Mechanism of the δ Wrench in Opening the β Sliding Clamp
2003; Elsevier BV; Volume: 278; Issue: 41 Linguagem: Inglês
10.1074/jbc.m305828200
ISSN1083-351X
AutoresChiara Indiani, Mike O’Donnell,
Tópico(s)Force Microscopy Techniques and Applications
ResumoThe β sliding clamp encircles DNA and tethers DNA polymerase III holoenzyme to the template for high processivity. The clamp loader, γ complex (γ3δδ′χψ), assembles β around DNA in an ATP-fueled reaction. The δ subunit of the clamp loader opens the β ring and is referred to as the wrench; ATP modulates contact between β and δ among other functions. Crystal structures of δ·β and the γ3δδ′ minimal clamp loader make predictions of the clamp loader mechanism, which are tested in this report by mutagenesis. The δ wrench contacts β at two sites. One site is at the β dimer interface, where δ appears to distort the interface by via a steric clash between a helix on δ and a loop near the β interface. The energy for this steric clash is thought to derive from the other site of interaction, in which δ binds to a hydrophobic pocket in β. The current study demonstrates that rather than a simple steric clash with β, δ specifically contacts β at this site, but not through amino acid side chains, and thus is presumably mediated by peptide backbone atoms. The results also imply that the interaction of δ at the hydrophobic site on β contributes to destabilization of the β dimer interface rather than acting solely as a grip of δ on β. Within the γ complex, δ′ is proposed to prevent δ from binding to β in the absence of ATP. This report demonstrates that one or more γ subunits also contribute to this role. The results also indicate that δ′ acts as a backboard upon which the γ subunits push to attain the ATP induced change needed for the δ wrench to bind and open the β ring. The β sliding clamp encircles DNA and tethers DNA polymerase III holoenzyme to the template for high processivity. The clamp loader, γ complex (γ3δδ′χψ), assembles β around DNA in an ATP-fueled reaction. The δ subunit of the clamp loader opens the β ring and is referred to as the wrench; ATP modulates contact between β and δ among other functions. Crystal structures of δ·β and the γ3δδ′ minimal clamp loader make predictions of the clamp loader mechanism, which are tested in this report by mutagenesis. The δ wrench contacts β at two sites. One site is at the β dimer interface, where δ appears to distort the interface by via a steric clash between a helix on δ and a loop near the β interface. The energy for this steric clash is thought to derive from the other site of interaction, in which δ binds to a hydrophobic pocket in β. The current study demonstrates that rather than a simple steric clash with β, δ specifically contacts β at this site, but not through amino acid side chains, and thus is presumably mediated by peptide backbone atoms. The results also imply that the interaction of δ at the hydrophobic site on β contributes to destabilization of the β dimer interface rather than acting solely as a grip of δ on β. Within the γ complex, δ′ is proposed to prevent δ from binding to β in the absence of ATP. This report demonstrates that one or more γ subunits also contribute to this role. The results also indicate that δ′ acts as a backboard upon which the γ subunits push to attain the ATP induced change needed for the δ wrench to bind and open the β ring. Chromosomal replicases of both eukaryotes and prokaryotes derive their high processivity during synthesis from a ring-shaped clamp that encircles DNA and binds to the chromosomal replicase (1Kelman Z. O'Donnell M. Annu. Rev. Biochem. 