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• W2020836403 abstract "The phage T4 gp45 sliding clamp is a ring-shaped replication accessory protein that is mounted onto DNA in an ATP-dependent manner by the gp44/62 clamp loader. In the preceding paper (Pietroni, P., Young, M. C., Latham, G. J., and von Hippel, P. H. (1997) J. Biol. Chem. 272, 31666–31676), two gp45 mutants were exploited to probe interactions of the sliding clamp ring with the gp44/62 loading machinery at various steps during the clamp loading process. In this report, these studies are extended to examine the polarity of the binding interaction between gp45 and gp44/62. Three different gp45 mutants containing a single cysteine in three topographically distinct positions were used. Several different reporter groups, including extrinsic fluorophores, a photo-cross-linker, and a biotin linker for use in a novel “streptavidin interference assay,” were covalently attached to these cysteine residues. Since gp45 is a trimeric protein, these three different mutations allowed us to probe up to nine distinct local environments along the surface of the sliding clamp in the presence and absence of other replication proteins. The results show that the gp44/62-ATP clamp loader complex binds exclusively to the C-terminal (S19C) face of the gp45 ring. The phage T4 gp45 sliding clamp is a ring-shaped replication accessory protein that is mounted onto DNA in an ATP-dependent manner by the gp44/62 clamp loader. In the preceding paper (Pietroni, P., Young, M. C., Latham, G. J., and von Hippel, P. H. (1997) J. Biol. Chem. 272, 31666–31676), two gp45 mutants were exploited to probe interactions of the sliding clamp ring with the gp44/62 loading machinery at various steps during the clamp loading process. In this report, these studies are extended to examine the polarity of the binding interaction between gp45 and gp44/62. Three different gp45 mutants containing a single cysteine in three topographically distinct positions were used. Several different reporter groups, including extrinsic fluorophores, a photo-cross-linker, and a biotin linker for use in a novel “streptavidin interference assay,” were covalently attached to these cysteine residues. Since gp45 is a trimeric protein, these three different mutations allowed us to probe up to nine distinct local environments along the surface of the sliding clamp in the presence and absence of other replication proteins. The results show that the gp44/62-ATP clamp loader complex binds exclusively to the C-terminal (S19C) face of the gp45 ring. DNA replication is an enormously complex process that requires the coordination of a number of specialized enzymatic activities involving many different proteins (1Baker T.A. Kornberg A. DNA Replication. 2nd Ed. W. H. Freeman and Co., New York1992Google Scholar). These proteins include: (i) the replication helicase, which unwinds the duplex DNA to create single-stranded regions to serve as templates for DNA synthesis; (ii) the single-stranded binding protein, which protects the exposed single strands of DNA from nucleases and acts to stabilize the replication machinery; (iii) the primase, which synthesizes oligoribonucleotide primers that nucleate DNA polymerization on the leading and lagging strands; (iv) the DNA polymerase, which extends the RNA primers continuously against the leading strand template and discontinuously against the lagging strand template by means of a deoxynucleotide transferase mechanism; (v) the trimeric ring-shaped sliding clamp complex, which binds to the DNA polymerase to stabilize protein-DNA interactions and promotes highly processive DNA synthesis; and (vi) the clamp loader accessory proteins subassembly, which uses ATP hydrolysis to drive the loading of the sliding clamp onto the DNA (2Young M.C. Reddy M.K. von Hippel P.H. Biochemistry. 1992; 31: 8675-8690Crossref PubMed Scopus (89) Google Scholar, 3Kelman Z. O'Donnell M. Annu. Rev. Biochem. 1995; 64: 171-200Crossref PubMed Scopus (360) Google Scholar, 4Stillman B. Cell. 1994; 78: 725-728Abstract Full Text PDF PubMed Scopus (221) Google Scholar). Recent structural studies have demonstrated that hexameric helicases and sliding clamp proteins from several different organisms adopt a ring-like structure (5Egelman E.H. Structure. 