Crystal Structure of the Helicase Domain from the Replicative Helicase-Primase of Bacteriophage T7

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provided by Elsevier - Publisher Connector Cell, Vol. 99, 167–177, October 15, 1999, Copyright 1999 by Cell Press Crystal Structure of the Helicase Domain from the Replicative Helicase-Primase of Bacteriophage T7

Michael R. Sawaya, Shenyuan Guo, Stanley Tabor, the bacteriophage T7 replication system (Debyser et al., Charles C. Richardson, and Tom Ellenberger* 1994; Lee et al., 1998; Park et al., 1998) where only four Department of Biological Chemistry proteins are required at the replication fork (Richardson, and Molecular Pharmacology 1983). These proteins are the DNA helicase-primase (en- Harvard Medical School coded by gene 4 of the phage), a single-stranded DNA- Boston, Massachusetts 02115 binding protein (encoded by gene 2.5), the DNA polymerase catalytic subunit (encoded by gene 5), and the processivity factor Escherichia coli thioredoxin. Summary Several high-resolution structures have been reported for monomeric/dimeric helicases that are distantly re- Helicases that unwind DNA at the replication fork are lated to the replicative T7 helicase. A common protein ring-shaped oligomeric enzymes that move along one architecture has been revealed by crystal structures of strand of a DNA duplex and catalyze the displacement two DNA helicases, Bacillus stearothermophilus PcrA of the complementary strand in a reaction that is cou- (Subramanya et al., 1996; Velankar et al., 1999) and E. pled to nucleotide hydrolysis. The helicase domain of coli Rep (Korolev et al., 1997), and an RNA helicase, the the replicative helicase-primase protein from bacte- hepatitis C virus NS3 protein (Yao et al., 1997; Cho et riophage T7 crystallized as a helical filament that re- al., 1998; Kim et al., 1998). These helicases consist of sembles the Escherichia coli RecA protein, an ATP- three or four subdomains linked by flexible hinges, giv- dependent DNA strand exchange factor. When viewed ing the appearance of a crab claw with one large and in projection along the helical axis of the crystals, six one small pincer. ATP binds in the cleft between pincers, protomers of the T7 helicase domain resemble the at the interface between two subdomains that are struc- hexameric rings seen in electron microscopic images turally similar to RecA (Story et al., 1992), an ATP-depen- of the intact T7 helicase-primase. Nucleotides bind dent DNA strand exchange factor. PcrA, Rep, and NS3 at the interface between pairs of adjacent subunits helicases belong to two large superfamilies that are where an arginine is near the ␥-phosphate of the nu- characterized by seven conserved helicase motifs (Gor- cleotide in trans. The bound nucleotide stabilizes the balenya and Koonin, 1993), which consist of residues folded conformation of a DNA-binding motif located located near the nucleotide-binding site and interdo- near the center of the ring. These and other observa- main cleft. The T7 helicase-primase and other hexameric tions suggest how conformational changes are cou- DNA helicases such as E. coli DnaB belong to another, pled to DNA unwinding activity. smaller family of proteins (Ilyina et al., 1992) that have only five motifs (H1, H1a, H2, H3, and H4), of which only two (H1, H2) are similar to the conserved motifs of the Introduction two large superfamilies. The approximate shape and arrangement of subunits The transient unraveling of duplex DNA or RNA by heli- in the T7 helicase-primase and several other hexameric cases is essential for the replication and repair of DNA, helicases have been revealed by electron microscopic transcription, RNA splicing, and other vital cellular pro- studies (Egelman, 1996). The T7 helicase-primase is a cesses (Wassarman and Steitz, 1991; Matson et al., hexamer that forms a topologically closed ring (Patel 1994; Baker and Bell, 1998). Genetic defects in helicases and Hingorani, 1993; Egelman et al., 1995; Notarnicola have been linked to several inherited diseases in hu- et al., 1995) that encircles one strand of duplex DNA (Yu mans, including xeroderma pigmentosum, Bloom’s syn- et al., 1996; Hacker and Johnson, 1997), whereas the drome, and Werner’s syndrome (Ellis, 1997). Despite other DNA strand is diverted to the outside of the protein their diverse biological roles, all helicases studied to ring (Brown and Romano, 1989; Ahnert and Patel, 1997; date unwind duplex DNA or RNA by a vectorial move- Hacker and Johnson, 1997). A direct interaction of the ment along one strand with the concomitant displace- helicase with both of the separating DNA strands is ment of the complementary strand, in a reaction coupled suggested by the strict requirement for single-stranded with the hydrolysis of nucleoside triphosphates. regions on both strands of the forked DNA substrate The DNA helicases that function in replication are inti- (Matson et al., 1983; Ahnert and Patel, 1997). These mately associated with other proteins of the replication findings are consistent with a strand exclusion model of fork. Together these proteins constitute a macromolecu- helicase action in which the ring-shaped protein moves lar machine that copies both strands of DNA with re- processively along the encircled DNA strand, displacing markable speed and accuracy. In addition to unwinding the complementary strand. duplex templates for DNA synthesis, the replicative heli- The T7 helicase-primase protein consists of two do- cases also coordinate the activities of other replication mains that can be separated by proteolytic cleavage proteins associated with the leading and lagging strands (Rosenberg et al., 1992; Bird et al., 1997). A three-dimen- of the replication fork. The cooperative interactions of sional reconstruction from electron microscopic images replication proteins have been studied extensively in of the hexameric T7 helicase-primase (Egelman et al., 1995) reveals two stacked rings, one small and one large, * To whom correspondence should be addressed (e-mail: tome@ which correspond to the primase and helicase domains, hms.harvard.edu). respectively (Washington et al., 1996). The isolated Cell 168