1995; 64: 171-200Crossref PubMed Scopus (361) Google Scholar, 2Baker T.A. Bell S.P. Cell. 1998; 92: 295-305Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 3Davey M.J. O'Donnell M. Curr. Opin. Chem. Biol. 2000; 4: 581-586Crossref PubMed Scopus (36) Google Scholar, 4Jeruzalmi D. O'Donnell M. Kuriyan J. Curr. Opin. Struct. Biol. 2002; 12: 217-224Crossref PubMed Scopus (116) Google Scholar, 5O'Donnell M. Jeruzalmi D. Kuriyan J. Curr. Biol. 2001; 11: 935-946Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The closed circular structure of the clamp necessitates a clamp loader to crack open the ring and place it around the primed DNA in an ATP driven reaction. In Escherichia coli, the β-clamp is a ring-shaped dimer formed by two crescent-shaped protomers that encircle the duplex (6Stukenberg P.T. Studwell-Vaughan P.S. O'Donnell M. J. Biol. Chem. 1991; 266: 11328-11334Abstract Full Text PDF PubMed Google Scholar). Each β protomer consists of three domains, each of which have the same chain fold (7Kong X.P. Onrust R. O'Donnell M. Kuriyan J. Cell. 1992; 69: 425-437Abstract Full Text PDF PubMed Scopus (638) Google Scholar). This gives the β dimer a 6-fold appearance. The β ring binds to the replicative DNA polymerase III core (Pol III) 1The abbreviations used are: Pol III, polymerase III; DTT, dithiothreitol; ssDNA, single-stranded DNA; RFC, replication factor C; PCNA, proliferating cell nuclear antigen. and tethers it to the template for high processivity. The clamp is opened and closed around the DNA by the γ complex clamp loader. The minimal γ complex clamp loader machine (reviewed in Ref. 5O'Donnell M. Jeruzalmi D. Kuriyan J. Curr. Biol. 2001; 11: 935-946Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) consists of five different subunits: δ, δ′, and three copies of γ (τ) that are arranged as a circular heteropentamer (Fig. 1A) (8Jeruzalmi D. O'Donnell M. Kuriyan J. Cell. 2001; 106: 429-441Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). In addition, two other subunits, χ and ψ, are associated with the clamp loader, but they are not required for clamp loading in vitro (9Onrust R. O'Donnell M. J. Biol. Chem. 1993; 268: 11766-11772Abstract Full Text PDF PubMed Google Scholar). In order for this clamp loader to bind two molecules of DNA polymerase III core, two of the γ subunits are replaced by two τ subunits. τ and γ are encoded by the same gene (dnaX); τ is the full-length product, and γ is a truncated version produced by a translational frameshift (10Blinkowa A.L. Walker J.R. Nucleic Acids Res. 1990; 18: 1725-1729Crossref PubMed Scopus (181) Google Scholar, 11Flower A.M. McHenry C.S. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3713-3717Crossref PubMed Scopus (204) Google Scholar, 12Tsuchihashi Z. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2516-2520Crossref PubMed Scopus (225) Google Scholar). The unique 24-kDa C terminus of τ, absent in γ, binds DNA polymerase III core (13Studwell-Vaughan P.S. O'Donnell M. J. Biol. Chem. 1991; 266: 19833-19841Abstract Full Text PDF PubMed Google Scholar, 14McHenry C.S. J. Biol. Chem. 1982; 257: 2657-2663Abstract Full Text PDF PubMed Google Scholar) and DnaB helicase (15Kim S. Dallmann H.G. McHenry C.S. Marians K.J. J. Biol. Chem. 1996; 271: 21406-21412Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 16Yuzhakov A. Turner J. O'Donnell M. Cell. 1996; 86: 877-886Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar), thereby acting to organize the replisome machinery. The γ (τ) subunits of the γ complex are the only subunits that hydrolyze ATP (1Kelman Z. O'Donnell M. Annu. Rev. Biochem. 1995; 64: 171-200Crossref PubMed Scopus (361) Google Scholar) and therefore constitute the motor of the clamp loading machine. The δ subunit is referred to as the wrench of the clamp loader, since it can open the β dimer at one interface on its own (17Turner J. Hingorani M.M. Kelman Z. O'Donnell M. EMBO J. 1999; 18: 771-783Crossref PubMed Scopus (157) Google Scholar, 18Naktinis V. Onrust R. Fang L. O'Donnell M. J. Biol. Chem. 1995; 270: 13358-13365Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 19Stewart J. Hingorani M.M. Kelman Z. O'Donnell M. J. Biol. Chem. 2001; 276: 19182-19189Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 20Jeruzalmi D. Yurieva O. Zhao Y. Young M. Stewart J. Hingorani M. O'Donnell M. Kuriyan J. Cell. 2001; 106: 417-428Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). The energy for ring opening is not derived from ATP (neither δ nor β bind ATP) but from the energy of protein-protein interaction between δ and β (17Turner J. Hingorani M.M. Kelman Z. O'Donnell M. EMBO J. 1999; 18: 771-783Crossref PubMed Scopus (157) Google Scholar). In the absence of ATP, the γ complex does not bind β (18Naktinis V. Onrust R. Fang L. O'Donnell M. J. Biol. Chem. 1995; 270: 