Structural and Functional Domains of the Large Subunit of the Bacteriophage T3 DNA Packaging Enzyme: importance of the C-Terminal Region in Prohead Binding

JMB—MS 291 Cust. Ref. No. GO 34/94

[SGML] J. Mol. Biol. (1995) 245, 635–644

Structural and Functional Domains of the Large Subunit of the Bacteriophage T3 DNA Packaging Enzyme: importance of the C-Terminal Region in Prohead Binding Miyo Morita, Masao Tasaka and Hisao Fujisawa* Department of Botany Faculty of Science, Kyoto University, Kyoto 606, Japan

*Corresponding author

During head assembly of phage T3, DNA is packaged into a preformed protein shell, called the prohead, with the aid of non-capsid packaging proteins, the products of genes 18 and 19 (gp18 and gp19). We have developed a defined system, composed of purified gp18, gp19 and proheads for in vitro packaging of T3 DNA. Our previous results using the defined in vitro system indicate the sequential events in DNA packaging: the packaging proteins, gp18 and gp19, bind DNA and proheads, respectively. These complexes associate to form a direct precursor complexes for DNA translocation into the head. The formation of the precursor complexes requires ATP as an allosteric effector. Subsequent DNA translocation is driven by ATP hydrolysis. gp19 is an ATP binding protein that plays multiple roles in DNA packaging through interaction with ATP. gp19 changes its conformation by binding to ATP, as judged from the analysis of limited proteolysis. Sites cleaved by limited proteolysis were determined and mapped on the gp19 polypeptide (586 amino acid residues) to image the conformational change of gp19 induced by ATP. C-Terminal fragments generated by trypsin digestion bound the prohead and inhibited DNA packaging by intact gp19 in a competitive manner. On the other hand, N-terminal fragments did not bind the prohead nor did they inhibit DNA packaging. These results define a prohead binding domain at the C terminus of gp19. To identify the prohead binding domain more precisely, deletion mutants lacking the last 10 and 15 amino acids (gp19-DC10 and gp19-DC15, respectively) of the extreme C terminus of gp19 were constructed. Limited tryptic digestion patterns of these mutant proteins in the presence or absence of ATP were basically the same as those of gp19-wt, indicating that the conformation and its ATP response were not changed by these deletions. gp19-DC15 lacked prohead binding activity and, therefore, DNA packaging activity. gp19-DC10 had significant DNA packaging activity although it was reduced to one-tenth of that of gp19-wt. These results indicate that a C-terminal region of residues L571 to D576 of gp19 is crucial for prohead binding and that the last ten residues D577 to W586 of the C terminus seems to be important in stable binding of gp19 to the prohead. Keywords: T3 head assembly; packaging enzyme; the large subunit; prohead binding

Introduction During head assembly of most double-stranded (ds) DNA bacteriophages, DNA is packaged into the Abbreviations used: wt, wild type; ds, double-stranded; peq, phage equivalents; P.F.U., plaque forming units; DIFP, diisopropyl fluorophosphate; PTH, phenyl thiohydantoin. 0022–2836/95/050635–10 $08.00/0

cavity of a preformed protein shell, called a prohead, with the aid of non-capsid proteins, called terminases or packaging enzymes. DNA is synthesized as concatemers, in which unit-length molecules are joined together in a head-to-tail fashion through the terminally redundant sequences. During packaging of DNA, mature monomers are cut from the concatemer. DNA packaging is thought to proceed by a common 7 1995 Academic Press Limited

