title: Modulating the DNA affinity of Elk-1 with computationally selected mutations.
abstract: 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 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 P(c-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 P(c-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.
authors.affiliation: Makineni Theoretical Laboratories, Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, PA 19104, USA.
authors.forename: Sheldon
authors.initials: S
authors.lastname: Park
authors.forename: Eric T
authors.initials: ET
authors.lastname: Boder
authors.forename: Jeffery G
authors.initials: JG
authors.lastname: Saven
mesh_terms: D000595:Amino Acid Sequence; D003198:Computer Simulation; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050807:ets-Domain Protein Elk-1; D050808:ets-Domain Protein Elk-4
chemical_list: D004268:DNA-Binding Proteins; C494112:ELK1 protein, human; C494193:ELK4 protein, human; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D050807:ets-Domain Protein Elk-1; D050808:ets-Domain Protein Elk-4; D004247:DNA