Sliding clamp of the bacteriophage T4 polymerase has open and closed subunit interfaces in solution

Abstract

The sliding clamps of bacteriophage T4 (gp45), Escherichia coli (beta clamp), and yeast (PCNA) are required for processive DNA synthesis by their cognate DNA polymerases. The X-ray crystal structures of all three of these clamps have been shown to be closed, circular complexes. This paper reports investigations of the solution structure of bacteriophage T4 gp45 by analytical ultracentrifugation, fluorescence, and hydrodynamic modeling. Mutants of gp45 with inter- and intrasubunit disulfide bonds were created to alter the solution structure of gp45, with additional mutagenesis used to investigate the importance of the proline-rich loop region found between the two domains of each gp45 monomer. The wild-type gp45 trimer assembles from monomers cooperatively with a dissociation constant of 0.21 microM2 and values between 0.088 and 0. 32 microM2 for the mutants. Velocity ultracentrifugation experiments showed that wild-type gp45 possesses a sedimentation coefficient strongly dependent on concentration, typical of asymmetric or elongated molecules, that when extrapolated to zero concentration yields a sedimentation coefficient of 4.0 S. The loop and the disulfide mutants exhibited sedimentation coefficients with little concentration dependence, typical of symmetric or spherical molecules, that when extrapolated to zero concentration yielded sedimentation coefficients of 4.4-4.8 S. The lower sedimentation coefficient in the former case is consistent with wild-type gp45 being more asymmetric or elongated than the mutant forms. Fluorescence-resonance energy-transfer experiments were used to measure the distance between two amino acids (W91 and V162C-coumarin) on opposite sides of the gp45 subunit interface. For an intrasubunit disulfide mutant, the distance between these two amino acids was determined to be 19 A (14 A in the X-ray crystal structure), consistent with a closed complex. For the mutants without intrasubunit disulfides, the efficiency of fluorescence-resonance energy transfer was in accord with a model of gp45 being an open complex composed of two closed subunit interfaces and a third open interface separated by a distance of 35-38 A. The collective data supplemented with hydrodynamic modeling were consistent with gp45 subunit separation achieved within the plane of the gp45 ring.