JMB—MS 291 636 mechanism for most dsDNA phages (Murialdo & Becker, 1978; Earnshaw & Casjens, 1980), and packaging proteins play a central role in DNA packaging for many phages (Feiss, 1986). Phage l terminase is a multimer of the products of gene Nu1 and gene A (gpNu1 and gpA, respectively). The N-terminal region of gpNu1 determines DNA binding specificity related to DNA packaging, its C terminus interacts with the N terminus of gpA, and the C terminus of gpA interacts with the prohead (Frackman et al., 1984, 1985). For f-29, a packaging protein, gp16, has been shown to bind to a connector structure located at the portal vertex of the prohead (Guo et al., 1987a). To elucidate the DNA translocation mechanism, we have developed a defined in vitro system for efficient packaging of mature phage T3 DNA (Hamada et al., 1986b). This system is composed of purified proheads, two packaging proteins gp18 and gp19, and mature T3 DNA. Analysis of DNA packaging in the defined in vitro system indicates that gp18 and gp19 form separate complexes with DNA and proheads, respectively (Shibata et al., 1987a). These complexes associate to form a direct precursor complex (50 S complex) for DNA translocation into the head. The formation of the gp19–prohead complex and the 50 S complex requires ATP as an allosteric effector (Shibata et al., 1987b). The DNA translocation is driven by hydrolysis of ATP. Actually the defined system displays an ATPase that is composed of DNA packaging-dependent and -independent ATPases (Morita et al., 1993). gp19 is the only component that interacts with ATP in the defined in vitro system (Hamada et al., 1987). gp19 has putative ATP binding consensus sequences and reveals multiple activities affected by interactions with ATP, which include specific and non-specific DNA cleavage, prohead binding, DNA translocation and ATP hydrolysis. Genetic and biochemical experiments have shown the localization of some of these functions on gp19 polypeptide (Morita et al., 1994). Our previous study showed that gp19 changes its conformation by binding to ATP, as judged from the analysis of limited proteolysis (Fujisawa et al., 1991). This conformational change is prerequisite for gp19 to form functional complexes with proheads, because proheads are inactivated by gp19 in the absence of ATP. gp19 binds to the portal vertex of a prohead at a molar ratio of 6 or 20 to 30 in the presence or absence of ATP, respectively (Fujisawa et al., 1991). gp18 binds linear DNA and is stimulative, but not essential, for DNA packaging in the defined in vitro system (Hamada et al., 1986b; Fujisawa et al., 1991). This finding suggests that the gp19–prohead complex is the main DNA packaging machinery. In the present paper, we have analyzed structural domains of gp19 by determining cleavage sites in limited proteolysis and have mapped the structural domains resistant to proteases on gp19

Prohead Binding Domain of Phage T3

Figure 1. Effect of ATP on the cleavage of gp19 by thermolysin or trypsin. Limited proteolysis of gp19 (2.5 mg) was performed with thermolysin (A) or trypsin (B) in 20 ml of pac buffer at 20°C for 30 min as described in Materials and Methods. (A) Lane 0, intact gp19; gp19 was exposed to 0.1 mg (lanes 1 and 4), 0.2 mg (lanes 2 and 5), 0.5 mg (lanes 3 and 6) of thermolysin in the absence (lanes 1 to 3) or presence (lanes 4 to 6) of ATP (50 mM). (B) gp19 was exposed to 0.1 mg (lanes 1 and 5), 0.2 mg (lanes 2 and 6), 0.5 mg (lanes 3 and 7), or 1.0 mg (lanes 4 and 8) of trypsin in the absence (lanes 1 to 4) or presence (lanes 5 to 8) of ATP. Samples were subjected to SDS/polyacrylamide gel electrophoresis. The positions of major fragments are indicated by arrowheads. Positions of molecular mass markers (in kDa) are indicated at the right.

polypeptide. A prohead binding domain is defined by functional analyses of proteolytic fragments and mutant proteins deleted for the C-terminal region. Based on these results, the structure–function relationship of gp19 in gp19–prohead complex formation and in DNA translocation will be discussed at a molecular level.

Results Analysis of structural domains of gp19 gp19 has ATP-binding consensus motifs (Kimura & Fujisawa, 1991) and requires ATP to form a functional complex with a prohead. In a previous paper (Fujisawa et al., 1991), we showed that cleavage patterns of gp19 by several proteases were altered by the addition of ATP, indicating that ATP induces a conformational change in gp19. To study the conformational change in gp19 further, sites cleaved by proteases were determined. As shown in Figure 1, digestion patterns of gp19 by thermolysin (A) or trypsin (B) were drastically changed by the addition of ATP. In the case of thermolysin, gp19 protein (66.7 kDa) was converted to major fragments with molecular masses of 34.0 kDa (Th) or of 59.6 kDa (ThA I) and 55.7 kDa (ThA II) in the absence or presence of ATP, respectively. With tryptic digestion,

JMB—MS 291 637

Prohead Binding Domain of Phage T3

Figure 2. Determination of the sites cleaved by limited proteolysis. The major proteolytic fragments were isolated from SDS/polyacrylamide gel, and the N-terminal 5 amino acid sequences were determined by the protein sequencer as described in Materials and Methods. Th or Tr fragments were generated by limited proteolysis with thermolysin or trypsin, respectively, in the absence of ATP. ThA I and ThA II fragments or TrA I and TrA II fragments were generated by limited proteolysis with thermolysin or trypsin, respectively, in the presence of ATP. Since two or three peaks of PTH-amino acids were detected in each cycle of sequence, the Th band was thought to be a mixture of three fragments with indicated N-terminal amino acid sequences. The orders of amino acid of each fragments were inferred from ratios of amounts of PTH-amino acids. The sequences shown at the top are the sequences of corresponding regions of gp19 and amino acid residues are numbered from the initiation M1 predicted from DNA sequences (Yamada et al., 1986). The C terminus of each peptide fragment was estimated by determining the molecular mass, and the approximate length of each fragment is indicated by a bar.