Table 1. Data Collection and Refinement Statistics Data Collection

Native HgϩSe Se dTTP dATP dTDP Resolution limit (A˚ ) 2.3 2.2 2.4 2.3 2.3 2.5

Rsym (%) 7.3 6.2 7.8 5.0 6.3 5.7

Rsym (%, last shell) 39.9 47.6 43.2 18.4 32.7 45.3 I/sigma (last shell) 4 3 6 7 10 3 Total observations 144,142 250,429 177,892 147,526 153,764 27,233 Unique reflections 13,523 16,120 12,533 13,707 13,072 10,706 Completeness (%) 97.8 99.5 99.6 98.0 96.1 96.5 Completeness (%, last shell) 99.9 99.9 100.0 97.1 100.0 99.6 Phase Determination

Nativea HgϩSe Se (reference)

Rcullis (%, 20–2.6 A˚ , acent/cent, iso) 0.98/0.98 0.87/0.83

Rcullis (%, 20–2.6 A˚ , anom) 0.66 Phasing power (20–2.6 A˚ , acent/cent) 0.54/0.45 0.89/0.68 No. of sites 7 1 Hg, 7 Se Mean overall figure of merit 0.510 Model Refinement Native dTTP dATP dTDP

Rfree (%, 20 A˚ -upper limit) 30.2 29.6 29.6 29.1

Rwork (%, 20 A˚ -upper limit) 24.2 24.8 24.7 24.4 PDB ID code 1CR0 1CR1 1CR2 1CR4 Model Refinement—dTTP rmsd B Values No. Res. Aver. B Bonds (A˚ ) Angles (Њ)(A˚ 2 bonded) Protein 248 32.1 0.014 2.0 2.0 Water 46 37.9 a The Se data set was treated as a reference for phasing calculations and the native data set treated as a derivative. See Experimental Procedures section.

N-terminal primase domain (residues 1–272) is mononucleotide-binding site at the interface of two RecA-like meric, and it catalyzes the template-directed synthesis domains is also a feature of the PcrA, Rep, and NS3 of short oligoribonucleotides that are used as primers for helicases, and it could be of universal importance for lagging strand synthesis by T7 DNA polymerase (Frick et the mechanism of DNA unwinding activity. A comparison al., 1998; Guo et al., 1999). Several C-terminal fragments of the T7 helicase domain with RecA and other structur- of the T7 helicase-primase have been identified that ally similar nucleotide hydrolases suggests possible form stable hexamers that have helicase activity at high mechanisms for coupling the energy of nucleotide hy- protein concentrations (Bird et al., 1997). A minimal C-ter- drolysis to a change in protein conformation that un- minal fragment of the T7 helicase-primase comprising winds DNA. all five of the conserved helicase motifs (residues 272– 566) is predominately monomeric and it forms oligomers only at high protein concentrations (Guo et al., 1999). Results and Discussion Although this minimal domain is defective as a helicase, it hydrolyzes nucleotides rapidly at high protein concen- The crystal structure of the T7 DNA helicase domain trations (Guo et al., 1999) and hydrolysis is further stimu- was determined by multiple isomorphous replacement lated by addition of DNA (S. G., S. T., and C. C. R., followed by model refinement with diffraction data ex- unpublished results). Furthermore, it binds tightly to the tending to 2.3 A˚ resolution (Table 1). Structures of the T7 DNA polymerase. As an initial step toward a high- protein complexed to 2Ј-deoxythymidine triphosphate resolution study of a hexameric helicase, we have deter- (dTTP), 2Ј-deoxyadenosine triphosphate (dATP), and mined a crystal structure of the C-terminal helicase do- 2Ј-deoxythymidine diphosphate (dTDP) are isomor- main (residues 272–566) from the T7 helicase-primase phous with the unliganded structure and they were re- in the presence and absence of nucleotide cofactors. fined by difference Fourier methods. The T7 helicase The structure of the T7 helicase domain and its inter- domain has a globular fold with mixed ␣, ␤ structure actions with neighboring subunits in the crystal resem- having dimensions of 30 A˚ by 38 A˚ by 46 A˚ . The T7 ble those of the RecA protein. A nucleotide binds in the helicase domain did not crystallize with the subunits crevice formed between adjacent subunits, where an packed in a closed ring structure analogous to that ob- arginine that is analogous to the arginine finger motif served in electron microscopic images of the intact heli- of GTPase-activating proteins could trans-activate the case-primase protein (Egelman et al., 1995). Instead, bound nucleotide for hydrolysis. This arrangement of a the six molecules in the crystallographic unit cell are T7 DNA Helicase 169

arranged along a 61 screw axis, creating a helical filament (pitch ϭ 86 A˚ per 6 subunits, outer radius ϭ 60 A˚ ) (Figure 1a). Although previously unobserved for T7 helicase-primase, the helical filament is quite similar to the filament observed in the crystal structure of E. coli RecA (pitch ϭ 82 A˚ per 6 subunits; outer radius ϭ 60 A˚ ) (Story et al., 1992). RecA’s strand exchange activity is thought to be catalyzed by the protein filament, although RecA can also form hexameric rings resembling those of the hexameric DNA helicases (Yu and Egelman, 1997).

