Electrostatics and hydration at the homeodomain-DNA interface: chemical probes of an interfacial water cavity

Electrostatics and hydration of a homeodomain-DNA complex are dissected by chemical modification. Selective neutralization of phosphate charges by methylphosphonate substitution demonstrates the differential importance of short- and long-range electrostatic interactions. Whereas the footprint of direct contacts is in accord with crystal structures, interference is also observed at non-contacted sites. Such sites adjoin a novel interfacial water cavity in the major groove. Non-contacted phosphodiester groups in the cavity are proposed to contribute to long-range ordering of an extended protein-water-DNA interface. Use of isolated S(p) and R(p) methylphosphonate diastereomers demonstrates that interference at this extended interface is stereoselective and charge-independent. Attenuation of protein binding presumably reflects groove-specific reorganization of bound water. Surprisingly, such attenuation can exceed that due to neutralization of a direct phosphate-side-chain salt bridge. These results support the hypothesis that hydration of an interfacial cavity functions as a non-covalent extension of the DNA surface. Stereo-specific interrogation of bound water by chemical synthesis provides a general method to assess the coupling between solvation and DNA recognition.