a major 37.5 kDa fragment (Tr) and two fragments with molecular masses of 57.0 kDa (TrA I) and 49.2 kDa (TrA II) were generated in the absence and presence of ATP, respectively. Proteolytic fragments, Th, ThA I, ThA II, Tr, TrA I and TrA II, were purified by SDS/polyacrylamide gel electrophoresis and five amino acid residues from their N termini were determined as described in Materials and Methods. These sequences were compared with the predicted amino acid sequence of gp19 (Figure 2). The Th band contained three peptides that were produced by cleavage at very close sites (M288, L289 and L293). The Tr fragment was produced by tryptic cleavage between K280 and A281. The N termini of ThA I and ThA II fragments were identical (S2). TrA I and TrA II fragments also had the same N terminus, N8. Observed cleavage sites of Th, Tr, TrA I and TrA II fragments were consistent with the recognition sites of thermolysin or trypsin. Although the N-terminal amino acid of ThA I and ThA II fragments was S2, a peptide bond of M–S is not a recognition site for thermolysin. The first M might be removed from nascent gp19 polypeptides as predicted by Dunn & Studier (1983). It should be noted that the thermolysin and trypsin cleavage sites were located at similar positions in gp19, in both the presence and absence of ATP. The C terminus of each peptide fragment was estimated by judging the fragment molecular mass (Figure 2). In the absence of ATP, the C-terminal half of gp19 appears to form a structure inaccessible to proteases while the N-terminal half is easily digested. On the other hand, in the presence of ATP, most of the gp19 appears to be protected from protease digestion, while about 100 amino acid residues of the C-terminal region are easily accessible to proteases.

Functional analysis of the tryptic fragments gp19 performs multiple functions through interaction with ATP. The first step of the DNA packaging process is ATP binding to gp19, which allows gp19 to form a functional complex with a prohead. As shown in Figure 2, either N-terminal or C-terminal peptide fragments of gp19 were obtained by limited proteolysis. To examine whether these fragments retain functions of gp19, tryptic digests were added to the defined in vitro system as described in Materials and Methods. Tryptic digests containing mainly TrA II or Tr fragments had a low packaging activity by itself, which could be due to the presence of undigested gp19 (Figure 3). When a tryptic digest of gp19 done in the absence of ATP, containing Tr fragments, was added to the defined in vitro system containing intact gp19, DNA packaging was inhibited in a competitive manner. On the other hand, a tryptic digest of gp19 done in the presence of ATP, containing TrA II fragments, did not affect DNA packaging in the defined in vitro system (Figure 3). The inhibitory effect of the former mixture suggests that the Tr fragments bind proheads to form abortive complexes. To examine whether the Tr fragments retained prohead binding activity, the tryptic digest was added to complete pac buffer containing proheads in the presence or absence of ATP and gp19–prohead complexes were isolated and subjected to SDS/polyacrylamide gel electrophoresis as described in Materials and Methods. As shown in Figure 4, the Tr fragments bound to the prohead in the presence or absence of ATP, but the TrA I and II fragments did not bind to the prohead at all (data not shown), even in a Western blotting experiment using anti-gp19 antiserum. These findings indicate that the Tr

JMB—MS 291 638

Prohead Binding Domain of Phage T3

(A)

Figure 3. Effects of tryptic fragments on DNA packaging activity in the defined in vitro system. gp19 (2.5 mg) was digested with trypsin (0.1 mg) in 20 ml of pac buffer in the presence (q, Q) or absence (w, W) of ATP at 20°C for 30 min, followed by the addition of soybean trypsin inhibitor (1 mg) to terminate the reaction. Subsequently, indicated amounts (ml) of the digests were added to the defined in vitro system (q, w) or the system from which gp19 was omitted (Q, W). The reaction mixtures were incubated at 30°C for 30 min and filled heads were assayed as described in Materials and Methods. P.F.U., plaqueforming units.

(B)

fragment has a region responsible for prohead binding. The C-terminal analysis of the Tr fragment Although the Tr fragment bound to a prohead, it

Figure 4. SDS/polyacrylamide gel electrophoresis of the Tr fragment–prohead complexes. gp19 (2.5 mg) was digested with trypsin (0.1 mg) in 20 ml of pac buffer in the absence of ATP at 20°C for 30 min, followed by the addition of soybean trypsin inhibitor (1 mg) to terminate the reaction. Proheads were incubated with the tryptic digest in complete pac buffer with or without ATP at 30°C for 30 min. The prohead fraction was separated and subjected to SDS/polyacrylamide gel electrophoresis as described in Materials and Methods. Lane 1, proheads; lane 2, intact gp19; lane 3, the prohead fraction incubated with the tryptic digest without ATP; lane 4, the prohead fraction incubated with the tryptic digest with ATP; lane 5, tryptic digest in the absence of ATP.