Viewed in projection along the helical 61 axis of the crystals, the T7 helicase domain has the appearance of a 6-fold symmetric ring that is 120 A˚ in diameter with a central hole of 35 A˚ diameter (Figure 1b), dimensions that closely match the larger of the two stacked rings seen in an electron microscopic reconstruction of the intact T7 helicase-primase (Egelman et al., 1995). The subunit packing arrangement in the crystal structure of the T7 helicase domains is readily converted to a 6-fold symmetric ring by collapsing the filament onto a plane

perpendicular to the crystallographic 61 axis, followed by an 18Њ rotation and a 10 A˚ translation of each subunit. The resulting model of the ring-shaped helicase closely resembles the size and shape of the helicase domain of the helicase-primase reconstructed from electron microscopic images (Egelman et al., 1995). The packing interactions of subunits in the modeled ring involve most of the same residues in orientations slightly modified from those in the crystal structure. Moreover, energy minimization of the model relieves the few bad contacts present by simply readjusting side chain conformations. We conclude that the subunit interactions in the crystal structure are closely related to those that stabilize the ring-shaped helicase-primase. The core of the T7 helicase domain is a mononucleotide-binding fold that consists of a central, mostly parallel ␤ sheet with flanking helices (Figure 2a). The structure of the T7 helicase domain closely resembles that of RecA (122 C␣ atoms superimposed with an rmsd of 1.6 A˚ ) (Story

et al., 1992) and the F1 ATPase (Abrahams et al., 1994), confirming earlier predictions based on sequence ho- mology (Washington et al., 1996; Bird et al., 1998). Four of the five conserved helicase motifs of the DnaB family (Figure 2c) (Ilyina et al., 1992) are located at the C-terminal ends of adjacent ␤ strands of the T7 helicase Figure 1. Subunit Interactions of the T7 Helicase Domain domain (H1, H1a, H2, H3) and are implicated in nucleo- (a) The crystal packing arrangement of the T7 helicase domain, tide binding and hydrolysis (Figure 2b). Residues within motif H4 (purple) are implicated in the DNA binding activ- viewed perpendicular to the 61 screw axis (black line), shows the helical filament formed by 6 molecules (colored differently) in the ity of the T7 helicase-primase. Their location near the crystallographic unit cell. dTTP binds at the interface between sub- center of the hexamer is consistent with electron micro- units and it is represented here by van der Waals spheres. scopic images showing single-stranded DNA passing (b) The view parallel to 61 crystallographic axis (rotated 90Њ away through the central hole of the ring-shaped helicase- from the view shown in [a]) reveals the ring-like appearance of the primase (Figure 1b) (Egelman et al., 1995). Structural 6 molecules in the crystallographic unit cell. The filament has an outer diameter of approximately 120 A˚ with a central hole that is elements flanking either end of the central ␤ sheet of about 35 A˚ in diameter. The N-terminal helix (shaded gray) of each the T7 helicase domain (helices A, D1, D2, and D3) form subunit packs against the neighboring subunit of the hexamer, in a most of the subunit interface (Figures 1 and 2b). swapping arrangement. The conserved sequence motifs of the DnaB An ␣-helical segment spanning residues 545–553 pro- family of helicases (Figure 2c) are represented by the following col- trudes from the surface near the C-terminal end of the T7 ors: red (H1), yellow (H1a), green (H2), blue (H3), and purple (H4). helicase domain (Figures 1a and 2a). Residues 554–566 Residues that are disordered and not included in the crystallo- comprise an acidic C-terminal tail of the helicase that graphic model are represented by the black loops (boldface) at the center of the diagram. The bound dTTP is cradled by residues within is critical for its physical interaction with the T7 DNA motifs H1, H1a, H2, and H3. DNA-binding motif H4 is located near polymerase during replication. This protein–protein in- the central hole of the ring. The subunits enclosed by the box are teraction, which is required for phage growth, coordi- shown in more detail in Figure 2b. Figures 1, 2a, 2b, 4, and 5 were nates DNA unwinding activity with synthesis of the lead- created with the program SETOR (Evans, 1993). ing strand of the replication fork (Notarnicola et al., Cell 170

