Protein-DNA contacts in the structure of a homeodomain-DNA complex determined by nuclear magnetic resonance spectroscopy in solution.

Abstract

The 1:1 complex of the mutant Antp(C39––S) homeodomain with a 14 bp DNA fragment corresponding to the BS2 binding site was studied by nuclear magnetic resonance (NMR) spectroscopy in aqueous solution. The complex has a molecular weight of 17,800 and its lifetime is long compared with the NMR chemical shift time scale. Investigations of the three‐dimensional structure were based on the use of the fully 15N‐labelled protein, two‐dimensional homonuclear proton NOESY with 15N(omega 2) half‐filter, and heteronuclear three‐dimensional NMR experiments. Based on nearly complete sequence‐specific resonance assignments, both the protein and the DNA were found to have similar conformations in the free form and in the complex. A sufficient number of intermolecular 1H‐1H Overhauser effects (NOE) could be identified to enable a unique docking of the protein on the DNA, which was achieved with the use of an ellipsoid algorithm. In the complex there are intermolecular NOEs between the elongated second helix in the helix‐turn‐helix motif of the homeodomain and the major groove of the DNA. Additional NOE contacts with the DNA involve the polypeptide loop immediately preceding the helix‐turn‐helix segment, and Arg5. This latter contact is of special interest, both because Arg5 reaches into the minor groove and because in the free Antp(C39––S) homeodomain no defined spatial structure could be found for the apparently flexible N‐terminal segment comprising residues 0‐6.

Structural rearrangements on HIV‐1 Tat (32–72) TAR complex formation

“…The present results suggest that the interaction of BP1 peptide with HIV-1 TAR RNA involves noticeable conformational changes of both components, as observed earlier for other protein-nucleic acid interactions [30][31][32]. BP1 comprises the highly flexible basic region and the adjacent compact core sequence region of the full-length HIV-1 Tat protein [33].…”

Kinetics of binding of Antp homeodomain to DNA analyzed by measurements of surface plasmon resonance

“…However, major differences observed between specific and non-specific complexes might be a tight complex versus a loose complex. Although some other changes such as bending could be induced in the conformation of either DNA or the protein, the structural analyses of the complexes of the homeodomain and the specific DNA did not indicate such conformational changes [15][16][17][18][19]. Therefore, the reasons why the enthalpy term contributed more than the entropy term to the association of the homeodomain with the specific DNA could be that many contacts such as hydrogen bonds and van der Waals' contacts formed at the boundary should contribute favorably to such binding.…”

“…The Antp homeodomain used in the present study was described in detail by Otting et al [15]. It is a polypeptide of 68 amino acids that includes the entire Antp homeodomain, with a serine residue at the 39th position replacing a cysteine residue.…”

“…The interactions between homeodomains and DNA have been extensively analyzed by foot-printing methods and gel shift assays [6][7][8][9][10][11][12][13][14]. Direct structural analysis of the complexes has also been performed with NMR and X-ray [15][16][17][18][19]. The results indicated that a homeodomain binds to a specific sequence of less than 10 bp.…”

A proline-rich transcriptional activation domain in murine HOXD-4 (HOX-4.2)

“…Transcriptional activation by p4.2 and its mutated derivatives was expressed as the ratio of standardized luciferase activity over that obtained with p4.2FS. Transcriptional activation by mutant HOXD-4 proteins Site-specific binding by the homeodomain is in part mediated by the third a helix (recognition helix) which makes contacts in the major groove (25)(26)(27)(28)(29)(30)(31)(32). A key determinant of specificity is the residue at position 50 of the homeodomain, within the recognition helix (33 -35).…”