1996; 4: 759-762Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 6Egelman E.H. Yu X. Wild R. Hingorani M.M. Patel S.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3869-3873Crossref PubMed Scopus (251) Google Scholar, 7Kong X.-P. Onrust R. O'Donnell M. Kuriyan J. Cell. 1992; 69: 425-437Abstract Full Text PDF PubMed Scopus (631) Google Scholar, 8Krishna T.S. Kong X.-P. Gary S. Burgers P.M. Kuriyan J. Cell. 1994; 79: 1233-1243Abstract Full Text PDF PubMed Scopus (749) Google Scholar), raising a number of questions regarding the relation of form and function. Why is a ring-shaped structure necessary for the function(s) of these proteins? What is the structural basis of the interactions of protein rings with other macromolecules? Are opposite sides of the protein ring functionally equivalent?In the case of the sliding clamp proteins, their toroidal structure is generally believed to be essential for proper function. Prior to the determination of the first high resolution structure of a sliding clamp (the β dimer of Escherichia coli), considerable biochemical evidence existed to support the notion that the β-clamp must be topologically associated with the DNA (9Stukenberg P.T. Studwell-Vaughan P.S. O'Donnell M. J. Biol. Chem. 1991; 266: 11328-11334Abstract Full Text PDF PubMed Google Scholar). Thus, it was particularly gratifying to learn from crystallographic studies that the β-clamp is shaped like a doughnut, with a central hole of sufficient diameter to accommodate double-stranded DNA (7Kong X.-P. Onrust R. O'Donnell M. Kuriyan J. Cell. 1992; 69: 425-437Abstract Full Text PDF PubMed Scopus (631) Google Scholar). Although the dimeric β-clamp has no meaningful sequence homology with the trimeric sliding clamp proteins (proliferating cell nuclear antigen) of eukaryotes that have been investigated, nor with the trimeric phage T4 gp45 1The abbreviations used are: gp45, T4 gene 45 protein; gp44/62, a 4:1 complex of the T4 gene 44 protein and gene 62 protein; gp43, T4 gene 43 protein; IA, 5-((((2-iodoacetyl)amino)ethyl)amino)napthalene-1-sulfonic acid; TFPAM-3 or T3, N-(1-pyrene)maleimide; PM,N-(4-azido-2,3,5,6-tetrafluorobenzyl)-3-maleimidopropionamide; SA, streptavidin; DTT, dithiothreitol; Biotin-BMCC, 1-biotinamido-4-[4′-(maleimidomethyl)cyclohexane-carboxamido]butane.1The abbreviations used are: gp45, T4 gene 45 protein; gp44/62, a 4:1 complex of the T4 gene 44 protein and gene 62 protein; gp43, T4 gene 43 protein; IA, 5-((((2-iodoacetyl)amino)ethyl)amino)napthalene-1-sulfonic acid; TFPAM-3 or T3, N-(1-pyrene)maleimide; PM,N-(4-azido-2,3,5,6-tetrafluorobenzyl)-3-maleimidopropionamide; SA, streptavidin; DTT, dithiothreitol; Biotin-BMCC, 1-biotinamido-4-[4′-(maleimidomethyl)cyclohexane-carboxamido]butane. clamp protein, structural studies have now revealed that these proteins, like β, exist as protein rings (8Krishna T.S. Kong X.-P. Gary S. Burgers P.M. Kuriyan J. Cell. 1994; 79: 1233-1243Abstract Full Text PDF PubMed Scopus (749) Google Scholar). 2I. Moarefi and J. Kuriyan, manuscript in preparation.2I. Moarefi and J. Kuriyan, manuscript in preparation. This result is consistent with the fact that β, proliferating cell nuclear antigen, and gp45 have nearly identical functions. All three proteins are essential for highly processive DNA replication, can slide one-dimensionally on double-stranded DNA, and are lost to solution if the DNA is linearized. Significantly, gp45 is also known to act as a mobile enhancer (i.e. a DNA-tracking protein) in late transcription in phage T4 by stimulating the RNA polymerase machinery (11Herendeen D.R. Kassavetis G.A. Geiduschek E.P. Science. 1992; 256: 1298-1303Crossref PubMed Scopus (108) Google Scholar, 12Tinker R.L. Williams K.P. Kassavetis G.A. Geiduschek E.P. Cell. 1994; 77: 225-237Abstract Full Text PDF PubMed Scopus (54) Google Scholar).From a structural perspective, one of the more interesting questions in DNA replication is how the sliding clamp is loaded onto the template-primer DNA at the replication fork. In the phage T4 system, gp45 loading is an ATP-dependent event that requires the action of the gp44/62 accessory proteins complex (13Jarvis T.C. Paul L.S. Hockensmith J.W. von Hippel P.H. J. Biol. Chem. 1989; 264: 12717-12729Abstract Full Text PDF PubMed Google Scholar). Both gp45 and, separately, template-primer DNA, stimulate the rate of ATP hydrolysis of the gp44/62 clamp