Figure 2. The Structure and Topology of the T7 Helicase Domain (a) The T7 helicase domain consists of a mononucleotide-binding fold that is remarkably similar to E. coli RecA. The helices and ␤ strands of the T7 helicase domain are labeled in correspondence with the structurally analogous elements of RecA. Helices G1 and G2 comprise motif H4 (purple), which is part of the DNA-binding surface of the helicase. This segment is disordered in the unliganded helicase, and it becomes folded when a nucleotide (dTTP) is bound to the nearby P loop (labeled) and motif H2, located at the N-terminal end of helix E. Solid black lines indicate segments that remain disordered in the crystal structure of the complex with nucleotide. These disordered loops include residues 429–438, 468–492, and 505–509. Residues 554–571 at the C terminus of the helicase domain are also disordered and they are not included in the model. (b) Interactions between neighboring subunits of the T7 helicase domain. The two subunits shown here correspond to the boxed subunits in Figure 1b. The interaction of helix A with helices D1, D2, and D3 of the neighboring subunit account for 80% (1500 A˚ 2) of the total contact surface. Several loops around the nucleotide-binding site account for the remaining contact surface between subunits. Arg522 is positioned for an interaction with the nucleotide bound by the neighboring subunit, analogous to the arginine finger of the GTPase activating proteins (see text). Disordered loops are not represented in this panel. (c) Sequence alignment of the DnaB family of helicases. The secondary structure of T7 helicase domain is superimposed above the aligned T7 DNA Helicase 171

1997). The very weak electron density in this area of the just as satisfactorily. The lack of base-specific interac- structure has the appearance of a pair of antiparallel tions is consistent with the ability of the T7 helicase to helices; however, this C-terminal segment could not be hydrolyze a variety of nucleotide substrates (Matson modeled with certainty and it was not included in the and Richardson, 1983; Patel et al., 1992). However, only final model. The protein surface around the disordered dTTP and to a lesser extent dATP hydrolysis supports C-terminal segment is also negatively charged (Figure efficient DNA unwinding activity by the helicase-primase 3), and it might contribute additional contacts that stabi- (S. T. and C. C. R., unpublished results; Hingorani and lize the interaction with the polymerase. Patel, 1996). The basis for the selective coupling of hydrolysis of dTTP to helicase activity in contrast to other The Nucleotide-Binding Site nucleotide cofactors is not revealed by the crystal The crystal structure of T7 helicase reveals a number structure. of protein interactions with the bound dTTP/dATP that Residues from helicase motif H1, also known as the suggest a mechanism of nucleotide hydrolysis. How- Walker A motif or phosphate-binding loop (P loop; Fig- ever, mechanistic proposals must be viewed with cau- ure 4), contact all three phosphates of the bound nucleo- tion because the divalent metal ions that are required for tide (Figure 5a). The ␣ and ␤ phosphates of the bound nucleotide hydrolysis (Kolodner and Richardson, 1977) nucleotide are hydrogen bonded to backbone nitrogens were not included in the crystallization conditions, and at the N-terminal end of helix C (Ser319 and Thr320). the packing interactions in the crystal deviate from those The P loop is located at the N-terminal end of helix C predicted to stabilize the protein hexamer in a closed where it wraps around the ␤ and ␥ phosphates and the ring. Nonetheless, some features of nucleotide binding highly conserved Lys318 contacts a nonbridging oxygen are conserved between T7 helicase and the distantly of the ␤-phosphate (Figures 2a and 5a). The importance related monomeric/dimeric helicases. Nucleotides bind of Lys318 in catalyzing hydrolysis is reflected by a 200- at the interface between subunits of the T7 helicase fold decrease in dTTPase activity and a complete loss (Figure 2b), analogous to the nucleotide-binding site of of helicase activity incurred by an alanine substitution the PcrA and Rep helicases, which is located between at this position (Patel et al., 1994; Washington et al., two RecA-like subdomains that bind ATP in a similar 1996). Similar effects were found for the double mutant orientation (Subramanya et al., 1996; Korolev et al., G317V, K318M (Notarnicola and Richardson, 1993). 1997). Furthermore, motifs H1, H1a, H2, and H3 of the Ser314 contacts the ␥-phosphate, as does His465 (Fig- T7 helicase domain superimpose well on motifs I, Ia, II, ure 5a). In the PcrA helicase a glutamine residue occu- and III of PcrA, Rep, and NS3 helicases (not shown) pies the position analogous to His465, and it contacts despite their different oligomeric state and distant evolu- the ␥-phosphate in the ternary complex with a nonhy- tionary relationship to the T7 helicase. Thus, the cata- drolyzable ATP analog and DNA (Velankar et al., 1999). lytic functions of residues within the nucleotide-binding Notably, glutamine occupies this position in the se- pocket of the T7 helicase are suggested by the known quences of other DnaB family helicases (Figure 2c). roles of structurally homologous residues of other nucle- Magnesium is required for efficient hydrolysis of nu- otide hydrolases. cleotides and the DNA unwinding activity of the T7 heli- Clear electron density for the bound nucleotide was case (Kolodner and Richardson, 1977) and other heli- seen after the crystals were equilibrated with dTTP or cases. In the crystal structure of the PcrA helicase dATP (Figure 4). Residues from the conserved helicase complexed to a nonhydrolyzable ATP analog (Velankar motifs H1, H1a, H2, and H3 (Ilyina et al., 1992) cradle et al., 1999), a magnesium ion is ligated by a threo- the bound nucleotide at the subunit interface (Figure nine corresponding to Ser319 of motif H1 of T7 helicase 5a), where most of the contacts are made by residues and by the highly conserved aspartic acid of motif H2 from motifs H1 and H2. These residues correspond to (Asp424 of T7 helicase; Figure 5a). A bound water mole- the strictly conserved nucleotide-binding motifs A and cule occupies the expected metal-binding site in the B present in all helicases (Gorbalenya and Koonin, 1993), crystal structure of the T7 helicase domain. The water as well as G proteins, motor proteins (myosin, kinesin, is hydrogen bonded to conserved residues of the nucle- dynein), and the F1 ATPase (Schulz, 1992; Vale, 1996). otide-binding site and to the nonbridging oxygens of The conserved residues within these motifs position and the ␤ and ␥ phosphates (Figure 4). A divalent metal activate the nucleotide cofactors for hydrolysis, as dis- bound to this site could position the scissle phosphate cussed below. Motifs H1a and H3 are less conserved for nucleophilic attack and stabilize the developing among the members of different helicase families, al- negative charge of the transition state. The proposed though they are located in similar positions within the involvement of Asp424 in metal ion ligation is consis- active sites of the T7 helicase domain and the PcrA tent with the finding that the D424N mutant of the T7 and Rep helicases. Arg504 and Tyr535 are conserved helicase-primase retains only 0.3% of the wild-type residues (Figure 2c) that sandwich the base of the bound dTTPase activity (Washington et al., 1996). nucleotide (Figure 4). These interactions would not The structural environment of motif H2 of the T7 heli- specify a particular base, and the complex with dATP case domain is more similar to that of the hexameric shows this pocket can accommodate other nucleotides RecA and the F1 ATPase than it is to the ATP-binding