Figure 5. The C-terminal analysis of the Tr fragment. (A) gp19 (0.125 mg) was digested with trypsin (0.025 mg) in 1 ml of pac buffer in the absence of ATP at 20°C for 30 min, followed by the addition of a trypsin inhibitor (5 mM DIFP) to terminate the reaction. The mixture was applied to an anhydrotrypsin agarose column. A through fraction and a 5 mM HCl fraction were collected as described in Materials and Methods. Lane 1, the sample before application to column; lane 2, the through fraction; lane 3, the 5 mM HCl fraction. Positions of Tr fragment and molecular mass markers are indicated at the right. (B) Recognition sites of trypsin in the C-terminal region of gp19 are underlined. An arrowhead indicates the position of the C terminus of the Tr fragment as judged from its molecular mass (see the text).

remained to be determined whether the Tr fragment had an intact C terminus or not. To determine the C-terminal amino acid residue, tryptic gp19 digests were applied to an anhydrotrypsin agarose column as described in Materials and Methods. If a peptide has K or R at its C terminus that is generated by tryptic digestion, it adsorbs to the anhydrotrypsin agarose column (Kumazaki et al., 1987). Since the C-terminal amino acid residue of gp19 is W, it would pass through the column if the C terminus of the Tr fragment remained intact. While intact gp19 passes through the column (data not shown), the Tr fragment was adsorbed by the column and eluted with 5 mM HCl (Figure 5(A)). The results indicate that the Tr fragment has R or K as the C-terminal amino

JMB—MS 291 Prohead Binding Domain of Phage T3

639

Figure 6. The C-terminal amino acid sequences of C-terminal deletion mutant proteins and their DNA packaging activities in the defined in vitro system. The C-terminal amino acid sequences of intact gp19-wt and deletion mutant proteins (gp19-DC10 and gp19-DC15) are presented. Deletion mutations were introduced into gene 19 by oligonucleotide-directed mutagenesis and mutant proteins were purified as described in Materials and Methods. gp19-DC10 and gp19-DC15 deleted 10 and 15 amino acids in their C termini, respectively. The packaging activities of gp19-DC10 and gp19-DC15 were assayed by adding the proteins to the defined in vitro system in place of gp19-wt.

acid. R583 and K533 are the first and second recognition sites for trypsin from the C terminus, respectively (Figure 5B). The molecular masses of peptide fragments from A281 to K533 and to R583 are calculated to be 29.3 kDa and 34.4 kDa from the predicted amino acid sequence of gp19, respectively. The molecular mass of the Tr fragment was estimated to be 37.5 kDa by SDS/polyacrylamide gel electrophoresis analysis. Judging from these results, we conclude that R583 is likely the C-terminal amino acid of the Tr fragment. Construction and characterization of C-terminal deletion mutants of gp19 Since the Tr fragment retained prohead binding activity in spite of lacking the last four amino acid residues at its C terminus, a region from R583 to W586 appeared not to be necessary for gp19 to bind a prohead. To define a region involved in prohead binding, mutant proteins lacking the last 10 and 15 amino acid residues (gp19-DC10 and gp19-DC15, respectively) were constructed by oligonucleotidedirected mutagenesis (Figure 6) as described in Materials and Methods (and see Kunkel et al., 1987), and characterized.

To confirm the truncations in their C termini, gp19-DC15 and gp19-DC10 were subjected to limited tryptic digestion in the presence or absence of ATP. As shown in Figure 7, gp19-DC15 and gp19-DC10 generated the fragments corresponding to the Tr fragments of gp19-wt with expected molecular masses of 36.5 kDa and 36 kDa in the absence of ATP, respectively. The digestion patterns of deletion mutant proteins were almost the same as those of gp19-wt in both the presence and absence of ATP, indicating that the conformation of gp19-DC15 and gp19-DC10 and their interactions with ATP were not severely affected by the deletions of the C terminus. gp19-wt, gp19-DC15 or gp19-DC10 was added to the defined in vitro system to assay the DNA packaging activity. As shown in Figure 6, gp19DC15 had no DNA packaging activity. To characterize the defect in the DNA packaging activity of gp19-DC15, gp19–prohead complexes were isolated by sedimentation and subjected to SDS/polyacrylamide gel electrophoresis as described in Materials and Methods. Figure 8 shows that gp19-wt bound to a prohead at molar ratios of 6 or more than 20 in the presence or absence of ATP, respectively, as described in a previous paper (Fujisawa et al., 1991). On the other hand, gp19-DC15 was not detected in

Figure 7. Effect of ATP on the cleavage of gp19 by trypsin. gp19 (2.5 mg) was exposed in the presence (lanes 1 to 3) or absence (lanes 4 to 6) of ATP (50 mM) to 0.5 mg or 0.1 mg of trypsin, respectively, in 20 ml of pac buffer at 20°C for indicated times. Lane 0, 0 min; lanes 1 and 4, 10 min; lanes 2 and 5, 30 min; lanes 3 and 6, 60 min. Samples were subjected to SDS/polyacrylamide gel electrophoresis. Positions of molecular mass markers are indicated at the right.

