- 2:18 PM

Modulating the DNA Affinity of Elk-1 with Computationally Selected Mutations

Sheldon Park, Eric T. Boder, and Jeffery G. Saven. Chemistry, University of Pennsylvania, 231 S. 34th Street, Philadelphia, PA 19104

In order to regulate gene expression, transcription factors must first bind their target DNA sequences. The affinity of this binding is determined by both the network of interactions at the interface and the entropy change associated with the complex formation. To study the role of structural fluctuation in fine-tuning DNA affinity, we performed molecular dynamics (MD) simulations of two highly homologous proteins, Elk-1 and SAP-1, that exhibit different sequence specificity. Simulation studies show that several residues in Elk have significantly higher main chain root mean square deviations than their counterparts in SAP. In particular, a single residue D69 may contribute to Elk's lower DNA affinity for Pc-fos by structurally destabilizing the carboxy terminus of the recognition helix. While D69 does not contact DNA directly, the increased mobility in the region may contribute to its weaker binding. We measured the ability of single point mutants of Elk to bind Pc-fos in a reporter assay, in which D69 of wild type Elk has been mutated to other residues with higher helix propensity in order to stabilize the local conformation. The gains in transcriptional activity and the free energy of binding suggested from these measurements correlate well with stability gains computed from helix propensity and charge-macrodipole interactions. The study suggests that residues that are distal to the binding interface may indirectly modulate the binding affinity by stabilizing the protein scaffold required for efficient DNA interaction.