N Jamin, F. Toma/ Progress in Nuclear Magnetic Resonance Spectroscopy 38(2001)83-114 Fig. 6. Snapshot of the protein-DN after 1148 ps of MD simulation of the Antp HD-DNA complex(Fig 4 from Ref. [44D). Al atoms of the protein are represented ept for the side-chains of lle 47(yellow), Gin50(pink), Asn51(gray)and Met54(green). The a strand of the DNA is colored orange - strand red. The water molecules at the interface are represented by dark blue sphere. Reprinted with the permission of K. wuthrich (1996)held by Cell Press. sequence specific DNA binding, numerous NMR and labeled Antp(C39S)and unlabeled DNA to assign X-ray studies have been carried out. the resonances. As shown in Fig. 7, these experiments The solution structure of two homeodomain -dna discriminate along the 2-frequency axis between reso- complexes have been solved by NMR: the Drosophila nances of protons bound to N from others. The sum antennapedia mutant homeodomain (Antp(C39S)) spectrum(Fig. 7A) contains the diagonal peaks and bound to a 14-mer duplex DNA containing the BS2 cross peaks with all DNA resonances and with those site [1] and the Drosophila ventral nervous system protons of the protein not bound to N while the differ- (vnd)-NK2 homeodomain bound to a 16-mer duplex ence spectrum(Fig. 7B )contains the diagonal peaks DNA containing the vnd/NK2 binding site [48] and cross peaks with the amide protons of the protein. Antennapedia is probably the most well-known The assumption is made and verified that the homeodomain and its overexpression in the Droso- conformations of both the protein and the dNa are phila embryo leads in a fly with an extra pair of feet similar in the free state and in the complex. Using this instead of antennae. The determination of the solution strategy, all proton resonances of the polypeptide structure of the Antp(C39S)-DNA complex(molecu backbone(except MetO and Arg 1),B-protons for 60 lar weight about 18,000)was made possible by the residues, y-protons for 40 residues, all non- development of isotope edited and filtered techniques. exchangeable side-chain protons for 34 residues as Due to the poor quality of the COsY and ToCSY well as all nonexchangeable base protons, all I spectra of the Antp(C39S)-DNA complex, Otting protons and with two exceptions all 2H, 2H and coworkers [49 used a strategy based on NOE 3H resonances of the dna were assigned. Four data obtained from 2D [H, H NOESY with intermolecular protein-DNA NOEs involving PN(o2)-half-filter and 3D N correlated ['H, H] amino acid residues Arg5, Tyr8, Tyr25, lle 47, NOESY on a sample containing uniformly Gln50 and Met54 were identified and these allowedsequence speci®c DNA binding, numerous NMR and X-ray studies have been carried out. The solution structure of two homeodomain±DNA complexes have been solved by NMR: the Drosophila antennapedia mutant homeodomain (Antp(C39S)) bound to a 14-mer duplex DNA containing the BS2 site [1] and the Drosophila ventral nervous system (vnd)-NK2 homeodomain bound to a 16-mer duplex DNA containing the vnd/NK2 binding site [48]. Antennapedia is probably the most well-known homeodomain and its overexpression in the Droso￾phila embryo leads in a ¯y with an extra pair of feet instead of antennae. The determination of the solution structure of the Antp(C39S)±DNA complex (molecu￾lar weight about 18,000) was made possible by the development of isotope edited and ®ltered techniques. Due to the poor quality of the COSY and TOCSY spectra of the Antp(C39S)±DNA complex, Otting and coworkers [49] used a strategy based on NOE data obtained from 2D [1 H,1 H] NOESY with 15N(v2)-half-®lter and 3D 15N correlated [1 H,1 H] NOESY on a sample containing 15N uniformly labeled Antp(C39S) and unlabeled DNA to assign the resonances. As shown in Fig. 7, these experiments discriminate along thev2-frequency axis between reso￾nances of protons bound to 15N from others. The sum spectrum (Fig. 7A) contains the diagonal peaks and cross peaks with all DNA resonances and with those protons of the protein not bound to 15N while the differ￾ence spectrum (Fig. 7B) contains the diagonal peaks and cross peaks with the amide protons of the protein. The assumption is made and veri®ed that the conformations of both the protein and the DNA are similar in the free state and in the complex. Using this strategy, all proton resonances of the polypeptide backbone (except Met0 and Arg1), b-protons for 60 residues, g-protons for 40 residues, all non￾exchangeable side-chain protons for 34 residues as well as all nonexchangeable base protons, all 10 sugar protons and with two exceptions all 20 H, 200H and 30 H resonances of the DNA were assigned. Four￾teen intermolecular protein±DNA NOEs involving amino acid residues Arg5, Tyr8, Tyr25, Ile 47, Gln50 and Met54 were identi®ed and these allowed N. Jamin, F. Toma / Progress in Nuclear Magnetic Resonance Spectroscopy 38 (2001) 83±114 93 Fig. 6. Snapshot of the protein±DNA interface after 1148 ps of MD simulation of the Antp HD±DNA complex (Fig. 4. from Ref. [44]). All atoms of the protein are represented in cyan except for the side-chains of Ile47 (yellow), Gln50 (pink), Asn51 (gray) and Met54 (green). The a￾strand of the DNA is colored orange and the b-strand red. The water molecules at the interface are represented by dark blue sphere. Reprinted with the permission of K. WuÈthrich. Copyright (1996) held by Cell Press