sequences, and the conserved sequence motifs of the DnaB family are boxed. Sequences from the following genomes are shown: T7 bacteriophage, Escherichia coli, Bacillus stearothermophilus, Chlamydia trachomatis, Heliobacter pylori, Borrelia burgdorferi, Haemophilus influenzae, Mycoplasma genitalium, and T4 bacteriophage. Cell 172

sites of the monomeric/dimeric helicases PcrA, Rep, and NS3. A ␤ bulge in this region is formed by a cis- peptide bond in the T7 helicase (Figure 4), RecA, and

the F1 ATPase, whereas a structurally similar ␤ bulge is formed by a trans-peptide bond in PcrA, Rep, and NS3. Furthermore, helicase motif H2 of the monomeric/ dimeric helicases may perform an additional role not performed by motif H2 of the hexameric enzymes. In the monomeric/dimeric helicases this motif includes two conserved acidic residues in the sequence DExx (Gor- balenya and Koonin, 1993; Korolev et al., 1998). In addition to the metal-liganding aspartic acid described above, the conserved glutamic acid is thought to activate the attacking water molecule for nucleotide hydrolysis (Glu224 of PcrA [Subramanya et al., 1996]; Glu215 of Rep [Korolev et al., 1997]; Glu291 of NS3 [Cho et al., 1998]). An analogous glutamic acid is not present in the T7 helicase-primase and other DnaB family members. Instead, a glutamic acid from motif H1a (Glu343) located on an adjacent strand occupies an equivalent location, where it could activate water for nucleophilic attack of the ␥-phosphorous (Figure 5a). It has been proposed that the analogous residue of RecA (Glu188) assists in the activation of the water nucleophile for ATP hydrolysis (Story and Steitz, 1992).

A Flexible Joint between Helicase Subunits The subunit interface of the T7 helicase domain consists of two regions—a helix that is swapped between subunits, and several loops located near the nucleotide- binding site (Figure 2b). The N-terminal helix of each subunit packs against the neighboring subunit (residues 345–388), completing a bundle of four helices on the outer periphery of the filament. The packing interface is stabilized by hydrophobic and electrostatic interactions over the full length of helix A. These interactions comprise most of the total contact surface between monomers (1500 A˚ 2 of buried surface per monomer). The swapped helix is linked to the rest of the helicase domain by an extended and presumably flexible segment of three residues (Figure 2a). Like helix A, the primase domain located immediately N-terminal to helix A might pack most intimately against a neighboring subunit of the hexameric helicase-primase, instead of packing against the same subunit that it is covalently linked to. This intertwining or swapping of adjacent domains has

Figure 3. Electrostatic Surface Potential of the T7 Helicase Domain Three views of the solvent-accessible surface of the T7 helicase domain, showing the electrostatic surface potential with positively charged regions colored blue and negatively charged regions colored red. (a) shows the N-terminal face of the T7 helicase domain that is expected to pack against the N-terminal primase domain of the primase-helicase protein. Positively charged regions around the DNA-binding motif H4 (colored purple in Figure 1b) are evident at the center of the ring. dTTP (yellow) binds in a crevice between subunits of the helicase. (b) is rotated 90Њ about a horizontal axis in comparison to (a). The C-terminal face of the helicase domain (c) is negatively charged. An acidic segment comprising 18 residues at the C terminus of the protein is required for its interaction with T7 DNA polymerase (Notarnicola et al., 1997). However, this acidic tail is disordered in the crystal structure, and it is not included in the electrostatic surface shown. This figure was prepared with the program GRASP (Nicholls et al., 1991). T7 DNA Helicase 173

means of communicating this change from one subunit to the other. The paucity of contacts near the nucleotide- binding site might allow the subunits to reorient within the hexameric ring by a hinge-like motion about the flexible joint associated with the N-terminal helix. Varia- tion in subunit packing is also suggested by analogy

