Probing the Interface of the trp Repressor–Operator Complex Using Operator Sequences Containing Isosteric Base-Pair Analogues

A series of base pair analogues, dI-dM and dD-dU, have been used in place of the native dA-dT and dG-dC residues to probe the interface between the trp repressor and its operator sequence. Both analogues maintain the interstrand hydrogen bond character that correlates with the corresponding native base pair, but alters the relative positions of the amino and carbonyl residues of the base pairs that extend into the major groove of B-form DNA. None of the sequences tested exhibit native-like binding with the trp repressor. Although functional group contacts between complementary hydrogen bond donors and acceptors could account for these differences, such a scenario is not consistent with the published crystal structures. Differences in binding by these analogue sequences is suggested to result from incremental differences in the van der Waals contacts at the interface between the protein and the nucleic acid. Although this surface would appear to be nonspecific in nature, previous studies have indicated that the state of hydration in the major groove for the free and complexed operators is in part conserved. These bound water molecules alter the nature of the van der Waals surface and could function as an extension of the van der Waals surface of the major groove. Either of the base pair analogues, dI-dM or dD-dU, results in either the loss of a conserved site of hydration, or the introduction of a new hydration site. Either effect would alter the van der Waals surface relative to the native operator and result in reduced affinity between the protein and nucleic acid. Additional analogues designed to alter the nature of the van der Waals surface by removing polar functional groups (adenine → purine) result in some cases in higher affinity binding than is observed with the native operator sequence.