JMB—MS 291 640

Prohead Binding Domain of Phage T3

Figure 8. Prohead binding activity of gp19-DC10 and gp19-DC15. After incubation of gp19s and proheads in complete pac buffer with or without ATP, the prohead fractions were separated and subjected to SDS/polyacrylamide gel electrophoresis as described in Materials and Methods. Lane 1, proheads; lane 2, gp19-wt; lanes 3 and 4, the prohead fraction was incubated with gp19-wt in the presence and absence of ATP, respectively; lanes 5 and 6, the prohead fraction was incubated with gp19-DC10 in the presence and absence of ATP, respectively; lanes 7 and 8, the prohead fraction was incubated with gp19-DC15 in the presence and absence of ATP, respectively.

the prohead fractions in the presence and absence of ATP (Figure 8), even by Western blotting using anti-gp19 antiserum (data not shown). There was a possibility, however, that the prohead binding activity of gp19-DC15 was so weak that the gp19–prohead complexes would dissociate during sedimentation. Therefore, competition experiments between gp19-wt and gp19-DC15 were carried out in the defined in vitro system (Figure 9(A)). gp19-DC15 did not inhibit DNA packaging activity of gp19-wt. When gp19-wt was preincubated with proheads in the absence of ATP, the prohead was inactivated by formation of inactive complexes with gp19 (Fujisawa et al., 1991). The preincubation of proheads with gp19-DC15 in the absence of ATP had no inhibitory effect on the DNA packaging activity of the prohead, in contrast to the case of gp19-wt (Figure 9(B)). These results indicate that gp19-DC15 is defective in prohead binding. gp19-DC10 was active in DNA packaging in the defined in vitro system, although the activity was 10% that of gp19-wt. gp19-DC10 bound the prohead in the absence of ATP to the same extent as gp19-wt, while its binding to the prohead was not significant in the presence of ATP (Figure 8). It is possible that gp19-DC10 might dissociate during sedimentation of gp19-DC10–prohead complexes. However, since gp19-DC10 had DNA packaging activity, it appears to be able to bind to the prohead, to interact with gp18–DNA complexes and to translocate DNA into the head in spite of lacking the last ten amino acid residues of its C terminus.

Figure 9. Effect of gp19-DC15 on DNA packaging activity. (A) DNA packaging reactions were performed in the defined in vitro system containing proheads (2 × 1010 peq), gp18 (20 pmol), gp19-wt (0.8 pmol) and indicated amounts (molecules) of gp19-DC15 per gp19-wt. After incubation of the reaction mixtures at 30°C for 30 min, filled heads were assayed as described in Materials and Methods. (B) Proheads (2 × 1010 peq) and indicated amounts (molecules) of gp19-wt (w, W) or gp19-DC15 (q, Q) per prohead were preincubated in 10 ml of complete pac buffer with (w, q) or without (W, Q) ATP. The gp19–prohead complex activities were assayed by a second reaction after diluting the preincubation mixtures 10-fold into complete pac buffer (20 ml) containing 50 mM of ATP, gp18 (20 pmol), gp19 (3 pmol) and T3DNA (1010 peq). After the second incubation at 30°C for 30 min, filled heads were assayed as described in Materials and Methods.

Discussion DNA packaging is thought to proceed by a common mechanism for most of the dsDNA phages (Murialdo & Becker, 1978; Earnshaw & Casjens, 1980). DNA is packaged into the prohead with the aid of a packaging enzyme or terminase. In general, the packaging enzyme is composed of two subunits: the small subunit has DNA binding activity and stimulates DNA packaging; the large subunit has prohead binding activity and appears to be critical for DNA packaging (f29: Guo et al., 1987b; l: Becker et al., 1977; T3: Shibata et al., 1987a). However, the role of the large subunits has not been discussed in the current models for the mechanism of DNA translocation into proheads (Hendrix, 1978; Serwer,

JMB—MS 291 Prohead Binding Domain of Phage T3

641

Figure 10. Functional domains of gp19. Three ATP binding consensus motifs are indicated as I, II and III. The importance of G61 and G429 in ATP interaction and in coupling ATP hydrolysis to DNA translocation, respectively, is described in a previous paper (Morita et al., 1994). Mapping of regions resistant to trypsin in the presence (open box) or absence (stippled box) of ATP and the importance of L571–D576 in prohead binding are described in the present paper.

