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

The sliding clamps of bacteriophage T4 (gp45), Escherichia coli (beta clamp ), and yeast (PCNA) are required for processive DNA synthesis by their cogn ate DNA polymerases. The X-ray crystal structures of all three of these cla mps have been shown to be closed, circular complexes. This paper reports in vestigations of the solution structure of bacteriophage T4 gp45 by analytic al ultracentrifugation, fluorescence, and hydrodynamic modeling. Mutants of gp45 with inter- and intrasubunit disulfide bonds were created to alter th e solution structure of gp45, with additional mutagenesis used to investiga te the importance of the proline-rich loop region found between the two dom ains of each gp35 monomer. The wild-type gp45 trimer assembles from monomer s cooperatively with a dissociation constant of 0.21 mu M-2 and values betw een 0.088 and 0.32 mu tM(2) for the mutants. Velocity ultracentrifugation e xperiments showed that wild-type gp35 possesses a sedimentation coefficient strongly dependent on concentration, typical of asymmetric or elongated mo lecules, that when extrapolated to zero concentration yields a sedimentatio n coefficient of 4.0 S. The loop and the disulfide mutants exhibited sedime ntation coefficients with little concentration dependence, typical of symme tric or spherical molecules, that when extrapolated to zero concentration y ielded sedimentation coefficients of 4.4-4.8 S. The lower sedimentation coe fficient in the former case is consistent with wild-type gp35 being more as ymmetric or elongated than the mutant forms. Fluorescence-resonance energy- transfer experiments were used to measure the distance between two amino ac ids (W91 and V162C-coumarin) on opposite sides of the gp45 subunit interfac e. For an intrasubunit disulfide mutant, the distance between these two ami no acids was determined to be 19 Angstrom(14 Angstrom in the X-ray crystal structure), consistent with a closed complex. For the mutants without intra subunit disulfides, the efficiency of fluorescence-resonance energy transfe r 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 distanc e of 35-38 Angstrom. The collective data supplemented with hydrodynamic mod eling were consistent with gp45 subunit separation achieved within the plan e of the gp45 ring.