM (2006) Evolutionary relationships and structural mechanisms 46. Schneider TD, Stephens RM (1990) Sequence logos: A new way to display consensus of AAA+ proteins. Annu Rev Biophys Biomol Struct 35:93–114. sequences. Nucleic Acids Res 18(20):6097–6100. 30. Glynn SE, Martin A, Nager AR, Baker TA, Sauer RT (2009) Structures of asymmetric 47. Svergun DI, Petoukhov MV, Koch MH (2001) Determination of domain structure of ClpX hexamers reveal nucleotide-dependent motions in a AAA+ protein-unfolding proteins from X-ray solution scattering. Biophys J 80(6):2946–2953. machine. Cell 139(4):744–756. 48. Baker NA, Sept D, Joseph S, Holst MJ, McCammon JA (2001) Electrostatics of nano- ’ 31. Kelch BA, Makino DL, O Donnell M, Kuriyan J (2011) How a DNA polymerase clamp systems: Application to microtubules and the ribosome. Proc Natl Acad Sci USA 98(18): – loader opens a sliding clamp. Science 334(6063):1675 1680. 10037–10041. 32. DeLaBarre B, Brunger AT (2003) Complete structure of p97/valosin-containing protein 49. Liu H, Naismith JH (2008) An efficient one-step site-directed deletion, insertion, single – reveals communication between nucleotide domains. Nat Struct Biol 10(10):856 863. and multiple-site plasmid mutagenesis protocol. BMC Biotechnol 8:91. 33. Wang J (2004) Nucleotide-dependent domain motions within rings of the RecA/AAA 50. Kornberg A, Pricer WE, Jr (1951) Enzymatic phosphorylation of adenosine and (+) superfamily. J Struct Biol 148(3):259–267. 2,6-diaminopurine riboside. J Biol Chem 193(2):481–495. 34. Cox JM, Abbott SN, Chitteni-Pattu S, Inman RB, Cox MM (2006) Complementation of 51. Lee TJ, Zhang H, Liang D, Guo P (2008) Strand and nucleotide-dependent ATPase one RecA protein point mutation by another. Evidence for trans catalysis of ATP activity of gp16 of bacterial virus phi29 DNA packaging motor. Virology 380(1):69–74. hydrolysis. J Biol Chem 281(18):12968–12975. 52. Zwart PH, et al. (2008) Automated structure solution with the PHENIX suite. Methods 35. Pierce B, Tong W, Weng Z (2005) M-ZDOCK: A grid-based approach for Cn symmetric Mol Biol 426:419–435. multimer docking. Bioinformatics 21(8):1472–1478. 53. Strong M, et al. (2006) Toward the structural genomics of complexes: Crystal structure 36. Pierce BG, et al. (2014) ZDOCK server: Interactive docking prediction of protein-pro- of a PE/PPE protein complex from Mycobacterium tuberculosis. Proc Natl Acad Sci USA tein complexes and symmetric multimers. Bioinformatics 30(12):1771–1773. 103(21):8060–8065. 37. Zhao M, et al. (2015) Mechanistic insights into the recycling machine of the SNARE 54. Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data. Methods Enzymol complex. Nature 518(7537):61–67. – 38. Bailey S, Eliason WK, Steitz TA (2007) Structure of hexameric DnaB helicase and its 276:307 326. complex with a domain of DnaG primase. Science 318(5849):459–463. 55. Emsley P, Cowtan K (2004) Coot: Model-building tools for molecular graphics. Acta – 39. Vafabakhsh R, et al. (2014) Single-molecule packaging initiation in real time by a viral Crystallogr D Biol Crystallogr 60(Pt 12 Pt 1):2126 2132. DNA packaging machine from bacteriophage T4. Proc Natl Acad Sci USA 111(42): 56. Adams PD, et al. (2010) PHENIX: A comprehensive Python-based system for macro- – 15096–15101. molecular structure solution. Acta Crystallogr D Biol Crystallogr 66(Pt 2):213 221. 40. Fujisawa H, Shibata H, Kato H (1991) Analysis of interactions among factors involved 57. Hura GL, et al. (2009) Robust, high-throughput solution structural analyses by small in the bacteriophage T3 DNA packaging reaction in a defined in vitro system. Virol- angle X-ray scattering (SAXS). Nat Methods 6(8):606–612. ogy 185(2):788–794. 58. Petoukhov MV, Konarev PV, Kikhney AG, Svergun DI (2007) ATSAS2.1—Towards 41. Yang Q, Catalano CE, Maluf NK (2009) Kinetic analysis of the genome packaging automated and web-supported small-angle scattering data analysis. J Appl Cryst reaction in bacteriophage lambda. Biochemistry 48(45):10705–10715. 40(Suppl):s223–s228. 42. Rao VB, Mitchell MS (2001) The N-terminal ATPase site in the large terminase protein gp17 59. Pettersen EF, et al. (2004) UCSF Chimera—A visualization system for exploratory re- is critically required for DNA packaging in bacteriophage T4. J Mol Biol 314(3):401–411. search and analysis. J Comput Chem 25(13):1605–1612. 43. Moffitt JR, et al. (2009) Intersubunit coordination in a homomeric ring ATPase. Nature 60. Zhang Y (2008) I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 457(7228):446–450. 9:40.

8of8 | www.pnas.org/cgi/doi/10.1073/pnas.1506951112 Hilbert et al. Downloaded by guest on October 1, 2021