1988; Turnquist et al., 1992). In previous papers (Shibata et al., 1987b; Morita et al., 1993), we have proposed a ratchet model for DNA translocation in which the gp19–connector complex located at the portal vertex would translocate DNA into the prohead by a cyclic change in the conformation of gp19 mediated by ATP. Ordered conformational changes in proteins that are driven by nucleotide triphosphate hydrolysis provide explanations for ATP-driven biological mechanisms (Alberts & Miyake-Lye, 1992). To elucidate the mechanism of DNA translocation, the function(s) of the large subunits has to be defined. For l, some information on the functional domains of terminase, gpNu1 and gpA, is accumulating through genetic studies using hybrid phages between l and a related phage 21. Sippy & Feiss (1992) have reported the accumulation of unused proheads in lysates of gpA amber mutant deleting the last five amino acids in the C terminus. However, direct interactions between truncated terminases and proheads have not been shown. A prohead binding domain of gp19 can be defined from the results of molecular studies presented in this paper. Structural domains of gp19 Previously, we have demonstrated by limited proteolysis experiments that ATP induces a drastic conformational change in gp19 (Fujisawa et al., 1991). ATP binds to a K residue located in ATP binding consensus sequences for several ATPases (Fry et al., 1986; Hinz & Kirley, 1990; Maruyama & MacLennan, 1988). We tentatively eliminated the possibility that tryptic digestion patterns are changed by ATP covering K residue(s) because the changes in the cleavage patterns of gp19 caused by ATP were obtained using several proteases with different specificities. In the present paper, we used thermolysin, which does not recognize a K residue as a target site and has a broad substrate

specificity. Patterns of thermolysin digestion of gp19 were altered by the addition of ATP. There were several common structural domains that were resistant to trypsin and thermolysin. We determined cleavage sites by amino acid sequencing of N termini of peptide fragments generated by digestion with trypsin or thermolysin to image the conformational change in gp19, in detail (Figure 2). Limited proteolysis with trypsin gave rise to major fragments with molecular masses of 37.5 kDa (Tr) and 42.9 kDa (TrA II) in the absence and presence of ATP, respectively. The N termini of the Tr and TrA II fragments were N281 and N8, respectively. The N termini of thermolysinresistant fragments mapped quite close to those of the tryptic fragments produced in the presence or absence of ATP, in spite of the different substrate specificities of trypsin and thermolysin. These results indicate that the accessibility of rather broad region(s), including cleavage sites to several proteases, are changed by a structural change induced by ATP. The terminal regions of proteins are generally located on the surface of the protein, accessible to solvent and flexible. Thornton & Sibanda (1983) suggested that in many globular proteins the terminal regions fulfil a structural role, stabilizing the tertiary or quaternary structure by providing links between domains or subunits. In the presence of ATP, about 100 amino acid residues of the C-terminal region containing a prohead binding domain of gp19 were digested by proteases, while the remaining region was not accessible to proteases. The C-terminal region of gp19 would be exposed on the surface of the protein and be flexible in the presence of ATP, ensuring functional interaction with the prohead. In the absence of ATP, however, this region would be not accessible to proteases, judging from the results of limited proteolysis. In contrast to the rigid C-terminal structure, the N-terminal half was much more accessible to protease attacks in the

JMB—MS 291 642 absence of ATP. These results are summarized in Figure 10. A prohead binding domain of gp19 One of the tryptic fragments, Tr, was mapped to the C-terminal half of gp19 and its C terminus was inferred to be R582 by measuring its molecular mass (Figure 5). The Tr fragment bound to the prohead and inhibited packaging activity of gp19-wt in a competitive manner (Figures 3 and 4). These results indicate that the N-terminal half and the last four amino acids in the C terminus of gp19 are not necessary for binding to a prohead. To define a region involved in prohead binding more precisely, deletions were introduced into the C-terminal region of gp19 by site-directed mutagenesis. gp19-DC15– prohead complexes could not be isolated and gp19-DC15 did not inhibit the packaging activity of gp19-wt at all, indicating that the defect of gp19-DC15 in DNA packaging is due to a defect in prohead binding. Although it is possible that the deletions would cause some local conformational changes that were not able to be detected by limited proteolysis experiments with trypsin, protease cleavage patterns of gp19-DC15 were basically identical to those of gp19-wt in the presence or absence of ATP (Figure 7). In addition, gp19-DC15 retained another function of gp19; non-specific endonuclease activity suppressed by ATP (unpublished data). These results indicate that gp19-DC15 is specifically defective in prohead binding activity. gp19-DC10 showed a significant packaging activity in the defined in vitro system in spite of a deletion of the last ten amino acid residues (Figure 6). gp19-DC10 bound to a prohead in the absence of ATP as efficiently as gp19-wt, while gp19-DC10–prohead complexes were not detected in the presence of ATP. gp19-DC10 might dissociate from the complexes during sedimentation. These results indicate that a restricted region from Y572 through D576 at the C-terminal region of gp19 is involved in prohead binding and that the extreme C-terminal region is necessary for stable binding to the prohead. For l, Sippy & Feiss (1992) suggested that the last five amino acid residues are involved in prohead binding of gpA from genetic analyses. The last six amino acid residues of gpA (L-S-G-E-D-E) are similar to that of L571 to D576 of gp19 (L-Y-W-E-D-D). Both have the first hydrophobic residue and the last three acidic residues. Wu et al. (1988) proposed that the carboxyl 32 amino acids of gpA were concerned with the prohead binding specificity between l and 21. Much work on the structure–function relationship of the C-terminal of the large subunits is required to understand the common and specific aspects of the prohead binding. The structure–function relationship of gp19 On the basis of the information obtained in the present study, we propose that three structural