loader (13Jarvis T.C. Paul L.S. Hockensmith J.W. von Hippel P.H. J. Biol. Chem. 1989; 264: 12717-12729Abstract Full Text PDF PubMed Google Scholar, 14Young M. Weitzel S.E. von Hippel P.H. J. Mol. Biol. 1996; 264: 440-452Crossref PubMed Scopus (36) Google Scholar). This stimulation is synergistically activated when both gp45 and template-primer DNA are combined with gp44/62-ATP (13Jarvis T.C. Paul L.S. Hockensmith J.W. von Hippel P.H. J. Biol. Chem. 1989; 264: 12717-12729Abstract Full Text PDF PubMed Google Scholar, 14Young M. Weitzel S.E. von Hippel P.H. J. Mol. Biol. 1996; 264: 440-452Crossref PubMed Scopus (36) Google Scholar). Biochemical studies indicate that one gp44/62 molecule can act catalytically to load many gp45 molecules onto DNA (15Kaboord B.F. Benkovic S.J. Curr. Biol. 1995; 5: 149-157Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 16Kaboord B.F. Benkovic S.J. Biochemistry. 1996; 35: 1084-1092Crossref PubMed Scopus (49) Google Scholar), with the release of ADP from gp44/62 probably representing the rate-limiting step in the reaction (14Young M. Weitzel S.E. von Hippel P.H. J. Mol. Biol. 1996; 264: 440-452Crossref PubMed Scopus (36) Google Scholar, 17Latham G.J. Pietroni P. Dong F. Young M.C. von Hippel P.H. J. Mol. Biol. 1996; 264: 426-439Crossref PubMed Scopus (30) Google Scholar). In the preceding paper (18Pietroni P. Young M.C. Latham G.J. von Hippel P.H. J. Biol. Chem. 1997; 272: 31685-31692Abstract Full Text Full Text PDF PubMed Google Scholar) we have used photochemical cross-linking experiments with specific mutant cysteine residues of gp45 that had been covalently labeled with a photochemical cross-linker to probe specific interactions of the gp45 trimer ring with the gp44/62 clamp loading machinery at various stages of the ATP-driven loading process. These experiments have permitted us to begin to build some topographical details into the clamp loading reaction pathway that we (14Young M. Weitzel S.E. von Hippel P.H. J. Mol. Biol. 1996; 264: 440-452Crossref PubMed Scopus (36) Google Scholar, 17Latham G.J. Pietroni P. Dong F. Young M.C. von Hippel P.H. J. Mol. Biol. 1996; 264: 426-439Crossref PubMed Scopus (30) Google Scholar) and others (16Kaboord B.F. Benkovic S.J. Biochemistry. 1996; 35: 1084-1092Crossref PubMed Scopus (49) Google Scholar, 19Berdis A.J. Benkovic S.J. Biochemistry. 1996; 35: 9253-9265Crossref PubMed Scopus (55) Google Scholar) had previously tried to define largely from a kinetic perspective. In this paper, we build on these results to further examine the interaction polarity and relative positioning of the two accessory proteins subassemblies during the clamp loading process.In an attempt to better understand how the gp45 DNA sliding clamp interacts with other DNA replication proteins and DNA itself, we previously described the creation of a gp45 mutant that contains a single cysteine residue (gp45-S19C) (17Latham G.J. Pietroni P. Dong F. Young M.C. von Hippel P.H. J. Mol. Biol. 1996; 264: 426-439Crossref PubMed Scopus (30) Google Scholar). This mutant protein was labeled at the S19C position with an extrinsic fluorophore, which in turn reported ATP-dependent environmental changes when gp45-S19C was bound to the gp44/62 clamp loader or template-primer DNA. More specifically, we discovered that the fluorescence of the 5-((((2-Iodoacetyl)amino)ethyl)amino)napthalene-1-sulfonic acid (IA) fluorophore, covalently coupled to the S19C residue of gp45, increased dramatically upon binding gp44/62-ATP. This fluorescence enhancement could be quantified as a function of the concentration of gp44/62-ATP to yield a dissociation constant of ∼8 nm for the complex (17Latham G.J. Pietroni P. Dong F. Young M.C. von Hippel P.H. J. Mol. Biol. 1996; 264: 426-439Crossref PubMed Scopus (30) Google Scholar). This result suggested either that the S19C residue was located at or near the gp45 binding site for gp44/62-ATP or that this site was sensitive to a conformational change induced by the binding of the gp44/62-ATP complex to form the gp45-gp44/62-ATP loading complex. Significantly, the recent crystal structure of gp452 has revealed that the S19C residue of gp45 is located near the hole of the torus, on the C-terminal face of the ring (see Fig. 1).To determine if the S19C face of gp45 is indeed involved in binding to the gp44/62-ATP complex in clamp