13358-13365Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Study of the δ′ subunit shows that it modulates the ability of δ to bind β even in the absence of other γ complex subunits (17Turner J. Hingorani M.M. Kelman Z. O'Donnell M. EMBO J. 1999; 18: 771-783Crossref PubMed Scopus (157) Google Scholar, 19Stewart J. Hingorani M.M. Kelman Z. O'Donnell M. J. Biol. Chem. 2001; 276: 19182-19189Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Thus, δ′ is proposed to obscure the δ subunit within γ complex from binding to β when ATP is not present (17Turner J. Hingorani M.M. Kelman Z. O'Donnell M. EMBO J. 1999; 18: 771-783Crossref PubMed Scopus (157) Google Scholar, 20Jeruzalmi D. Yurieva O. Zhao Y. Young M. Stewart J. Hingorani M. O'Donnell M. Kuriyan J. Cell. 2001; 106: 417-428Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). However, when ATP binds to the γ subunits, the complex undergoes a conformational change in which it is hypothesized that a portion of δ′ separates from δ, allowing δ to bind and open the β ring (18Naktinis V. Onrust R. Fang L. O'Donnell M. J. Biol. Chem. 1995; 270: 13358-13365Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 20Jeruzalmi D. Yurieva O. Zhao Y. Young M. Stewart J. Hingorani M. O'Donnell M. Kuriyan J. Cell. 2001; 106: 417-428Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 22Hingorani M.M. O'Donnell M. J. Biol. Chem. 1998; 273: 24550-24563Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Only one δ binds to the β2 dimer (18Naktinis V. Onrust R. Fang L. O'Donnell M. J. Biol. Chem. 1995; 270: 13358-13365Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Moreover, δ binds to a monomer mutant of β (β1) with 50-fold higher affinity than to β2 (19Stewart J. Hingorani M.M. Kelman Z. O'Donnell M. J. Biol. Chem. 2001; 276: 19182-19189Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). This result indicates that when δ interacts with one member of the β2 dimer, it uses a portion of its binding energy to perform work (i.e. to part one of the dimer interfaces), thus lowering the observed affinity of δ to β2. Furthermore, only one δ subunit binds β2, suggesting that only one of the dimer interfaces is disrupted by δ (17Turner J. Hingorani M.M. Kelman Z. O'Donnell M. EMBO J. 1999; 18: 771-783Crossref PubMed Scopus (157) Google Scholar, 18Naktinis V. Onrust R. Fang L. O'Donnell M. J. Biol. Chem. 1995; 270: 13358-13365Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 19Stewart J. Hingorani M.M. Kelman Z. O'Donnell M. J. Biol. Chem. 2001; 276: 19182-19189Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Opening at only one β2 interface is also consistent with the observation that β2, cross-linked at only one dimer interface, is efficiently loaded onto DNA by γ complex (17Turner J. Hingorani M.M. Kelman Z. O'Donnell M. EMBO J. 1999; 18: 771-783Crossref PubMed Scopus (157) Google Scholar). The crystal structure of δ in complex with a β monomer mutant (δ·β1), combined with the structure of the minimal clamp loader (γ3δδ′), provides further details and allows predictions about how δ opens β2 (8Jeruzalmi D. O'Donnell M. Kuriyan J. Cell. 2001; 106: 429-441Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Of the three domains of δ, only the N-terminal domain (domain I) interacts with β (Fig. 1B), and it contacts the clamp in two different places. The first site of the δ-β interaction involves hydrophobic contacts between residues Leu73 and Phe74 of δ and a hydrophobic pocket of β located between domains II and III (Fig. 1, B and C). We refer to this as "site 1." Both Leu73 and Phe74 of δ protrude out to form a hydrophobic plug that fits into the hydrophobic pocket on the surface of β. This hydrophobic interaction is presumed to be responsible for the majority of the binding energy. Interestingly, δ residues Leu73 and Phe74 are the most highly conserved residues among δ subunits of different bacteria (20Jeruzalmi D. Yurieva O. Zhao Y. Young M. Stewart J. Hingorani M. O'Donnell M. Kuriyan J. Cell. 2001; 106: 417-428Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 23Dalrymple B.P. Kongsuwan K. Wijffels G. Dixon N.E. Jennings P.