Structural code for DNA recognition revealed in crystal structures of papillomavirus E2-DNA targets

Abstract

Transcriptional regulation in papillomaviruses depends on sequence-specific binding of the regulatory protein E2 to several sites in the viral genome. Crystal structures of bovine papillomavirus E2 DNA targets reveal a conformational variant of B-DNA characterized by a roll-induced writhe and helical repeat of 10.5 bp per turn. A comparison between the free and the protein-bound DNA demonstrates that the intrinsic structure of the DNA regions contacted directly by the protein and the deformability of the DNA region that is not contacted by the protein are critical for sequence-specific protein/DNA recognition and hence for gene-regulatory signals in the viral system. We show that the selection of dinucleotide or longer segments with appropriate conformational characteristics, when positioned at correct intervals along the DNA helix, can constitute a structural code for DNA recognition by regulatory proteins. This structural code facilitates the formation of a complementary protein-DNA interface that can be further specified by hydrogen bonds and nonpolar interactions between the protein amino acids and the DNA bases.

Figure 1

A stereoview of the hydrated E2-R3 dodecamer. Water molecules are shown in red, the octahedrally coordinated magnesium ions in green, and the attached water molecules in orange. Only the 157 water molecules of the asymmetric unit are shown. Three hydrated Mg²⁺ ions are in the major groove and two are in the minor groove. The picture was produced with molscript 2.0 (13).

Figure 2

A view of a hexahydrated magnesium ion (in green) bridging three DNA duplexes with the corresponding electron density (2Fo-Fc) map at 1.6 Å resolution contoured at 1.5σ. Hydrogen bonds between the Mg-coordinated water molecules (shown in red) and base or backbone donor/acceptor atoms from three helices (shown in blue, orange, and magenta) are drawn as yellow lines. The figure was prepared with o 5.10 (10).

Figure 3

The five unique E2 helices of the free DNA crystal structures. From left to right: E2-R3 (cyan), the three E2-P1 molecules (magenta, orange, and red), and E2-P4 (green). Despite of differences in crystal packing and base sequence, all five duplexes exhibit similar global structures with rms deviations ranging from 0.7 to 1.0 Å using all common atoms. The figure was drawn with setor (14).

Figure 4

Two views of (from left to right) the protein-bound E2-DNA (only the central 12 bp are shown), the E2-R3 dodecamer, and fiber B-DNA of the same sequence (20). (a) View perpendicular to the 2-fold axis of the double helix. (b) View along the 2-fold axis. The green line represents the best-fitted global axis and the red, short vectors represent the local axes of the base pairs. The global and local axes were calculated by curves 5.2 (25). In the free and bound E2 targets, the writhe of the local axes around the global axis is illustrated by the inclined red arrows precessing around the green line. The images were prepared with curves 5.2 (25) and rasmol 2.6 (26).

Figure 5

Comparison of local parameters between the free E2 dodecamer (■) and the bound E2 16-mer (▴). End-to-end average values of the free dodecamer are also shown (□). Parameters shown are roll (a), helix twist (b), and slide (c). The parameters were derived by freehelix (15) The various definitions are given in Table 2 and indicated by the corresponding drawings at the left. The consensus ACCG/CGGT regions are shaded.