loading, we have constructed an additional gp45 mutant, gp45-S45C, in which the inserted cysteine residue lies on the N-terminal face of the gp45 ring, nearly directly opposite to the S19C mutation. In addition, we have continued to use a third mutated form of gp45, the previously described gp45-K81C (17Latham G.J. Pietroni P. Dong F. Young M.C. von Hippel P.H. J. Mol. Biol. 1996; 264: 426-439Crossref PubMed Scopus (30) Google Scholar), which lies on the outside edge of the protein ring. Since the gp45 clamp is a trimeric protein, these three mutations allow us to probe up to nine different local environments, depending on the symmetry of the gp45 interaction with the loading proteins. For example, if the protein environment at each of these nine sites is nonequivalent in the trimer, then the nine positions would consist of three sites on one face of the gp45 ring (S19C), three on the opposite face (S45C), and three on the outside edge of the ring (K81C) (Fig. 1). In contrast, if the gp45 trimer interacts with other proteins with a predisposed symmetry, fewer than nine unique sites would exist, since some sites would share the same environment. By monitoring the environment of reporter groups covalently linked to each of these mutation sites in steady-state fluorescence assays, photo-cross-linking studies, and a novel “streptavidin interference assay,” we have determined that gp44/62-ATP binds to a single, defined face on the gp45 ring. We also present fluorescence evidence that gp45 binds asymmetrically in the gp45-gp44/62-ATP accessory protein complex, further supporting the detailed photochemical findings on the same point that are presented in the preceding paper (18Pietroni P. Young M.C. Latham G.J. von Hippel P.H. J. Biol. Chem. 1997; 272: 31685-31692Abstract Full Text Full Text PDF PubMed Google Scholar).DISCUSSIONIn this report, we describe our efforts to “map” the surface regions on the gp45 sliding clamp that are important for binding to the gp44/62 clamp loader. The recently determined three-dimensional crystal structure of gp452 has proved invaluable in guiding our selection of residues that lie in different regions of the protein. In this regard, it is important to note that gp45 monomers self-assemble into trimers in solution, and thus each amino acid change is represented in triplicate in the biologically relevant structure. As a result, efficient labeling of the three mutated residues (S19C, S45C, and K81C) by reporter groups has allowed us to characterize up to nine potentially unique sites within the gp45 trimer.Protein-protein interactions between gp45 and gp44/62-ATP were probed by three distinct methods. First, extrinsic fluorophores were covalently attached to the lone cysteine residue, the positions of which differ within the trimer ring for the three gp45 mutants, and the steady-state fluorescence intensity and fluorescence polarization properties of these labeled gp45 trimers were examined. Second, photo-cross-linking groups were bound to the cysteine residues, and the nature of the protein-protein cross-links was analyzed by SDS-polyacrylamide gel electrophoresis. Finally, a novel streptavidin interference assay was developed that permits the occlusion of large regions of the gp45 surface using the streptavidin tetramer as a molecular surface “blocker.” The consequence of this steric perturbation upon gp45-gp44/62-ATP binding was quantified by measuring the ability of the various SA-gp45 mutant trimer rings to stimulate the gp44/62 ATPase. For each method, the results are consistent with the interpretation that gp44/62-ATP binds to gp45 on the C-terminal (S19C) side of the ring and, further, that no significant gp44/62-ATP interactions occur with the gp45 protein surface either at the edge of the torus or on the N-terminal (S45C) face.Fluorescence Methods for Determining Binding between the Clamp Loader and the Sliding ClampExtrinsic fluorophores such as IA and PM are known to be very sensitive indictors of local polarity change (22Haugland R.P. Molecular Probes: Handbook of Fluorescent Probes and Research Chemicals. 