Predicting indirect readout effects in protein-DNA interactions

Abstract

Recognition of DNA by proteins relies on direct interactions with specific DNA-functional groups, along with indirect effects that reflect variable energetics in the response of DNA sequences to twisting and bending distortions induced by proteins. Predicting indirect readout requires knowledge of the variations in DNA curvature and flexibility in the affected region, which we have determined for a series of DNA-binding sites for the E2 regulatory protein by using the cyclization kinetics method. We examined 16 sites containing different noncontacted spacer sequences, which vary by more than three orders of magnitude in binding affinity. For 15 of these sites, the variation in affinity was predicted within a factor of 3, by using experimental curvature and flexibility values and a statistical mechanical theory. The sole exception was traced to differential magnesium ion binding.

Fig. 1.

Illustration of the E2 protein-mediated mini-DNA looping system. (A) The conserved binding sites for all E2 proteins, where the 4-bp spacer sequence (N4) can be any base. (B) Crystal structure of HPV-18 E2 DBD complexed with CAACCGAATTCGGTTG (16). E2 DBD is a homodimer, with its two monomers colored red and blue, respectively. Each monomer inserts an α-helix in the major groove of ACCG, holding the DNA in an arc and leaving the spacer AATT uncontacted by the E2 protein. (C) Calculated equilibrium DNA axes in the free states in solution and in the constrained states in E2–DNA complexes. The scaffold conformation is derived from measured global DNA curvature and twist-induced by HPV-16 E2 DBD binding. All of the DNA-binding sites are aligned by global translation and rotation to overlap their base pairs at one end and then deformed with minimum energy such that the base pairs at the other end coincide with that of the scaffold. The strained state is modeled with six constraints at this end, which is similar to the case in DNA cyclization, with its corresponding J factor calculated. The bending angles shown here are amplified by 1.5-fold for better clarity.

Fig. 2.

J factors for DNA constructs of variable overall length containing E2-binding site AAAC or AGCT in the presence or absence of E2 DBD. The best-fit parameters for E2 bound DNA are given in Table 1.

Fig. 3.

Variation of the J factor with each parameter for global DNA structure and mechanical properties. Unless indicated as variable, the default parameters used for calculations shown here are 0° kink, 34.45° twist, 4.678° bending flexibility, and 4.388° twisting flexibility, respectively. The kink is a roll at the middle of the binding site. Note the difference in scale for the J factor between the two images.