Mapping protein-protein interactions in the bacteriophage T4 DNA polymerase holoenzyme using a novel trifunctional photo-cross-linking and affinity reagent
Mapping protein-protein interactions in the bacteriophage T4 DNA polymerase holoenzyme using a novel trifunctional photo-cross-linking and affinity reagent
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DOI：
10.1021/ja000591t
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发表时间：
2000-06-28
影响因子：
15
通讯作者：
Benkovic, SJ
中科院分区：
文献类型：
--
作者：
Alley, SC;Ishmael, FT;Benkovic, SJ
DNA replication requires the formation of multiprotein complexes, whose functions1 and structures2 have been conserved during evolution. In bacteriophage T4, the DNA polymerase holoenzyme forms the core of the DNA replication system, consisting of the DNA polymerase (gp43), a trimeric, circular processivity factor called the sliding clamp (gp45), and the clamp loader (a 4: 1 complex of gp44 and gp62) that hydrolyzes ATP to assemble the holoenzyme. 3 The holoenzymes of prokaryotic and eukaryotic DNA replication systems contain analogous proteins. 1a, 4 The structures of gp455 and gp436 have been individually solved by X-ray crystallography, but neither the gp44/62 complex nor any of the other multiprotein complexes such as the final holoenzyme have been solved. Models of the holoenzyme based on the individual gp45 and gp43 X-ray crystal structures have been proposed. 5a, 6 However, these models have not taken into account solution evidence that points to a holoenzyme that has undergone ATP hydrolysis-dependent conformational changes relative to the individual ground-state structures. 7 Site-specific incorporation of photo-cross-linking reagents has been extensively used to investigate the protein-protein and protein-nucleic acid interactions in solution in the bacteriophage T4 holoenzyme, including the organization of the holoenzyme, 8 the conformationally dynamic nature of the holoenzyme assembly process, 7c and the location of the interaction between the C-terminus of gp43 and gp45. 7g To streamline investigations such as these and make them amenable to even more complex multiprotein systems, we have synthesized 3, a novel, trifunctional photo-cross-linking and affinity reagent. 9 The three functional groups in 3 are (1) a thiol-reactive 2-thiopyridine mixed disulfide for conjugation to a bait protein,(2) a photoactivatable aryl azide for photo-cross-linking to a target protein (the target protein may be the same protein as the bait, resulting in intra-or intersubunit photo-cross-links), and (3) the affinity probe biotin, which can be used for purification and visualization of photo-cross-linked proteins. The succinimide 1 conjugates to lysine, 10 but it does not allow site-specific attachment, a requirement for mapping protein-protein interactions. The mixed disulfide 3 conjugates to cysteine, allowing site-specific attachment on many proteins. The resulting conjugate (following the loss of 2-thiopyridine; eg, 4) has a mixed disulfide at the surface of the bait protein that is exchangeable in the presence of free thiols and allows the transfer of biotin from the bait to the target upon reduction of photocross-links.The utility of 3 was demonstrated with the gp45 mutant I107C, 11 which contains a single cysteine per monomer. I107C is at the midpoint of the interdomain connecting loop of gp45, a region of the protein suggested to play a role in DNA loading by gp44/62. 7f The point of attachment of 3 was verified by digesting the I107C-3 conjugate with trypsin and then purifying biotinylated peptides on a monomeric avidin column. 12 Analysis by matrixassisted laser desorption ionization (MALDI) mass spectrometry (Figure 1) resulted in an ion at m/z 3889.0, corresponding to the mass of the predicted gp45 tryptic fragment containing I107C