5th Ed. Molecular Probes, Inc., Eugene, OR1992Google Scholar). Gp45-S19C and gp45-S45C were labeled with both of these reagents to determine the changes in fluorescence, if any, that might be observed upon adding gp44/62-ATP. Both proteins were efficiently modified by IA (2.5 IA labels/trimer for gp45-S19C, and 2.2 IA/trimer for gp45-S45C), consistent with the prediction from the gp45 crystal structure that both of these cysteine residues should be solvent-exposed. In agreement with previous work (17Latham G.J. Pietroni P. Dong F. Young M.C. von Hippel P.H. J. Mol. Biol. 1996; 264: 426-439Crossref PubMed Scopus (30) Google Scholar), IA-gp45-S19C reported a 20% increase in fluorescence and a slight blue shift in the emission maximum in the presence of saturating concentrations of gp44/62-ATP (Fig. 3). This result suggested that the environment of the IA label becomes more hydrophobic in the presence of gp44/62-ATP, as would be expected if S19C is at or near the binding site for the clamp loader. In contrast, IA-gp45-S45C failed to report any measurable change in fluorescence intensity upon the addition of gp44/62-ATP (Fig.4). The results were essentially the same when a second fluorophore, PM, was used to label the two proteins. Fluorescently labeled gp45-K81C also failed to show a fluorescence change in the presence of gp44/62-ATP, suggesting that the local environment of K81C, like that of S45C, is not perturbed in the clamp loading interaction.To ensure that binding between IA-gp45-S19C and gp44/62-ATP and IA-gp45-S45C and gp44/62-ATP is identical, the anisotropy of both fluorescent gp45 molecules was measured in the presence of increasing concentrations of gp44/62-ATP. As shown in Fig. 5, the dissociation constants calculated from curve fits (∼1 nm) were equivalent for both IA-gp45-S19C and IA-gp45-S45C. This result clearly shows that both IA-derivatized proteins were completely bound to gp44/62-ATP at the concentrations used in the fluorescence intensity assays. We note also that our determination of the gp45-gp44/62-ATP dissociation constant yielded a value that is in good agreement with the previously reported value (17Latham G.J. Pietroni P. Dong F. Young M.C. von Hippel P.H. J. Mol. Biol. 1996; 264: 426-439Crossref PubMed Scopus (30) Google Scholar), although the experimental set up was quite different.Photo-cross-linking Methods for Determining Binding between the Clamp Loader and the Sliding ClampAlthough the fluorescence intensity assays suggested that the S45C and K81C positions are not near the gp44/62-ATP binding site, we employed a photo-cross-linking technique to test this hypothesis. Our cross-linker of choice was T3, which is 13 Å in length and contains both a maleimide group (for attachment to sulfhydryl groups) and an azide moiety (which is converted to a highly reactive nitrene by irradiation with UV light). The results (Fig. 7) show that T3-gp45-S19C cross-links to both gp62 and gp44 in the presence and absence of ATP (see also Ref. 18Pietroni P. Young M.C. Latham G.J. von Hippel P.H. J. Biol. Chem. 1997; 272: 31685-31692Abstract Full Text Full Text PDF PubMed Google Scholar).In contrast to the cross-linking observed with T3-gp45-S19C, the T3-gp45-S45C mutant failed to show any evidence of cross-linking, even at elevated protein concentrations. As a result, although the T3 label was covalently linked to about three S45C residues per gp45 trimer, none of these cross-linkers was positioned within 13 Å of either gp44 or gp62. We note that the Benkovic laboratory has also been unable to cross-link gp45 to gp44/62 when cross-linkers are positioned at the T7C residue of gp45, which lies on the same face as S45C. 4D. Sexton, personal communication. Neither T3-gp45-S19C nor T3-gp45-S45C formed intermolecular gp45 cross-links (data not shown), nor would any be expected, since interatomic distance measurements taken from the gp45 crystal structure indicate that the S19C and S45C residues are >13 Å removed from the closest amino acid residue in the neighboring gp45 monomer.The results of T3-gp45-K81C cross-linking to gp44/62-ATP are described in the first paper in this series (18Pietroni P. Young M.C. Latham G.J. von Hippel P.H. J. Biol. Chem. 1997; 272: 31685-31692Abstract Full Text Full Text PDF PubMed Google Scholar). In summary, photo-cross-linking with T3-gp45-K81C revealed protein-protein cross-links between T3-gp45-K81C and gp44 but no significant cross-links between T3-gp45-K81C and gp62 in the absence of ATP. Although T3-gp45-K81C did cross-link to gp62 in the presence of ATP, the cross-linking efficiency between these two proteins was poor compared with the efficiency of cross-links formed between T3-gp45-S19C and gp62. Since gp62 is required to observe gp45 (but not DNA) stimulation of the ATPase activity of the gp44/62 complex (23Rush J. Lin T.