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. Because many proteins deform DNA upon binding (1, 2), it is reasonable to expect that protein-DNA association might be facilitated by enhanced ease of DNA deformation and a match between intrinsic DNA shape in solution and the strained DNA conformation in the complex. The lack of a simple correspondence code between amino acids and DNA bases in hydrogen bonding, or direct readout, makes a general prediction of protein-DNA affinity impossible at this time. However, variations in contributions from indirect readout due to sequencedependent shape and mechanical properties may be predictable, yielding a partial structural code for protein-DNA interaction (3). Many attempts to test this idea have been performed during the last two decades with different proteins, such as nucleosome (4-6), cAMP-binding protein (7), 434 repressor (8, 9), TATA box-binding protein (10), and E2 protein (11).Among the impediments to realization of this objective is the difficulty of accurately determining the multiple DNA parameters involved, including magnitude and direction of curvature, helical twist, and bending and torsional flexibilities, for a variety of sequences corresponding to the region of indirect readout. The DNA cyclization method (1, 12, 13), in the high-throughput format we recently described (14), coupled with a statistical mechanical theory (15) for extracting the curvature and flexibility parameters from the data, provides a solution to this problem. Although the extent of variation of DNA flexibility with sequence remains controversial, recent results from the cyclization kinetics method show that a DNA sequence with high histone affinity (TATAAACGCC) has a nearly 2-fold smaller bending force constant and 35% less torsional rigidity than generic DNA (6). Also, an AT repeating sequence has 28% lower bending rigidity (14).Experimental testing of the accuracy of prediction of indirect readout requires that the nucleotides involved not be also engaged in direct interactions. A system that meets this requirement is the noncontacted spacer region in the DNA-binding site for the E2 protein encoded by the human papillomavirus (HPV) type 16 genome (11)....

The shape of the DNA minor groove directs binding by the DNA-bending protein Fis

“…However, unlike Fis, the E2 spacer region adapts by a combination of bending (up to 45°) into the minor groove and compression (down to 2.7 Å ) of the minor groove. Current evidence indicates that the presence of both a preformed bend (or bending flexibility) and an intrinsically narrow minor groove targets E2 binding (Hegde 2002;Zhang et al 2004).…”

Targeted Chemical Wedges Reveal the Role of Allosteric DNA Modulation in Protein−DNA Assembly

“…In this protein, the two contiguous DNA binding modules when expressed as separate peptides are able to reciprocally increase the affinity of each other at the composite DNA binding site, even in the absence of any direct contact (18). Intriguingly, in these two cases, as well as several others, allosteric modulation often positively contributes ~10-fold or ~1.5 kcal/mol to assembly of the partner proteins (9,11,(14)(15)(16)(17)(18)(19)(20)(21)(22). This is the same scale at which DNA deformation caused by hairpin polyamides contribute to Exd binding.…”

Switching Control of Expression ofptsGfrom the Mlc Regulon to the NagC Regulon

“…It is a common observation that proteins produce substantially stronger curvature by exploiting inherent bends present in the free DNA (2). For example, a strong correlation between DNA flexibility and the binding affinity of papillomavirus E2 proteins to consensus sites with various central linker sequences has been demonstrated both experimentally and by molecular dynamic simulations (5,49). Both CG and GC base pairs produce flexible dinucleotide steps, as calculated from nuclear magnetic resonance structures in solution or derived from analysis of X-ray structures (9,20), and thus are easily deformed upon protein binding.…”