DNA encodes the biological instructions for the functioning of living organisms by folding into a complex three-dimensional structure. By wrapping around histone proteins, eukaryotic DNA forms nucleosome particles, which in turn self-associate to form a chromatin fiber with a structure that changes with the cell-cycle. DNA packaged into nucleosomes (“nucleosomal DNA”) is less accessible for most DNA-binding transcription factors and, accordingly, less active for gene expression. Considerable research efforts are being devoted to studying chromatin structure at distinct resolutions and to exploiting the therapeutic potential of nucleosomal DNA. Some proteins can bind to the exposed face of nucleosomal DNA and drive biological processes associated with cancer and viral infection. From a therapeutic point of view, drugs that bind to nucleosomal DNA with specificity hold great potential for blocking the action of proteins involved in a number of diseases, as they alter the accessibility of DNA to regulatory proteins.
The design of nucleosome-binding molecules specific for a DNA sequence remains an outstanding challenge. Nevertheless, nucleosomal DNA binding specificity is hard to achieve by repurposing currently existing chromatin-interacting proteins due to the lack of suitable natural templates. Recent advances in computational protein design provide a route to custom-tailor protein structures and interactions, and here we set out to design, for the first time, proteins customized to bind nucleosomal DNA with specificity. Such protein customization underlies the challenge of identifying and/or building a protein structure, its optimal placement around the DNA target site, and an amino-acid sequence that stabilizes the target protein-DNA complex.
DesProtDNA comprised three objectives. First, development of a general computational method to de novo design protein-DNA binding interfaces in the nucleosome with specificity. Second, experimentally validate the method by testing proteins designed to target a nucleosome with high-resolution structure available. Third, use the method to design proteins binding specific nucleosomal DNA sites involved in diseases. Additionally, the project aims to provide the researcher with new computational and experimental skills to implement new protein-DNA design schemes and validate them.
In conclusion, we have developed three different computational approaches that range from redesigning currently existing proteins to full de novo design to achieve optimal binding to nucleosomal DNA. The designed proteins have been experimentally characterized by expressing them recombinantly and assessing their binding properties. Novel nucleosome binding proteins have been obtained that where designed to target specific DNA sites and have binding interfaces similar as designed. Yet, further high-resolution structural information is necessary to confirm the actual binding mode of the designs. This is the first time proteins have been designed to bind the exposed face of nucleosomal DNA. Obtaining high-resolution structural conformation of our designs will lay the groundwork for developing new chromatin-based therapeutic strategies as well as innovative research tools for chromatin studies using our computational design method.