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11627-11632Crossref PubMed Scopus (258) Google Scholar). Likewise, alignment of bacterial β subunits shows that residues comprising the hydrophobic pocket in β to which δ binds are highly conserved (20Jeruzalmi D. Yurieva O. Zhao Y. Young M. Stewart J. Hingorani M. O'Donnell M. Kuriyan J. Cell. 2001; 106: 417-428Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). There is a second interaction between δ and β that involves δ helix α4 and β loop α1″-β2″. This contact is referred to here as "site 2" (Fig. 1C). The interaction of δ with β at this site leads to an extensive conformational change of the β loop that is thought to be important for ring opening (20Jeruzalmi D. Yurieva O. Zhao Y. Young M. Stewart J. Hingorani M. O'Donnell M. Kuriyan J. Cell. 2001; 106: 417-428Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). This five-residue loop of β (residues 274-278) is near the interface and in fact connects to the interfacial α helix that contains the two residues (Ile272 and Leu273) that form the hydrophobic core of the β2 dimer interface. In contrast, the structure of the δ·β1 complex shows that the hydrophobic core residues of β are rotated out of position, thereby precluding formation of the dimer interface (Fig. 1C). Thus, it would appear that δ distorts one β2 interface by altering the conformation of the β 274-278 loop. This distortion at site 2 appears to be the result of a steric clash between δ and β, which pushes on the loop, rather than being due to specific side chain contacts between δ and β. Hence, the site 2 interaction/steric clash presumably requires an input of energy that is obtained from the binding energy at site 1. The δ subunit destabilizes the interface of β2 but does not explain how a gap opens up at the β interface for DNA to pass through. The shape of β1 in the δ·β1 structure relative to the β2 dimer suggests how the ring actually opens (20Jeruzalmi D. Yurieva O. Zhao Y. Young M. Stewart J. Hingorani M. O'Donnell M. Kuriyan J. Cell. 2001; 106: 417-428Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Superposition of monomeric β (i.e. of the δ·β1 complex) onto dimeric β reveals that the shape of the β monomer is less curved than in the dimer. This change in curvature is produced by rigid body motions between the three domains of β. The largest rigid body motion occurs distant from the δ-binding sites, suggesting that the change is intrinsic to β and that the closed β dimer is under spring tension between domains of β. After disruption of the dimer interface by the δ subunit, release of the spring tension results in the motions between domains that produce the gap at the broken interface (20Jeruzalmi D. Yurieva O. Zhao Y. Young M. Stewart J. Hingorani M. O'Donnell M. Kuriyan J. Cell. 2001; 106: 417-428Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). The γ3δδ′ structure fits nicely with biochemical data indicating that β cannot bind γ complex in the absence of ATP and that δ′ participates in modulating the δ-β interaction. The γ3δδ′ structure (8Jeruzalmi D. O'Donnell M. Kuriyan J. Cell. 2001; 106: 429-441Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar) shows that each of the subunits is composed of three domains having the same chain fold and is a member of the AAA+ family. The five subunits are arranged as a circular heteropentamer (Fig. 1A). The C-terminal domains of all five subunits form a tight closed circular connection, holding the subunits together. On the contrary, the connections between the N-terminal domains contain a gap between δ and δ′ (see Fig. 1A). Docking of β2 onto δ shows that β2 does not fit due to steric occlusion by δ′ and possibly some of the γ subunits as well. This is consistent with the fact that ATP is not present in the structure. It is proposed that as the ATP sites fill, conformation changes in γ are propagated around the pentamer to increase the gap between δ and δ′, thereby allowing δ to bind to β for clamp opening (8Jeruzalmi D. O'Donnell M. Kuriyan J. Cell. 2001; 106: 429-441Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 17Turner J. Hingorani M.M. Kelman Z. O'Donnell M. EMBO J. 1999; 18: 771-783Crossref PubMed Scopus (157) Google Scholar, 18Naktinis V. Onrust R. Fang L. O'Donnell M. J. Biol. Chem. 1995; 270: 13358-13365Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 