-C. Quinones M. Spicer E.K. Douglas I. Williams K.R. Konigsberg W.H. J. Biol. Chem. 1989; 264: 10943-10953Abstract Full Text PDF PubMed Google Scholar), gp62 clearly plays a pivotal role in coordinating interactions between the gp44/62 clamp loader and the gp45 sliding clamp. As a result, we argue that the lack of T3-gp45-K81C cross-linking to gp62 when ATP is absent can be interpreted to mean that K81C is situated within 13 Å of gp44, whose position in space is determined by the disposition of gp44/62 at the gp45 binding site. In contrast, when ATP is available and weak cross-linking between T3-labeled gp45-K81C and gp62 is observed, we propose that conformational changes within the accessory protein complex (18Pietroni P. Young M.C. Latham G.J. von Hippel P.H. J. Biol. Chem. 1997; 272: 31685-31692Abstract Full Text Full Text PDF PubMed Google Scholar) bring K81C close enough to the actual gp62-gp45 binding site to permit cross-linking between the two protein subunits. In short, the cross-linking observed between T3-gp45-K81C and gp44 in the absence of ATP (and T3-gp45-K81C and gp62 in the presence of ATP) probably reflects the ability of the T3 cross-linker to “reach around and grab” the clamp loader bound at a site distal to K81C.Utility of the Streptavidin Interference AssayAs an additional test for the hypothesis that the N-terminal (S45C) face of the gp45 does not provide a binding surface for gp44/62-ATP, we developed a streptavidin interference assay. The purpose of this assay was to tether SA to biotin-derivatized gp45 and monitor the formation of the accessory protein complex by measuring the gp45-dependent stimulation of the gp44/62 ATPase. Since SA is a tetrameric protein with dimensions ∼50 × 50 × 40 Å and the face of the gp45 trimer is ∼90 Å in diameter, even one SA tethered to one face of gp45 would occlude roughly half of the available surface of the ring. The presence of a molecular obstacle the size of SA, combined with the flexibility of the long (32 Å) biotin-BMCC linker, would almost certainly prohibit gp44/62-ATP from docking to gp45 on the SA-bound face if that face contained the binding site for the clamp loader. This assertion is supported by the results of the experiment (Fig. 8). SA tethered to the S19C and K81C positions reduced the stimulation of the ATPase of gp44/62 by the gp45 cofactor to roughly the unstimulated level (∼0.3 μm/min under these conditions; Ref. 14Young M. Weitzel S.E. von Hippel P.H. J. Mol. Biol. 1996; 264: 440-452Crossref PubMed Scopus (36) Google Scholar) In contrast, SA tethered to S45C had no statistically significant effect on the stimulation of the ATPase activity of gp44/62 by this mutant.Structural analyses based on an examination of physical models scaled to the dimensions of gp45, SA, and the biotin-BMCC linker offer the following insights. (i) SA tethered to S19C can block the C-terminal face and a sizable portion of the gp45 ring edge. (ii) SA linked to K81C can occlude the edge of the gp45 ring and a significant portion of both the C-terminal (S19C) and N-terminal (S45C) faces. (iii) SA tethered to S45C can block the N-terminal face and a significant portion of the edge of the gp45 ring. The fact that gp45-dependent stimulation of the gp44/62 ATPase is abolished when SA is linked to the K81C residue is consistent with the observation from model building that SA can swing around from the protein edge and block the binding of the clamp loader to the C-terminal face of gp45. In fact, S45C is actually closer to the edge of the protein ring than S19C. Consequently, it is difficult to imagine how the gp44/62-ATP subassembly could bind primarily to the edge of the gp45 ring if SA was bound to S45C. The only scenario that offers a satisfactory explanation for these results is that gp44/62-ATP binds primarily, if not exclusively, to the C-terminal (S19C) face of gp45. This conclusion is fully supported by the results of the fluorescence assays and photo-cross-linking studies discussed above.Fluorescence Evidence for Asymmetric Binding between the Clamp Loader and the Sliding ClampNearly