Structural basis for inhibition of DNA replication by aphidicolin

Abstract

Natural tetracyclic diterpenoid aphidicolin is a potent and specific inhibitor of B-family DNA polymerases, haltering replication and possessing a strong antimitotic activity in human cancer cell lines. Clinical trials revealed limitations of aphidicolin as an antitumor drug because of its low solubility and fast clearance from human plasma. The absence of structural information hampered the improvement of aphidicolin-like inhibitors: more than 50 modifications have been generated so far, but all have lost the inhibitory and antitumor properties. Here we report the crystal structure of the catalytic core of human DNA polymerase α (Pol α) in the ternary complex with an RNA-primed DNA template and aphidicolin. The inhibitor blocks binding of dCTP by docking at the Pol α active site and by rotating the template guanine. The structure provides a plausible mechanism for the selectivity of aphidicolin incorporation opposite template guanine and explains why previous modifications of aphidicolin failed to improve its affinity for Pol α. With new structural information, aphidicolin becomes an attractive lead compound for the design of novel derivatives with enhanced inhibitory properties for B-family DNA polymerases.

Figure 1.

The electron density maps. The 2F_o−F_c Fourier maps covering aphidicolin and surrounding residues in the first (A) and in the second (B) independent molecules are displayed at contour level of 1.0 σ. Aphidicolin and protein are drawn as sticks, the water molecules are drawn as balls and the electron density maps are drawn as mesh.

Figure 2.

Structure of the human Pol α–DNA/RNA–aphidicolin ternary complex. (A) The overall view of the ternary complex. Protein is represented as cartoon, DNA/RNA as sticks and aphidicolin as spheres. The N-terminal domain is colored orange, the nonfunctional exonuclease domain—red, the palm—magenta, the fingers—marine, the thumb—lime-green and the linkers between domains—gray. The disordered linker between the N-terminal and palm domains is shown by dashed line. Aphidicolin atoms are colored gray for carbon and red for oxygen. DNA and RNA atoms are colored blue for nitrogen, red for oxygen, orange for phosphorus, and green or gray for DNA or RNA carbons, respectively. (B) Close-up view of the aphidicolin-binding pocket. Aphidicolin is shown as sticks. The protein surface is represented by the vacuum electrostatic potential; the DNA/RNA surface is shown at 50% transparency. (C) Structure of aphidicolin with numbered carbon positions.

Figure 3.

Alignment of human Pol α–DNA/RNA ternary complexes containing aphidicolin or dCTP. (A) The overall view of aligned ternary complexes. Proteins are represented as cartoon, all other molecules as sticks. For the complex with aphidicolin, the protein is colored pale-green, aphidicolin—yellow and DNA/RNA carbons—gray. For the complex containing dCTP, the protein is colored salmon, DNA/RNA—cyan and dCTP—marine. (B) Close-up view of aligned aphidicolin, dCTP and surrounding DNA/RNA bases. All atoms in aphidicolin complex are shown as sticks, and in dCTP complex as lines. For the ternary complexes containing aphidicolin or dCTP, the carbon atoms of DNA/RNA are colored gray or cyan, respectively.

Figure 4.

Interaction of aphidicolin with human Pol α. (A) Close-up view of aphidicolin in the active site of Pol α. Protein is represented as cartoon with 80% transparency; additionally, the main chain and residues located near aphidicolin are shown as sticks. DNA/RNA and aphidicolin are represented as sticks. Pol α atoms are colored blue for nitrogen, red for oxygen, yellow for sulfur and cyan for carbon. The predicted hydrogen bonds between aphidicolin, guanine and protein are shown by pink dashed lines. (B) Close-up view of the helix region interacting with aphidicolin after an alignment of the fingers domains from two complexes of Pol α in open conformation. For the human Pol α–DNA/RNA–aphidicolin complex, the main-chain atoms of the fingers domain are represented as sticks and colored cyan for carbon. Additionally, this finger helix is represented as cartoon with 80% transparency. For yeast Pol α–DNA/RNA complex (PDB code 4FXD (7)), the main-chain atoms of the fingers domain are represented as lines and colored wheat for carbon. Magenta arrows show the shift of N954 O and G958 N induced by aphidicolin binding.

Figure 5.

Mechanism of aphidicolin selectivity to template guanine. (A) The position of twisted guanine is stabilized by interaction with a side chain of Ser955. The color scheme for DNA/RNA and Pol α atoms is the same as on Figure 3A. Side-chain atoms of Arg784 from the ternary complex containing dCTP are colored blue for nitrogen and green for carbon. Aphidicolin atoms are omitted for clarity. (B) The modeled cytosine does not fit into the pocket due to sterical clash with Ser955. The hydrogen atom at C4 is shown and colored orange. Pol α surface is represented by the vacuum electrostatic potential. The modeling of cytosine in place of guanine was done with the PyMOL by keeping the position of the ribose and connected with it nitrogen from the nucleotide base unaltered.

Figure 6.

Modeling of aphidicolin derivatives. (A) Sterical hindrance between 3β-OH of 3-epiaphidicolin and C6, O6 of template guanine. The surfaces of guanine and 3β-OH are shown at 80% transparency. (B) Alignment of aphidicolin and its 15-ene-16-deoxy derivative. Pol α is represented as cartoon with 40% transparency. Aphidicolin is shown as lines; its derivative and the main-chain atoms of Pol α are shown as sticks. The carbons of aphidicolin, its derivative and Pol α are colored green, gray and cyan, respectively. The hydrogen bond between O17 of aphidicolin and Y865 N as well as the predicted hydrogen bond between O17 of 15-ene-16-deoxy-aphidicolin and Leu864 N are shown by red dashed lines.