22Hingorani M.M. O'Donnell M. J. Biol. Chem. 1998; 273: 24550-24563Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). In this state, with β and ATP bound to γ complex, a tight affinity for DNA is established (22Hingorani M.M. O'Donnell M. J. Biol. Chem. 1998; 273: 24550-24563Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 24Bertram J.G. Bloom L.B. Turner J. O'Donnell M. Beechem J.M. Goodman M.F. J. Biol. Chem. 1998; 273: 24564-24574Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Upon recognizing a primed site, the ATP is hydrolyzed and the γ subunits may move δ back into proximity with δ′, forcing the β ring off the δ wrench and allowing the β ring to close around the DNA. The δ′ structure was the first clamp loader subunit to be solved (25Guenther B. Onrust R. Sali A. O'Donnell M. Kuriyan J. Cell. 1997; 91: 335-345Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). δ′ appears to be more rigid than the γ and δ subunits (8Jeruzalmi D. O'Donnell M. Kuriyan J. Cell. 2001; 106: 429-441Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). This conclusion derives from the observation that the three domains of δ′ in γ complex are oriented nearly the same as in the δ′ alone. In contrast, the relative orientations of domain III relative to domains I/II of all three γ subunits are different in γ3δδ′, and the same is true for δ in δ·β1 compared with δ in the γ3δδ′ structure. Consistent with a rigid structure, δ′ has additional connections between domains compared with the few connections between the domains in either γ or δ. This rigid conformation of δ′ has earned it the title of stator, the stationary part of a machine upon which the other parts move (8Jeruzalmi D. O'Donnell M. Kuriyan J. Cell. 2001; 106: 429-441Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Perhaps the rigid δ′ stator serves as an anvil for the β interactive element of δ to strike, pushing β2 off of the γ complex following ATP hydrolysis. A recent report demonstrates that δ′, besides its role as stator, also plays an instrumental role in the motor function of γ complex, as predicted by the structure, by supplying a catalytic arginine into the ATP site at the intersubunit junction δ′/γ1 (8Jeruzalmi D. O'Donnell M. Kuriyan J. Cell. 2001; 106: 429-441Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 26Johnson A. O'Donnell M. J. Biol. Chem. 2003; 278: 14406-14413Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). In light of the γ3δδ′ and δ·β1 crystal structures and predictions of how these proteins function, we reexamine here the mechanism of clamp loading by the γ complex, in particular the δ-β interaction, and the role of δ′ in rendering the β interacting elements of δ accessible for binding the clamp. The studies contain surprises, but overall the results provide significant advancements in our understanding of how this complex machinery functions. Unlabeled deoxyribonucleoside triphosphates were from Amersham Biosciences; radioactive deoxyribonucleoside triphosphates were from PerkinElmer Life Sciences. Proteins were purified as described: α, ϵ, γ, τ (27Studwell P.S. O'Donnell M. J. Biol. Chem. 1990; 265: 1171-1178Abstract Full Text PDF PubMed Google Scholar), β (7Kong X.P. Onrust R. O'Donnell M. Kuriyan J. Cell. 1992; 69: 425-437Abstract Full Text PDF PubMed Scopus (638) Google Scholar), δ and δ′ (28Dong Z. Onrust R. Skangalis M. O'Donnell M. J. Biol. Chem. 1993; 268: 11758-11765Abstract Full Text PDF PubMed Google Scholar), χ and ψ (29Xiao H. Dong Z. O'Donnell M. J. Biol. Chem. 1993; 268: 11779-11784Abstract Full Text PDF PubMed Google Scholar), θ (30Studwell-Vaughan P.S. O'Donnell M. J. Biol. Chem. 1993; 268: 11785-11791Abstract Full Text PDF PubMed Google Scholar), SSB (31Yao N. Leu F.P. Anjelkovic J. Turner J. O'Donnell M. J. Biol. Chem. 2000; 275: 11440-11450Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Core polymerase and γ complex were reconstituted from wild-type and/or mutant subunits and purified as described (30Studwell-Vaughan P.S. O'Donnell M. J. Biol. Chem. 1993; 268: 11785-11791Abstract Full Text PDF PubMed Google Scholar). Samples of purified γ complex were analyzed on a 14% SDS-polyacrylamide gel stained with Coomassie Brilliant Blue G-250. M13mp18 ssDNA was purified as described (32Turner J. O'Donnell M. Methods Enzymol. 