DETERMINATION OF THE NMR SOLUTION STRUCTURE OF A SPECIFIC DNA COMPLEXOF THE MYB DNA-BINDING DOMAIN

The solution structure of a specific DNA complex of the minimum DNA-bi nding domain of the mouse c-Myb protein was determined by distance geo metry calculations using a set of 1732 nuclear Overhauser enhancement (NOE) distance restraints. In order to determine the complex structure independent of the initial guess, we have developed two different pro cedures for the docking calculation using simulated annealing in four- dimensional space (4D-SA). One is a multiple-step procedure, where the protein and the DNA were first constructed independently by 4D-SA usi ng only the individual intramolecular NOE distance restraints. Here, t he initial structure of the protein was a random coil and that of the DNA was a typical B-form duplex. Then, as the starting structure for t he next docking procedure, the converged protein and DNA structures we re placed in random molecular orientations, separated by 50 Angstrom. The two molecules were docked by 4D-SA utilizing all the restraints, i ncluding the additional 66 intermolecular distance restraints. The sec ond procedure comprised a single step, in which a random-coil protein and a typical B-form DNA duplex were first placed 70 Angstrom from eac h other. Then, using all the intramolecular and intermolecular NOE dis tance restraints, the complex structure was constructed by 4D-SA. Both procedures yielded the converged complex structures with similar qual ity and structural divergence, but the multiple-step procedure has muc h better convergence power than the single-step procedure. A model stu dy of the two procedures was performed to confirm the structural quali ty, depending upon the number of intermolecular distance restraints, u sing the X-ray structure of the engrailed homeodomain-DNA complex.