CRYSTAL STRUCTURES OF DNA TARGETS OF THE E2 PROTEIN FROM BOVINE PAPILLOMAVIRUS-1. H. Rozenberg1, D. Rabinovich1, R. H. Hegde2 and Z. Shakked1 1Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel 2Skirball Inst. of Biomolecular Medicine, NYU Medical center, New York, NY 10016, USA
The E2 protein is the dominant transcriptional regulator of papillomaviruses. In bovine papillomavirus-1 (BPV-1), E2 binds sequence-specifically to a consensus sequence ACCN6GGT found 17 times in the BPV-1 genome. We crystallized several DNA oligomers incorporating the consensus motif (underlined, see Table). The structure of the DNA binding domain of the bovine protein complexed to a 17-mer target (CCGACCGACGTCGGTCG) has been determined previously (Hegde et al., 1992).
The structures of three dodecamers: (1), (2) and (3), were solved by a novel modification of the classical molecular Fourier transform method ("MFT" available at http://www.weizmann.ac.il/~csrabin1/MFT/). The structures of (1) and (2) were refined to R-factors of 21 and 17% respectively. The refinement of (3) is in progress. The free targets adopt B-DNA structures with 10.5-10.7 bp/turn comparable to the complexed target. Sequence (2) is identical to the central 12 base-pair region of the bound sequence. The three unique duplexes of (2) adopt similar helical conformations. The conserved ACC/GGT motifs are relatively straight as in the complexed target whereas the central six base-pair region is bent by nearly 14deg. toward the major groove which is comparable in magnitude (20deg.) but in opposite direction to that of the complexed DNA. This observation indicates that the central GACGTC region is inherently flexible. Thus, the present system provides an example where the flexibility of the DNA region which is not contacted by the protein is an important determinant of sequence-specific recognition.
Crystal data: sequence, space group, unit-cell dimensions, no. of duplexes in the asymmetric unit and resolution (measured at 120K)
(1) ACCGGTACCGGT P43, (40.2, 40.2, 57.6 Å), 1, 2.8 Å
(2) ACCGACGTCGGT P1, (40.5,40.1,40.5Å, 82.6,116.2,80.7deg.),3,2.6Å
(3) ACCGACGTCGGT R3, (63.1, 63.1, 44.5 Å), 1, 2.1 Å
(4) ACCGACGIUCGGT P1, (40.3,40.3,40.2 Å, 87.1,87.3,68.0deg.),3,3.2Å
(5) CCGACCGACGTCGGTCG, R3, (100.7, 100.7, 79.8 Å), 3, 8.0Å