Crystal structure of an engineered cro monomer bound nonspecifically to DNA: Possible implications for nonspecific binding by the wild‐type protein

Abstract

The structure has been determined at 3.0 8, resolution of a complex of engineered monomeric Cro repressor with a seven-base pair DNA fragment. Although the sequence of the DNA corresponds to the consensus half-operator that is recognized by each subunit of the wild-type Cro dimer, the complex that is formed in the crystals by the isolated monomer appears to correspond to a sequence-independent mode of association. The overall orientation of the protein relative to the DNA is markedly different from that observed for Cro dimer bound to a consensus operator. The recognition helix is rotated 48" further out of the major groove, while the turn region of the helix-turn-helix remains in contact with the DNA backbone. All of the direct base-specific interactions seen in the wild-type Cro-operator complex are lost. Virtually all of the ionic interactions with the DNA backbone, however, are maintained, as is the subset of contacts between the DNA backbone and a channel on the protein surface. Overall, 25% less surface area is buried at the protein-DNA interface than for half of the wild-type Cro-operator complex, and the contacts are more ionic in character due to a reduction of hydrogen bonding and van der Waals interactions. Based on this crystal structure, model building was used to develop a possible model for the sequence-nonspecific interaction of the wild-type Cro dimer with DNA. In the sequence-specific complex, the DNA is bent, the protein dimer undergoes a large hinge-bending motion relative to the uncomplexed form, and the complex is twofold symmetric. In contrast, in the proposed nonspecific complex the DNA is straight, the protein retains a conformation similar to the apo form, and the complex lacks twofold symmetry. The model is consistent with thermodynamic, chemical, and mutagenic studies, and suggests that hinge bending of the Cro dimer may be critical in permitting the transition from the binding of protein at generic sites on the DNA to binding at high affinity operator sites.

Experimental snapshots of a protein-DNA binding landscape

“…However, the few kinetic mechanisms determined to date revealed a richer picture, with parallel routes and transient intermediates (4)(5)(6)(7)(8). In the absence of high-resolution kinetic work, structural studies suggested that multistate routes for protein-DNA-binding start with the formation of nonspecific interactions with the DNA backbone (1,(9)(10)(11)(12)(13)(14)(15)(16)(17), and that some dual-role residues may form transient nonnative interactions along the route (13-15, 17). E2C-DNA-binding is also two-state at equilibrium (21) but kinetically complex (4) ( Figure 4A).…”

“…It has been found that proteins contact the DNA using approximately the same binding site and orientation in all complexes (10)(11)(12)(13)(14)(15)(16)(17)(18). In nonspecific complexes, the interface between the two molecules is stabilized by electrostatic interactions between charged protein side chains and the DNA backbone (1,(9)(10)(11)(12)(13)(14)(15)(16)(17).…”

“…The kinetics of DNA sequence recognition (3,4) often involve multistate kinetic routes with populated intermediates (4)(5)(6)(7)(8). The build up of molecular interactions along multistate routes has been deduced indirectly from structural and thermodynamic studies (1)(2)(3)(9)(10)(11)(12)(13)(14)(15)(16)(17), but kinetic characterization of intermediates and transition state ensembles is still scarce.…”

“…The structures of several proteins in complex with target DNA sites, variants thereof and random DNA sequences, have been solved and used as models for transient intermediates of specific recognition (10)(11)(12)(13)(14)(15)(16)(17). It has been found that proteins contact the DNA using approximately the same binding site and orientation in all complexes (10)(11)(12)(13)(14)(15)(16)(17)(18).…”

“…In nonspecific complexes, the interface between the two molecules is stabilized by electrostatic interactions between charged protein side chains and the DNA backbone (1,(9)(10)(11)(12)(13)(14)(15)(16)(17). In complexes with target DNA sites, there are also interactions between side chains and bases (10)(11)(12)(13)(14)(15)(16)(17). Strikingly, some so-called "dual-role residues" interact with the DNA backbone in nonspecific complexes and with DNA bases in specific complexes (14,15,17) or form different interactions with bases in nonspecific and specific complexes (13).…”