Abstract
The energetic contributions of individual DNA-contacting side chains to specific DNA recognition in the human papillomavirus 16 E2C-DNA complex is small (less than 1.0 kcal mol(-1)), independent of the physical and chemical nature of the interaction, and is strictly additive. The sum of the individual contributions differs 1.0 kcal mol(-1) from the binding energy of the wild-type protein. This difference corresponds to the contribution from the deformability of the DNA, known as "indirect readout." Thus, we can dissect the energetic contribution to DNA binding into 90% direct and 10% indirect readout components. The lack of high energy interactions indicates the absence of "hot spots," such as those found in protein-protein interfaces. These results are compatible with a highly dynamic and "wet" protein-DNA interface, yet highly specific and tight, where individual interactions are constantly being formed and broken.
Highlights
The energetic contributions to the binding of proteins to specific DNA sequences is the result of the formation of an intricate network of contacts between the binding partners, often coupled to conformational rearrangements in either or both macromolecules [2]
The E2 transcriptional regulator (E2C)-DNA system is of particular interest because the interaction involves components of both direct and indirect readouts of the DNA sequence and, in contrast to many other protein-DNA systems, there is an established decoupling between the energetic contributions from both mechanisms [15]
We experimentally traced the origins of the direct readout energetics by means of a systematic rational perturbation approach of the DNA binding interface and determined that most of the binding energy comes from this recognition mode in this model system
Summary
The energetic contributions to the binding of proteins to specific DNA sequences is the result of the formation of an intricate network of contacts between the binding partners, often coupled to conformational rearrangements in either or both macromolecules [2]. The fact that mutations on DNA bases that are not directly contacted by the protein often affect the interaction energetics [5] implies that local or distant sequence-dependent conformational transitions of the DNA molecule affect protein-DNA binding, a mechanism termed indirect readout [6]. Experimental determination of the energetic contributions of direct and indirect readout requires modification of amino acid side chains and/or DNA bases, perturbations that would normally affect the structural and energetic properties of distant, non-targeted contacts [7, 8]. These mutually dependent effects preclude a straightforward assignment of the energetic contributions to DNA binding in most protein-DNA systems. We experimentally traced the origins of the direct readout energetics by means of a systematic rational perturbation approach of the DNA binding interface and determined that most of the binding energy comes from this recognition mode in this model system
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