Origins of DNA-binding specificity: role of protein contacts with the DNA backbone

Abstract

A central question in protein-DNA recognition is the origin of the specificity that permits binding to the correct site in the presence of excess, nonspecific DNA. In the P22 Arc repressor, the Phe-10 side chain is part of the hydrophobic core of the free protein but rotates out to pack against the sugar-phosphate backbone of the DNA in the repressor-operator complex. Characterization of a library of position 10 variants reveals that Phe is the only residue that results in fully active Arc. One class of mutants folds stably but binds operator with reduced affinity; another class is unstable. FV10, one member of the first class, binds operator DNA and nonoperator DNA almost equally well. The affinity differences between FV10 and wild type indicate that each Phe-10 side chain contributes 1.5-2.0 kcal to operator binding but less than 0.5 kcal/mol to nonoperator binding, demonstrating that contacts between Phe-10 and the operator DNA backbone contribute to binding specificity. This appears to be a direct contribution as the crystal structure of the FV10 dimer is similar to wild type and the Phe-10-DNA backbone interactions are the only contacts perturbed in the cocrystal structure of the FV10-operator complex.

Figure 1

(A) In the Arc-operator complex (2), the N-terminal arms and β-sheet of each Arc dimer are positioned to contact the operator DNA, and the side chains of Phe-10 and Phe-10′ interact with phosphate oxygen and ribose atoms of the DNA backbone. These contacts between Phe-10 and the DNA backbone contribute to both the specificity and affinity of operator binding as described in the text. This panel and Fig. 3A were prepared by using molscript (26). (B) Superimposition of the conformation of the Phe-10 side chain in unbound Arc (blue) and operator-bound Arc (yellow).

Figure 2

(A) Thermal stability monitored by circular dichroism. (B) Operator-DNA binding assayed by gel mobility-shift experiments. The dotted lines are theoretical fits of the data by using the K_u and K₂ values listed in Table 1.

Figure 3

(A) Superposition of backbone traces of the structures of the wild-type Arc dimer (cyan) and the mutant FV10 dimer (magenta). The C_α atoms of Phe-10 and Val-10 are shown as small spheres. (B) Superposition of the polypeptide backbones of residues 8–14 of wild-type Arc (magenta), the FV10 mutant (cyan), and wild-type Arc in the operator-bound complex (gold). The side chains of Phe-10 or Val-10, Leu-12, and Trp-14 also are shown.

Figure 4

Superimposition of the protein and DNA backbones from the protein-DNA cocrystal structures of wild-type Arc (yellow) and the FV10 mutant (orange). The wild-type Phe-10 side chains and mutant Val-10 side chains are shown in ball-and-stick representation.

Figure 5

The base-contact residues, Gln-9 and Asn-11, have similar conformations in the wild-type Arc (Right) and FV10 (Left) cocrystal structures. Residues from the Arc B subunit are shown, and the electron density (1 σ) is from simulated-annealing omit maps.

Figure 6

Competition of unlabeled specific and nonspecific DNA for wild-type Arc (Upper) or FV10 (Lower) binding to a 22-bp, ³²P-labeled oligonucleotide containing the left arc operator subsite. Competition was assayed by the reduction in the binding of protein to the labeled subsite DNA in gel mobility-shift experiments.