with F1 ATPase. The two enzymes share not only the same structural core, but also certain aspects of their kinetic pathways (Hingorani and Patel, 1996). Thus, the subunits of T7 helicase domain might shift in response to nucleotide binding and hydrolysis in a cyclic manner

as observed for F1 ATPase. The means of coupling hydrolysis to a rotation of subunits could be provided by Arg522. Arg522 resides on a loop at the subunit interface (Figure 2b). Although this side chain is characterized by high temperature factors, its guanido group appears 4 A˚ from the ␥-phosphate of dATP bound to the adjacent subunit (Figure 5a). A small adjustment of Arg522 side chain would easily allow hydrogen bond formation with the nucleotide. This trans- interaction is analogous to the “arginine finger” of GTPase-activating proteins (GAPs), which assists GTP hydrolysis by contacting the scissile phosphate (Figure Figure 4. The Nucleotide-Binding Site 5b) (Wittinghofer, 1998). The importance of Arg522 in

Electron density calculated from the final difference Fourier (2Fobs Ϫ enzyme activity is also suggested by analogy to Arg373

Fcalc) reveals the outline of the bound dATP (purple) and surrounding of the F1 ATPase. Arg373 of the F1 ␣ subunit superim- residues (orange). The nucleotide base stacks between Tyr535 and poses structurally on Arg522 of T7 helicase domain, and Arg504 side chains. This pocket can accommodate dATP or dTTP it similarly contacts the scissile phosphate of ATP bound (not shown) equally well. In both complexes, density for the base and sugar is relatively weak compared to the density of the triphosphate to the ␤ subunit (Abrahams et al., 1994). Mutation of moiety. The phosphates are anchored by residues of helicase motif Arg373 markedly decreases the rate of nucleotide hy- H1 (N terminus of helix C and P loop). The cis-peptide bond is shown drolysis by the F1 ATPase (Leslie et al., 1999). between Asp424 and His425 of motif H2. These residues form a ␤ bulge in the strand, analogous to F1 ATPase and RecA. Conformational Coupling of Nucleotide and DNA-Binding Sites Protein conformational changes are the primary means been observed in a number of oligomeric proteins (Ben- by which helicases transduce the energy of nucleotide nett et al., 1995). Biochemical evidence that the stability hydrolysis into a motion that unwinds DNA (Yong and of the T7 helicase hexamer is strongly dependent on a Romano, 1995; Lohman and Bjornson, 1996; Velankar segment preceding helix A (particularly residues 242– et al., 1999). Conformational changes in T7 helicase- 271) (Guo et al., 1999) suggests that at least this portion primase are evidenced by differences in its proteolytic of the primase domain packs against the neighboring sensitivity and DNA footprint in response to nucleotide subunit and is proximal to helix A. The second, smaller binding (Yong and Romano, 1995). Comparison of crys- component of the subunit interface is a series of loops tal structures of T7 helicase domain offer some evidence near the nucleotide-binding site at the center of the of conformational changes occurring upon nucleotide filament (Figures 1 and 2a). The contacts involving these binding in the crystal. loops account for less than 20% of the buried interface Upon binding to dTTP (or dTDP), a region around motif between subunits. H4 of the T7 helicase domain folds into a helical structure The structure of the subunit interface offers clues to (Figure 2a) that appears as a region of positive electron understanding the mechanism of helicase cooperativity. density in difference Fourier maps calculated from the Cooperative behavior between subunits is manifested nucleotide-bound and unliganded structures. Ten resi- in several aspects of helicase mechanism. For example, dues from motif H4 (483–492) become sufficiently well the addition of nucleotide (or nucleotide analogs) pro- ordered to be included in the structural model of the motes hexamer formation under dilute conditions other- protein complexed to nucleotide (Figures 1b and 2a). wise favoring protein monomers and dimers (Patel and These additional residues form a 310 helix that covers a Hingorani, 1993; Notarnicola et al., 1995). Cooperativity small hydrophobic patch on the central ␤ sheet near the is also manifested in both ATP hydrolysis (Patel et al., center of the filament. Two residues within this newly 1992) and nucleotide binding (Hingorani and Patel, 1996), folded segment (R487 and G488) have been implicated as well as the cooperative inhibition caused by the addition in the DNA binding activity of the helicase-primase pro- of a catalytically inactive T7 helicase-primase to the wild- tein (Washington et al., 1996). In the unliganded struc- type enzyme (Notarnicola and Richardson, 1993). ture, residues 468–492 are disordered, suggesting that

Examination of the subunit interface suggests two the 310 helix might move or undergo cycles of folding elements of a cooperative mechanism: the ability to ef- and unfolding in response to nucleotide binding and fect a conformational change between subunits, and a release, perhaps acting as a switch that controls DNA Cell 174

Figure 5. Comparison to p21Ras Suggests a Switching Mechanism for T7 Helicase (a) Conserved residues from the helicase motifs (colored as in Figure 1b) contact the phosphates of the bound dATP. The secondary structures are labeled as for Figure 2a. In the crystal structure a water molecule occupies the site where a divalent metal ion is expected to bind. Glu343 (H1A) is proposed to activate a water molecule for attack of the ␥-phosphate. His465 (H3) might act as the ␥-phosphate sensor and propagate conformational changes from the nucleotide-binding site to residues in the DNA-binding motif H4 (see Figure 2a). Arg522 from a neighboring monomer is in close proximity to the ␥-phosphate of dATP in a manner analogous to the “arginine finger” of the Ras GTPase–activating protein shown in (b) (see Figure 2b).

