Fig 1.
Location of the three double alanine substitutions in PCNA.
(A) Front and side views of the wild-type PCNA trimer (PDB entry 2OD8.pdb). The three subunits of PCNA are colored light blue, pale green, and pale yellow. The bound PIP motif is shown in the stick representation and colored red. (B) Close-up view of one subunit of PCNA with the location of each substituted amino acid residue shown in the sphere representation. The D41A/D42A, R61A/D63A, and L126A/I128A substitutions are colored light pink, wheat, and pale cyan, respectively. (C) Close-up view of one subunit of PCNA with the location of the residues comprising the surface cavity shown in the sphere representation.
Fig 2.
Native gel electrophoresis of wild-type and mutant PCNA proteins.
Solutions of the wild-type PCNA protein, the G178S mutant PCNA protein, the R61A/D63A mutant PCNA protein, the L126A/I128A mutant PCNA protein, and the D41A/D42A mutant PCNA protein (0.1–1.0 mg/ml) were run on a non-denaturing polyacrylamide gradient gel (4–20%) and stained with Coomassie blue. The positions of the PCNA monomer and PCNA trimer are indicated.
Fig 3.
X-ray crystal structure of the D41A/D42A mutant PCNA protein.
(A) Front and side views of the D41A/D42A mutant PCNA protein. All three subunits are colored light pink, and the locations of the substituted amino acid residues are shown in the sphere representation. (B) Close up of an overlay of the wild-type PCNA protein (light blue) and the D41A/D42A mutant PCNA protein (light pink) are shown in the ribbon representation (RMSD of 0.529 Å). The positions of the α carbons of residues 41 to 44 are indicated.
Table 1.
Data collection and refinement statistics.
Table 2.
Backbone α-carbon displacement.
Fig 4.
X-ray crystal structure of the R61A/D63A mutant PCNA protein.
(A) Front and side views of the R61A/D63A mutant PCNA protein. All three subunits are colored wheat, and the locations of the substituted amino acid residues are shown in the sphere representation. (B) Close up of an overlay of the wild-type PCNA protein (light blue) and the R61A/D63A mutant PCNA protein (wheat) are shown in the ribbon representation (RMSD of 0.317 Å). The positions of the α carbons of residues 21 to 24 and of residues 61 to 63 are indicated. (C) Close up of the structure of the R61A/D63A mutant PCNA protein shown in the cartoon representation. The N-terminus, β strand A1, α helix A1, and loop B are colored pale yellow.
Fig 5.
X-ray crystal structure of the L126/I128A mutant PCNA protein.
(A) Front and side views of the L126A/I128A mutant PCNA protein. All three subunits are colored pale green, and the locations of the substituted amino acid residues are shown in the sphere representation. (B) Close up of an overlay of the wild-type PCNA protein (light blue) and the L126A/I128A mutant PCNA protein (pale green) are shown in the ribbon representation (RMSD of 0.835 Å). The positions of the α carbons of residues 41 to 44 and of residues 124 to 128 are indicated. (C) Close up of the structure of the L126A/I128A mutant PCNA protein shown in the cartoon representation. The side chains of D42 and R44 are shown in the stick representation. A portion of a PEG molecule and a chloride ion are shown in the stick and sphere representation, respectively. The 2Fo-Fc map contoured at 1 σ is shown.
Fig 6.
Comparison of the surface cavity in the wild-type and mutant PCNA proteins.
(A) Close up of an overlay of the wild-type PCNA protein (light blue), the D41A/D42A mutant PCNA protein (light pink), the R61A/D63A mutant PCNA protein (wheat), and the L126A/I128A mutant PCNA protein (pale green) are shown in the ribbon representation. The positions of loop B, loop D, and the IDCL are indicated. The edge of the surface cavity is highlighted with a dashed ellipse. (B) Close up of the structure of the wild-type PCNA protein (light blue) is shown in the cartoon representation. The locations of residues D41 and D42 (light pink), residues R61 and D63 (wheat), and residues L126 and I128 (pale green) are shown in the sphere representation.