1995; 262: 442-449Crossref PubMed Scopus (27) Google Scholar) and primed with a 30-mer DNA oligonucleotide as described (27Studwell P.S. O'Donnell M. J. Biol. Chem. 1990; 265: 1171-1178Abstract Full Text PDF PubMed Google Scholar). Buffer A is 20 mm Tris-HCl (pH 7.5), 0.5 mm EDTA, 2 mm DTT, and 10% glycerol (v/v). Buffer B is Buffer A, except the pH was adjusted to 8.2. Buffer C is 10 mm sodium acetate (pH 7.5), 0.5 mm EDTA, 2 mm DTT. Buffer D is Buffer C, except the pH was adjusted to 6.1. Gel filtration buffer is Buffer A containing 100 mm NaCl, 1 mm ATP, and 10 mm MgCl2. Reaction buffer is 20 mm Tris-HCl (pH 7.5), 0.1 mm EDTA, 5 mm DTT, 4% glycerol (v/v), and 40 μg/ml bovine serum albumin. Tris-Sucrose buffer is 50 mm Tris-HCl (pH 7.5), 2 mm EDTA, and 10% sucrose. δ N62A, F65A and δ L73A, F74A—Two different double mutants of δ were constructed. In one, which we refer to here as δα4, the Asn62 and Phe65 residues located on helix α4 were mutated to alanines. In the other, referred to as δLF→AA, residues Leu73 and Phe74 were mutated to alanines. These mutants were constructed by DNA oligonucleotide site-directed mutagenesis of the pETδ expression plasmid (28Dong Z. Onrust R. Skangalis M. O'Donnell M. J. Biol. Chem. 1993; 268: 11758-11765Abstract Full Text PDF PubMed Google Scholar) and confirmed by sequence. Expression and purification of δα4 and δLF→AA were performed as follows. Expression plasmids were transformed into competent BL21 (DE3) cells (Novagen). Fresh transformants were grown in 12 liters of LB containing 200 μg of ampicillin/ml to a density of A 600 = 0.6 and induced with 1 mm isopropyl-1-thio-β-d-galactopyranoside. Cells were incubated 4 h with shaking at 37 °C, chilled to 15 °C, and incubated another 20 h with shaking at 15 °C. For cell lysis, cells were brought to a final volume of 300-400 ml with a final concentration of 30 mm spermidine, 100 mm NaCl, and 5 mm DTT in Tris-sucrose buffer. Cells were lysed by two passages through a French Press at 17,000 p.s.i. and insoluble material was removed by centrifugation at 12,000 rpm for 1 h at 4 °C in an SLA1500 rotor. The soluble cell lysate supernatant (Fraction I) was decanted and treated with 0.21 g/ml ammonium sulfate. After stirring for 30 min at 4 °C, the pellet was collected by centrifugation at 12,000 rpm for 30 min in a SLA1500 rotor. The resulting pellet was resuspended in Buffer B. The protein was diluted to a conductivity equal to 60 mm NaCl with Buffer B and then applied to a Heparin-agarose column (Bio-Rad) equilibrated in Buffer B. Protein was eluted with a 100-500 mm NaCl gradient in Buffer B. Fractions containing δ were pooled (Fraction II) and diluted with Buffer B to a conductivity equivalent to 100 mm NaCl. Particulate matter was removed by centrifugation at 10,000 rpm for 10 min at 4 °C in an SS34 rotor. The supernatant was applied to a MonoQ column equilibrated in Buffer B. The column was then washed with Buffer B before eluting the protein with a 100-500 mm NaCl gradient in Buffer B. Fractions containing δ were pooled (Fraction III, δLF→AA mutant: 45 ml, 1.2 mg/ml; δα4 mutant: 32 ml, 1.0 mg/ml) and then stored at -80 °C. δ′ΔN—Nucleotides encoding the N-terminal 206 residues of δ′ were deleted from the pETδ′ expression plasmid (28Dong Z. Onrust R. Skangalis M. O'Donnell M. J. Biol. Chem. 1993; 268: 11758-11765Abstract Full Text PDF PubMed Google Scholar) by PCR using the following primers: 5′-TG GCG TTG CAT ATG GGA GAT AAC TGG CAG GCT CG-3′, which introduces an NdeI site (CAT ATG) and inserts an initiating ATG codon for methionine at residue 207, and 5′-TTA TTG CTC AGC GGT GGC AGC AGC CAA CTC AGC TTC CTT TCG GG-3′. The product encodes the entire C-terminal domain (domain III) of δ′ but eliminates domains I and II. The resulting PCR product was placed into pETIIa at the NdeI and BamHI sites to yield pETδ′ΔN. BL21 (DE3) pETδ′ΔN cells were grown in 3 liters of LB containing 100 μg of ampicillin/ml to a density of A 600 = 0.6 and induced with 0.8 mm isopropyl-1-thio-β-d-galactopyranoside. Cells were incubated for 2 h with shaking at 37 °C. For cell lysis, cells were brought to a final volume of 300-400 ml with a