(b) Structure of p21Ras complexed to p120GAP and the transition state analog GDP-AlF3 (Scheffzek et al., 1997). Conserved sequence motifs of the G protein family are colored according to the key provided. The orientation of the bound nucleotide in the p21Ras structure is similar to that of dATP in the T7 helicase domain (a). Gln61 of the switch II loop is proposed to activate water for nucleotide hydrolysis and to sense the presence of the ␥-phosphate. Arg789 from the p120GAP protein donates an “arginine finger” to the active site of p21Ras, accelerating the rate of GTP hydrolysis. Arg522 could serve this role in the T7 helicase. binding affinity. This region of positive electrostatic po- T7 helicase, His465 of motif H3 is in position to act as tential is located on the inside surface of the filament ␥-phosphate sensor or conformational switch by form- (Figure 3) where it could form favorable interactions with ing a hydrogen bond with the ␥-phosphate of dATP or DNA passing through the center of the ring (Yu et al., dTTP (Figure 5a). The H3 motif is connected by a short 1996; Ahnert and Patel, 1997; Hacker and Johnson, loop to motif H4, which is part of the DNA-binding sur- 1997). face of the helicase (Washington et al., 1996). Conforma- The P loop also becomes more ordered in the nucleo- tional switching has also been proposed to effect the tide-bound complex than in the unliganded protein, as strand exchange activity of RecA (Story and Steitz, 1992) evidenced by a 20 A˚ 2 drop in the average temperature and the helicase activity of PcrA (Subramanya et al., factor of this segment in the complexes with a nucleotide 1996) and NS3 (Yao et al., 1997). cofactor. In the nucleotide complex structure, additional weak electron density extends from the last ordered residue of H4 (residue 483) toward the P loop. This unas- A Mechanism of Unwinding DNA signed electron density might correspond to a segment Two general models, the inchworm and active rolling of the H4 motif that directly contacts the P loop, a con- models, have been proposed for the mechanism of heli- nection that would physically link the DNA-binding sur- case-catalyzed unwinding of double-stranded nucleic face of the helicase to the nucleotide-binding site. acid (reviewed by Lohman and Bjornson, 1996; Bird et Conformational changes in the DNA-binding surface al., 1998). The inchworm-like mechanism proposed for might be augmented by a change in the conforma- PcrA (Velankar et al., 1999) could be employed by T7 tion of the His465 side chain associated with nucleotide helicase, in which a pair of adjacent RecA-like domains hydrolysis. His465 is implicated by analogy with the in the hexamer interacts with DNA substrate just like switching mechanism of G proteins. In this mechanism, the pair of RecA-like domains within the PcrA monomer. the loss of the ␥-phosphate following hydrolysis of a The similar juxtaposition of RecA domains in the T7 nucleoside triphosphate is detected through hydrogen helicase and PcrA lends support to this analogy; how- bonding interactions with nearby residues located on ever, the DNA-binding motif H4 of the T7 helicase do- two loops, called switch I and switch II (Figure 5b). These main is located in a different region than the DNA-bind- loops then splay wide apart in the GDP-bound state. In ing surface of PcrA. Thus, the means of coupling T7 DNA Helicase 175

