Conformational analysis of processivity clamps in solution demonstrates that tertiary structure does not correlate with protein dynamics

Abstract

The relationship between protein sequence, structure, and dynamics has been elusive. Here, we report a comprehensive analysis using an in-solution experimental approach to study how the conservation of tertiary structure correlates with protein dynamics. Hydrogen exchange measurements of eight processivity clamp proteins from different species revealed that, despite highly similar three-dimensional structures, clamp proteins display a wide range of dynamic behavior. Differences were apparent both for structurally similar domains within proteins and for corresponding domains of different proteins. Several of the clamps contained regions that underwent local unfolding with different half-lives. We also observed a conserved pattern of alternating dynamics of the α helices lining the inner pore of the clamps as well as a correlation between dynamics and the number of salt bridges in these α helices. Our observations reveal that tertiary structure and dynamics are not directly correlated and that primary structure plays an important role in dynamics.

Figure 1. Sliding Clamps Have Highly Conserved Structures and Low Sequence Similarity

(A) Structural alignment of T4 gp45 trimer (green; PDB ID: 1czd), E. coli β clamp dimer (blue; PDB ID: 1mmi), T. kodakaraensis TK0582 trimer (yellow; PDB ID: 3lx2), and human PCNA trimer (red; PDB ID: 1vym). (B) Superposition of all 17 sliding clamp domains in this study. Domains were extracted from the PDB files and superimposed. PDB IDs: T4 gp45, 1czd; β clamp, 1mmi; yPCNA, 1plq; TK0582, 3lx2, TK0535, 3lx1; AtPCNA1, 2zvv; AtPCNA2, 2zvw; hPCNA, 1vym. (C) Plot of the percent sequence identity versus the rmsd from the pairwise structural alignments. Filled diamonds, full-length proteins; open squares, domains; blue squares, domains within the same protein. See also Figure S1.

Figure 2. Sliding Clamps Have Different Dynamics

The relative percentage deuterium incorporation is shown using color gradients across different time points. Gray indicates regions that were not identified. Dashed lines indicate subunit interfaces. PDB IDs: T4 gp45, 1czd; β clamp, 1mmi; yPCNA, 1plq; TK0582, 3lx2; TK0535, 3lx1; AtPCNA1, 2zvv; hPCNA, 1vym. The melting temperatures were determined by a thermal shift assay as described in Experimental Procedures. The far right column highlights EX1 peptides and indicates proteins displaying only EX2 kinetics. HX MS data for AtPCNA2 (T_m = 51.6 °C) are shown in Figure S2. See also Figure S2

Figure 3. EX1 Kinetics in T4 gp45 and Human PCNA

(A) Mass spectra for a representative T4 gp45 peptide, residues 73-85 (m/z = 695.4, +2 charge state). The mass spectrum corresponding to the exchange time-point closest to the approximate half-life of the unfolding events is displayed in blue. (B) Time course of deuterium uptake of peptide 73-85. Each line represents an independent HX MS experiment. (C) All peptides showing EX1 kinetics in T4 gp45 (red) mapped onto the crystal structure (PDB ID: 1czd). (D) Mass spectra of the EX1 peptide in hPCNA, residues 170-182 (m/z = 630.3, +2 charge state). (E) Time course of deuterium uptake of peptide 170-182. (F) The EX1 peptide is part of the subunit interface and is shown in red on the crystal structure of hPCNA (PDB ID: 1vym). The two subunits are colored green and yellow, respectively.

Figure 4. Alternating Dynamics in the Inner Pore of Human PCNA

(A) Average number of salt bridges formed by the inner pore α-helices of hPCNA (PDB ID: 1vym) during 1 ns of MD simulation. (B) HX MS data for peptides that are part of the four α-helices in the inner pore of human PCNA. The fractional deuterium level at each time point is plotted as a function of the midpoint position of each peptide. The first and last residues of each peptide are indicated above each data set. See also Figure S3 and Table S1.