final concentration of 30 mm spermidine, 100 mm NaCl, and 5 mm DTT in Tris-sucrose buffer. Cells were lysed by two passages through a French press at 17,000 p.s.i., and insoluble material was removed by centrifugation at 12,000 rpm for 30 min at 4 °C in an SLA1500 rotor. The pellet was resuspended in 8 m urea and diluted to 2 m urea with buffer A. Urea was removed in the following step by application to a 40-ml Fast Flow Q-Sepharose column equilibrated in buffer A. Protein was eluted with a 400-ml, 0-500 mm NaCl gradient in Buffer A. Fractions containing δ′ΔN were pooled (108 mg) and diluted to a conductivity equivalent to 35 mm NaCl with Buffer A before being applied to a 50-ml Heparin-agarose column (Bio-Rad). Protein was eluted with a 500 ml, 50-600 mm NaCl gradient in Buffer A. Most of the protein was found in the flow-through and in the first few fractions. These were pooled together and applied to an 8-ml MonoQ column equilibrated in Buffer A. Protein was eluted using a 120-ml, 50-600 mm NaCl gradient in Buffer A. Fractions containing δ′ΔN were dialyzed against Buffer A containing 50 mm NaCl and then stored at -80 °C (15 ml, 1.4 mg/ml). β loop—An internal deletion of the dnaN gene encoding β amino acids 275-278 was made in the pETβ expression vector by PCR using the following primers: 5′-GAT GCC GGC CAC GAT GCG TCC GGC G-3′, which anneals to upstream vector sequence, and 5′-TTA TTG CTC AGC GGT GGC AGC AGC CAA CTC AGC TTC CTT TCG GG-3′, which includes a SacII site at nucleotide 834 of dnaN. The resulting PCR product was digested with MfeI/SacII, and the 287-bp fragment was inserted into pETβ digested with the same enzymes to produce pETβloop. BL21 (DE3) pETβloop cells were grown in 3 liters of LB containing 100 μg of ampicillin/ml to a density of A 600 = 0.8 and induced with 0.8 mm isopropyl-1-thio-β-d-galactopyranoside. Cells were incubated for 2 h with shaking in fluted flasks at 37 °C. For cell lysis, cells were brought to a final volume of 300-400 ml with a final concentration of 30 mm spermidine, 100 mm NaCl, and 5 mm DTT in Tris-sucrose buffer. Cells were lysed by two passages through a French press at 17,000 p.s.i., and insoluble material was removed by centrifugation at 12,000 rpm for 30 min at 4 °C in an SLA1500 rotor. Soluble lysate was treated with 0.436 g/ml of ammonium sulfate and stirred at 4 °C for 30 min. The pellet was resuspended and dialyzed overnight against Buffer A. The protein was applied to a Fast Flow Q-Sepharose column equilibrated in buffer A and then eluted with a 0-500 mm NaCl gradient in Buffer A. Peak fractions containing the βloop mutant were pooled and then dialyzed against Buffer C overnight. The protein was applied to an SP-Sepharose column equilibrated in Buffer C and eluted with a 0-500 mm NaCl gradient in the same buffer. The protein flowed through the column. The pH of the flow-through was lowered to pH 6.1 on ice by adding acetic acid, and the protein was reapplied to an SP-Sepharose column equilibrated in Buffer D. The βloop mutant was eluted with 0-500 mm NaCl gradient in Buffer D. The βloop mutant remained in the flow-through and was precipitated with 20% ammonium sulfate. The pellet was resuspended in Buffer A and dialyzed against Buffer A overnight. The protein was then applied to an 8-ml Mono Q column equilibrated in Buffer A. The column was washed extensively with Buffer A before eluting protein using a 160-ml gradient of 0-500 mm NaCl in Buffer A. Fractions containing the βloop mutant were pooled and dialyzed overnight against Buffer A. The preparation was passed over a 10-ml ATP-agarose column equilibrated in Buffer A to remove any possible contaminant of γ complex that binds this column tightly. The βloop protein flowed through the column and was stored at -80 °C (22 ml, 1.9 mg/ml). Interaction between β or the βloop mutant and wild-type and mutant γ complex was analyzed by gel filtration on an FPLC-Superose 12 column (Amersham Biosciences). The β subunit (25 or 30 μm dimer, as indicated) was incubated with 5 or 25 μm (as indicated) γ complex (or mutant γ complex) for 15 min at 15 °C in 200 μl of Buffer A containing 100 mm NaCl in the presence or absence of 1 mm ATP and
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