Experimentally based contact energies decode interactions responsible for protein–DNA affinity and the role of molecular waters at the binding interface

A major obstacle towards understanding the molecular basis of transcriptional regulation is the lack of a recognition code for protein–DNA interactions. Using high-quality crystal structures and binding data on the promiscuous family of C2H2 zinc fingers (ZF), we decode 10 fundamental specific interactions responsible for protein–DNA recognition. The interactions include five hydrogen bond types, three atomic desolvation penalties, a favorable non-polar energy, and a novel water accessibility factor. We apply this code to three large datasets containing a total of 89 C2H2 transcription factor (TF) mutants on the three ZFs of EGR. Guided by molecular dynamics simulations of individual ZFs, we map the interactions into homology models that embody all feasible intra- and intermolecular bonds, selecting for each sequence the structure with the lowest free energy. These interactions reproduce the change in affinity of 35 mutants of finger I (R2 = 0.998), 23 mutants of finger II (R2 = 0.96) and 31 finger III human domains (R2 = 0.94). Our findings reveal recognition rules that depend on DNA sequence/structure, molecular water at the interface and induced fit of the C2H2 TFs. Collectively, our method provides the first robust framework to decode the molecular basis of TFs binding to DNA

Electrostatic hot spot on DNA-binding domains mediates phosphate desolvation and the pre-organization of specificity determinant side chains

A major obstacle towards elucidating the molecular basis of transcriptional regulation is the lack of a detailed understanding of the interplay between non-specific and specific protein–DNA interactions. Based on molecular dynamics simulations of C2H2 zinc fingers (ZFs) and engrailed homeodomain transcription factors (TFs), we show that each of the studied DNA-binding domains has a set of highly constrained side chains in preset configurations ready to form hydrogen bonds with the DNA backbone. Interestingly, those domains that bury their recognition helix into the major groove are found to have an electrostatic hot spot for Cl− ions located on the same binding cavity as the most buried DNA phosphate. The spot is characterized by three protein hydrogen bond donors, often including two basic side chains. If bound, Cl− ions, likely mimicking phosphates, steer side chains that end up forming specific contacts with bases into bound-like conformations. These findings are consistent with a multi-step DNA-binding mechanism in which a pre-organized set of TF side chains assist in the desolvation of phosphates into well defined sites, prompting the re-organization of specificity determining side chains into conformations suitable for the recognition of their cognate sequence