conformational changes to the nucleotide-binding site parameters were refined using MLPHARE (CCP4, 1994), and initial would be different for the two helicases. phases were calculated from the isomorphous and anomalous dif- A modified form of the active rolling model proposed ferences for this derivative. The 7 sites of selenium substitution were then located by difference Fourier calculations. In the final phasing by analogy to F1 ATPase is also plausible (Hingorani et calculations, the selenomethionine derivative lacking mercury was al., 1997). As described above, the T7 helicase domain treated as the reference data set and the native data were included and the F1 ATPase have similar RecA-like folds, including with the 7 selenium sites given negative occupancies (Table 1). The a cis-peptide bond located in motif H2 (Figure 4), and experimental electron density map was further improved by density shared aspects of their kinetic pathways for nucleotide modification using the solvent-flattening algorithm in DM (reflection hydrolysis (Hingorani et al., 1997). It is notable that motif omit mode) (CCP4, 1994). Model building and adjustments were done with the graphics program TOM (Jones, 1985), first building H4 (DNA-binding motif) corresponds in sequence and into the solvent-flattened experimental electron density, and later location to a segment of the F1 ATPase that contacts incorporating model phase information weighted by the SIGMAA the ␥ subunit located at the center of the ␣3␤3 hexamer parameter (CCP4, 1994). The models were initially refined by torsion (Abrahams et al., 1994). Further insights into helicase angle molecular dynamics using the maximum likelihood target for mechanism await a crystal structure of the hexameric structure factor amplitudes in the program CNS (Bru¨ nger et al., enzyme complexed to a DNA substrate. 1998). After several rounds of manual rebuilding and model refinement by torsion angle molecular dynamics, it was recognized that the structure factors calculated from the model and the observed Experimental Procedures structure factor amplitudes did not scale together well at low resolution, even after the applying the solvent-scattering correction in Protein Expression and Purification the CNS program. We therefore applied a resolution-dependent bin The T7 helicase domain (residues 272–571) was overproduced as scaling of Fcalc and Fobs (Trikha et al., 1999) that better fit the model described (Guo et al., 1999). After sonication, the cell lysate was structure factors over the full range of resolution. After appropriate cleared by centrifugation and the T7 helicase domain was precipi- scaling of the diffraction data, the model was further refined by tated by the addition of polyethylene glycol 4000 (17.5% w/v). The conjugate gradient minimization using the programs TNT (Tronrud, precipitated protein was collected by centrifugation, resuspended, 1997) and CNS. The final working and free R factors, as well as PDB and applied to a DE52 anion exchange column that was developed ID codes, are given in Table 1. with a gradient of NaCl. The appropriate fractions from the DE52 chromatography step were applied to a Superdex 200 gel filtration column, followed by MonoQ anion exchange chromatography. This Acknowledgments procedure typically yielded 10–15 mg of homogeneous T7 helicase domain from 6 liters of bacterial culture. The final purity was greater We thank Tom Hollis, Hyock Joo Kwon, Albert Lau, and Dane Walther than 98% based on the appearance of a Coomassie blue–stained for their assistance with x-ray diffraction experiments, and the staff sample analyzed by SDS-PAGE. Selenomethionyl protein was pro- of the Macromolecular Diffraction Facility (MacCHESS) at the Cor- duced according to the procedure outlined in Van Duyne et al. (1993) nell High Energy Synchrotron Source (CHESS; Ithaca, NY) and the and purified by the procedures described above, except that MonoQ staff at beamline X-25 of the National Synchrotron Light Source chromatography was omitted from the purification. (NSLS; Upton, NY) for their advice with data collection. We thank David Filman for providing us with the bin scaling procedure, and Crystallization and Data Collection Edward Egelman, Dale Wigley, and Smita Patel for helpful com- Crystals of T7 helicase domain were grown by vapor diffusion in ments. Our work on the T7 helicase is funded by grants from the sitting drops at room temperature by adding equal volumes of a National Institutes of Health (T. E. and C. C. R.), the Giovanni Armen- protein solution and a reservoir solution. The native protein solution ise Foundation for Advanced Scientific Research, and the Harvard contained 25–35 mg mlϪ1 protein in 10 mM lithium sulfate, 20 mM Center for Structural Biology. M. R. S. is supported by a postdoctoral Tris (pH 7.5), and 5 mM DTT. The reservoir contained 1.4–1.6 M fellowship from the National Institutes of Health. ammonium sulfate and 100 mM ACES (pH 9.0). Hexagonal bipyrami- ˚ dal crystals belonging to space group P61 (a ϭ b ϭ 80.4 A,cϭ Received July 13, 1999; revised September 7, 1999. 86.1 A˚ ) appeared overnight and reached full size within a week. Crystals of the selenomethionyl-substituted helicase domain were References grown using a 20 mg mlϪ1 protein solution and a reservoir solution containing 1.7 M ammonium sulfate. Prior to freezing the crystals, Abrahams, J.P., Leslie, A.G., Lutter, R., and Walker, J.E. (1994). they were equilibrated in harvest solutions containing increasing Structure at 2.8 A˚ resolution of F -ATPase from bovine heart mito- concentrations of glycerol, to a final concentration of 18% glycerol. 1 chondria. Nature 370, 621–628. After equilibration in this cryoprotective solution, crystals were flash- frozen in a nitrogen stream at Ϫ160ЊC. A mercury derivative was Ahnert, P., and Patel, S.S. (1997). Asymmetric interactions of hexa- prepared by soaking a crystal of the selenomethionine-substituted meric bacteriophage T7 DNA helicase with the 5Ј- and 3Ј-tails of T7 helicase domain in the final cryoprotectant with 0.1 mM ethylmer- the forked DNA substrate. J. Biol. Chem. 272, 32267–32273. cury thiosalicylate for 2 hr. Complexes with dTTP, dATP, and dTDP Baker, T.A., and Bell, S.P. (1998). Polymerases and the replisome: were prepared by soaking the crystals in 5 mM nucleotide in the machines within machines. Cell 92, 295–305. final cryosolvent condition for 8 hr. Diffraction data (Table 1) from Bennett, M.J., Schlunegger, M.P., and Eisenberg, D. (1995). 3D do- the native crystal were collected at beamline F1 (␭ϭ0.9161 A˚ ) main swapping: a mechanism for oligomer assembly. Protein Sci. of the Cornell High Energy Synchrotron Source (CHESS), and the 4, 2455–2468. selenomethionyl and mercury derivatives were collected at beamline Bird, L.E., Hakansson, K., Pan, H., and Wigley, D.B. (1997). Charac- ˚ A1 (␭ϭ0.9350 A), both using an Area Detector Systems Corporation terization and crystallization of the helicase domain of bacterio- 2K ϫ 2K CCD detector. Data for the nucleotide complexes were phage T7 gene 4 protein. Nucleic Acids Res. 25, 2620–2626. collected at beamline X-25 (␭ϭ1.100 A˚ ) of the National Synchrotron Bird, L.E., Subramanya, H.S., and Wigley, D.B. (1998). 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Protein Data Bank ID Codes

See Table 1.