Prohead Binding Domain of Phage T3

domains are related to gp19 functions in DNA packaging: the N-terminal half-domain accessible to proteases in the absence of ATP (the N terminus to 280s amino acids); the ‘‘core’’ domain inaccessible to proteases in both the presence and absence of ATP (280s to 500s amino acids); and the C-terminal domain accessible to proteases in the presence of ATP (500s amino acids to the C terminus). The Tr fragment lacking the N-terminal half bound to a prohead in excess amounts even in the presence of ATP as intact gp19 in the absence of ATP (Figure 4). In a previous study (Morita et al., 1994), we undertook a molecular genetic approach to dissect the functional domains of gp19 by site-directed mutagenesis to three ATP binding consensus motifs (I, II, III; see Figure 10). One of the mutant proteins, gp19-G61D, with a G to D mutation at amino acid 61 in the ATP binding motif I located in the N-terminal half, showed some interesting characteristics; it had altered the gp19 conformation, resulting in defective interaction with ATP and in abortive binding to the prohead as gp19-wt in the absence of ATP. Preliminary experiments showed that gp19-DN lacking a region from L5 to F24 bound to a prohead even in the presence of ATP, like gp19-wt in the absence of ATP (unpublished data). Thus, both gp19-G61D and gp19-DN may have lost the ability to interact with ATP. These results suggest that ATP binds to the ATP binding consensus motif I and that this interaction between the N-terminal region of gp19 and ATP affects the prohead binding activity at the C-terminal region. gp19-G429R is a mutant gp19 in the ATP binding consensus motif III in the core domain and was inefficient in the coupling of ATP hydrolysis to DNA translocation (Morita et al., 1994), suggesting that the core domain would be concerned with coupling ATP hydrolysis to DNA translocation. Possibly, an ATP binding site for prohead binding would be different from that for ATP hydrolysis. The conformational change in gp19 shown in the present paper would be primarily concerned with prohead binding of gp19, and secondarily with DNA translocation. It is suggested that the large subunits change their conformation upon binding to the portal vertex (Fujisawa et al., 1991: f29; Guo et al., 1987a). The present studies indicate that gp19 is bound to the connector through its C-terminal region to form the DNA translocation machinery in the presence of ATP. Further investigations of the structure–function relationship of the DNA translocation machinery, composed of packaging enzymes and the connector, should provide some insight into the molecular mechanism of DNA packaging.

Materials and Methods Bacteria and plasmids Escherichia coli CJ 236 (dut 1 ung 1 thi 1 rel A1; pCJ105 (Cmr )) was used for preparation of M13 viral DNA containing uracil (Kunkel et al., 1987). E. coli JM109 was

JMB—MS 291 643

Prohead Binding Domain of Phage T3

used as a host (Ung+ ) for most of the plasmid constructs derived from M13 mp19 (Yanish-Perron et al., 1985). A host for an expression vector pNT45, which uses l PL promoter for expression of cloned genes, is described in a previous paper (Hamada et al., 1986a). pKH2 is a pNT45 derivative carrying a 2.32 kb fragment containing gene 19, which is an expression vector for gene 19 (Hamada et al., 1986a). Buffers Buffer PM is 10 mM potassium phosphate (pH 7.4) with 7 mM 2-mercaptoethanol; buffer PMG is a buffer PM with 5% (v/v) glycerol. pac buffer contains 20 mM Tris-HCl (pH 7.4), 50 mM NaCl, 1 mM spermidine, 5 mM MgCl2 , 7 mM 2-mercaptoethanol. Complete pac buffer is 5% (w/v) polyethylene glycol (6000), 50 mM ATP, pac buffer. Prohead buffer is 10 mM Tris-HCl (pH 7.4), 0.5 M NaCl, 1 mM MgCl2 . Sample buffer for agarose gel electrophoresis of DNA contains 20% (w/v) sucrose, 10 mM EDTA, 0.05% (w/v) bromophenol blue and 1% (w/v) SDS. Purification of phage proteins involved in DNA packaging Proheads and gp18 were purified as described (Hamada et al., 1986a).