all of the experiments described in this report have utilized gp45 molecules that were labeled efficiently by reporter groups (more than two labels/trimer). Thus, the absence of an effect, as reported by protein modification, simply indicates that none of the labels was able to make a macromolecular “contact.” These findings make the argument that a specific protein region is not involved in binding even more compelling, since more than one local environment was surveyed in each experiment. However, what can be said about the sites of interaction with gp44/62-ATP within the C-terminal (S19C) face itself? Since the S19C residue was labeled efficiently in each case, it is unclear whether gp44/62 contacts one, two, or all three monomers of the C-terminal face of the gp45 trimer ring.To address this question, we repeated the fluorescence intensity experiment described in Fig. 3 with IA-gp45-S19C labeled with fewer IA groups per gp45 trimer. When gp45-S19C contained an average of 1.4 IA labels/trimer (instead of the 2.5 labels/trimer used above), the fluorescence measured in the presence of gp44/62-ATP was enhanced by only 9%. Thus, a decrease in labeling efficiency (from 2.5 to 1.4 labels/trimer) caused a decrease in the relative percentage of fluorescence enhancement from 20 to 9% (data not shown). This finding suggests that each IA label on S19C does not make an equal contribution to the fluorescence increase upon binding gp44/62-ATP and that the loading complex interacts asymmetrically with the gp45 trimer, perturbing only one or two of the three available Cys-19 labeled fluorophores. This result is in good agreement with our photo-cross-linking studies (18Pietroni P. Young M.C. Latham G.J. von Hippel P.H. J. Biol. Chem. 1997; 272: 31685-31692Abstract Full Text Full Text PDF PubMed Google Scholar), which also suggest binding asymmetry between gp45 and gp44/62.Possible Sites of Recognition on the Surface of the Sliding Clamp ProteinIt is intriguing to speculate about where on the gp45 surface gp44/62-ATP might bind. Structurally speaking, the C-terminal face of gp45 differs from the N-terminal face in many ways, but one of the more obvious differences is that the C-terminal face has several protruding loops, or “handle” structures 5O'Donnell and colleagues (10Naktinis V. Turner J. O'Donnell M. Cell. 1996; 84: 137-145Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) have put forward a similar proposal in connection with binding interactions between the γ complex clamp loader and the β-clamp. (see Fig. 1) that are absent on the N-terminal face, which is nearly planar by comparison.2 Since these “handle” regions are clustered near the three monomer-monomer interfaces of gp45, it is reasonable to suggest that gp44/62-ATP might dock onto gp45 using some of these sites and, through conformational changes in the accessory protein complex (18Pietroni P. Young M.C. Latham G.J. von Hippel P.H. J. Biol. Chem. 1997; 272: 31685-31692Abstract Full Text Full Text PDF PubMed Google Scholar), disrupt the clamp interface(s) to open the ring. If this is true, it would also help to explain why the attachment of a bulky group such as PM to gp45 does not seem to compromise binding by gp44/62-ATP. Since the S19C residue does not extend into solution as far as do the “handle” structures,2 binding to these loop regions would also be consistent with the finding that Stern-Volmer plots failed to show any difference between the extent of acrylamide fluorescence quenching of the IA-gp45-S19C-gp44/62-ATP and IA-gp45-S45C-gp44/62-ATP accessory protein complexes (data not shown).ConclusionsElegant studies by O'Donnell and colleagues (10Naktinis V. Turner J. O'Donnell M. Cell. 1996; 84: 137-145Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) have shown that the C terminus of the E. coliβ-cl" @default.
• W2020836403 created "2016-06-24" @default.
• W2020836403 creator A5032218841 @default.
• W2020836403 creator A5035447333 @default.
• W2020836403 creator A5065959930 @default.
• W2020836403 creator A5072736112 @default.
• W2020836403 date "1997-12-01" @default.
• W2020836403 modified "2023-09-27" @default.
• W2020836403 title "Structural Analyses of gp45 Sliding Clamp Interactions during Assembly of the Bacteriophage T4 DNA Polymerase Holoenzyme" @default.
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