DETERMINATION OF THE STRUCTURE OF THE DNA-BINDING DOMAIN OF GAMMA-DELTA RESOLVASE IN SOLUTION

The DNA binding domain (DBD) of gamma delta resolvase (residues 141-18 3) is responsible for the interaction of this site-specific DNA recomb inase with consensus site DNA within the gamma delta transposable elem ent in Escherichia coli. Based on chemical-shift comparisons, the prot eolytically isolated DBD displays side-chain interactions within a hyd rophobic core that are highly similar to those of this domain when par t of the intact enzyme (Liu T, Liu DJ, DeRose EF, Mullen GP, 1993, J B iol Chem 268:16309-16315). The structure of the DBD in solution has be en determined using restraints obtained from 2-dimensional proton NMR data and is represented by 17 conformers. Experimental restraints incl uded 458 distances based on analysis of nuclear Overhauser effect conn ectivities, 17 phi and chi(1) torsion angles based on analysis of coup lings, and 17 backbone hydrogen bonds determined from NH exchange data . With respect to the computed average structure, these conformers dis play an RMS deviation of 0.67 Angstrom for the heavy backbone atoms an d 1.49 Angstrom for all heavy atoms within residues 149-180. The DBD c onsists of 3 alpha-helices comprising residues D149-Q157, S162-T167, a nd R172-N183. Helix-2 and helix-3 form a backbone fold, which is simil ar to the canonical helix-turn-helix motif. The conformation of the NH 2-terminal residues, G141-R148, appears flexible in solution. A hydrop hobic core is formed by side chains donated by essentially all hydroph obic residues within the helices and turns. Helix-1 and helix-3 cross with a right-handed folding topology. The structure is consistent with a mechanism of DNA binding in which contacts are made by the hydrophi lic face of helix-3 in the major groove and the amino-terminal arm in the minor groove. This structure represents an important step toward a nalysis of the mechanism of DNA interaction by gamma delta resolvase a nd provides initial structure-function comparisons among the divergent DBDs of related resolvases and invertases.