In vitro DNA packaging reaction in the defined system A standard reaction mixture (20 ml) contained 1010 phage equivalents (peq) of mature T3 DNA, 2 × 1010 peq of proheads, 20 pmol of gp18 and 3 pmol of gp19 in complete pac buffer (Shibata et al., 1987b). After the reaction, the mixture was treated with DNase I (0.2 mg/ml) for 20 minutes on ice to digest unpackaged DNA, if necessary, and mature heads were converted to infectious particles by incubation with a head-acceptor extract containing tail and tail fiber proteins, and phages were assayed by titration on E. coli BB as described previously (Shibata et al., 1987a). Limited proteolysis of gp19 gp19 (0.125 mg/ml) was incubated at 20°C for 30 minutes with either trypsin or thermolysin in 20 ml pac buffer in the presence or absence of ATP. The reactions were terminated by the addition of 5 ml of sample buffer for SDS/polyacrylamide gel electrophoresis, followed by heating at 100°C for two minutes or by the addition of 1 ml soybean trypsin inhibitor (2 mg/ml).

Determination of N-terminal amino acid sequences of proteolytic fragments Proteolytic fragments of gp19 were purified from SDS/polyacrylamide gels as following: after the gel was washed with blotting buffer (25 mM Tris-borate, 20% (v/v) methanol) to remove glycine in the electrophoresis buffer, peptide fragments were electrophoretically transferred from the gel onto a polyvinylidine difluoride (PVDF) membrane (Millipore) using a Trans Blot apparatus (BioRad) and stained with Ponceau S (Sigma). Peptide bands of interest were cut from the PVDF membrane and approximately 100 pmol of each peptide fragment was applied to an automated gas phase sequencer, 477A (Applied Biosystems). Phenylthiohydantoin (PTH-) amino acids were identified by HPLC.

Analysis of the C terminus of 37.5 kDa tryptic fragment gp19 (0.125 mg/ml) was digested at 20°C for 30 minutes with trypsin (0.025 mg/ml) in 1 ml of pac buffer. The reaction was terminated by the addition of diisopropyl fluorophosphate (DIFP, Sigma) at a final concentration of 5 mM and mixed with 0.3 ml of 1 M sodium acetate (pH 5.0) to adjust pH. The mixture was applied to a column (1 ml) of anhydrotrypsin agarose (TAKARA) previously equilibrated with a buffer (50 mM sodium acetate (pH 5.0), 20 mM CaCl2 , 5 mM DIFP). After washing with 3 ml of the same buffer (an unadsorbed fraction was saved as a through fraction), the column was eluted with 3 ml of 5 mM HCl and the eluate was saved as a 5 mM HCl fraction. Peptide fragments in both fractions were precipitated with 80% acetone and dissolved in sample buffer for SDS/polyacrylamide gel electrophoresis.

Oligonucleotide-directed mutagenesis Oligonucleotide-directed mutagenesis was carried out according to the protocol of Kunkel et al. (1987; see Kimura & Fujisawa, 1991). Primers (33 or 35 bases long) were synthesized by an Applied Biosystems 3804B-02 DNA synthesizer. Each oligonucleotide had one to three mismatched nucleotides that created both a termination codon and a new restriction site at the target region. The newly generated restriction sites were used in order to screen for candidates with expected mutations. The mutation sites were confined by DNA sequencing. Gene 19 nonsense mutations were cloned into an expression vector, pNT45, and mutant proteins were purified according to the method of Kimura & Fujisawa (1991).

Isolation of gp19–prohead complexes gp19 and proheads were incubated in complete pac buffer with or without ATP at 30°C for 30 minutes at a molar ratio of 40 gp19/prohead. After incubation, the reaction mixture was sedimented through 3 ml of 20% sucrose in pac buffer with or without ATP in a Hitachi RPS50 rotor at 26,000 revs/min for 90 minutes. Pellets were dissolved in sample buffer and subjected to SDS/polyacrylamide gel electrophoresis (Laemmli, 1970).

Acknowledgements We are grateful to Dr M. Feiss (The University of Iowa) for his invaluable help with the manuscript. We thank Drs K. Asada and A. Wada (Kyoto University) for their kind help in amino acid sequencing of proteolytic fragments. This work was supported by Grants-in-Aid for scientific research (no. 04454609 to H.F. and no. 3083 to M.M.) and a Grant-in-Aid for Creative Basic Research (Human

JMB—MS 291 644 Genome Program; to H.F.) from the Ministry of Education, Science and Culture of Japan. M. M. is a JSPS Research Fellow.

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Prohead Binding Domain of Phage T3

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Edited by M. Gottesman (Received 27 June 1994; accepted 20 October 1994)