The mechanical properties of PCNA: implications for the loading and function of a DNA sliding clamp

Abstract

Sliding clamps are toroidal proteins that encircle DNA and act as mobile platforms for DNA replication and repair machinery. To be loaded onto DNA, the eukaryotic sliding clamp Proliferating Cell Nuclear Antigen (PCNA) must be splayed open at one of the subunit-subunit interfaces by the ATP-dependent clamp loader, Replication Factor C, whose clamp-interacting sites form a right-handed spiral. Earlier molecular dynamics (MD) studies suggested that when PCNA opens, it preferentially adopts a right-handed spiral to match the spiral of the clamp loader. Here, analysis of considerably longer MD simulations shows that although the opened form of PCNA can achieve conformations matching the helical pitch of Replication Factor C, it is not biased toward a right-handed spiral structure. A coarse-grained elastic model was also built; its strong correspondence to the all-atom MD simulations of PCNA suggests that the behavior of the open clamp is primarily due to elastic deformation governed by the topology of the clamp domains. The elastic model was further used to construct the energy landscape of the opened PCNA clamp, including conformations that would allow binding to the clamp loader and loading onto double-stranded DNA. A picture of PCNA emerges of a rather flexible protein that, once opened, is mechanically compliant in the clamp opening process.

Figure 1

Overview of PCNA architecture. The crystal structure of PCNA from S. cerevisiae (PDB ID: 1PLQ) (5). PCNA contains three identical subunits assembled head-to-tail to form a ring. Each subunit contains two domains connected by a linker, giving the trimer pseudo sixfold symmetry.

Figure 2

The RMSDs in C^α positions from the crystal structure for two of the four simulations. The dimer rapidly diverges from its starting conformation in both simulations; however, individual domains display small deviations when superimposed individually. The RMSDs of domain 1B are <1 Å over the entire simulation. The other three domains display similar RMSDs.

Figure 3

In-plane and out-of-plane motions of PCNA. (A) In-plane and (B) out-of-plane order parameters from dimer simulation 1 were calculated at 40-ps intervals. PCNA relaxes in the in-plane direction, while simultaneously diverging from the planar conformation found in the crystal structure of the closed trimer. In panel B, positive values of the order parameter correspond to conformations consistent with a right-handed spiral, while negative values correspond to left-handed spirals. Over the course of the 92-ns simulation, PCNA takes on conformations consistent with both right- and left-handed spirals.

Figure 4

In-plane and out-of-plane conformations of PCNA. In-plane and out-of-plane order parameters from each of the four simulations were calculated at 40-ps intervals after removal of the first 5 ns of simulation. (A) Projection of in- and out-of-plane conformations. Each point represents a single conformation, colored by independent trajectory. (B) Conformational ensemble obtained using the anisotropic network model. (C) Histogram of out-of-plane displacements. (D) Histogram of in-plane distances. The data presented in panels C and D represent the aggregate of the MD simulations shown in panel A.

Figure 5

Comparison of all-atom MD simulations and anisotropic network model (ANM). (A) RMSF of C^α atoms from MD simulations (solid line) and ANM (shaded line). The ANM was fit to best reproduce the pattern and magnitude of the fluctuations. The linear correlation coefficient for the two sets of data is r = 0.93. (B) Grayscale plot showing the overlap between pairs of PCA modes calculated from the ANM (x axis) and MD simulations (y axis). The correspondence between modes is indicated by the shading of the block.

Figure 6

Coarse-grained free energy landscape for an open PCNA trimer. The conformational ensemble from the elastic network model is used to calculate the energetic cost of deforming an PCNA trimer with one disrupted subunit-subunit interface. Contour isolines are plotted in 1-k_BT increments. The open points overlaid on the free energy landscape correspond to conformations in the ensemble that are consistent with the FRET distance measured in the presence of ATP and the clamp loader RFC (15).