Structural Insight into the DNA Binding Function of Transcription Factor ERF.

ETS family transcription factors control development of different cell types in humans, whereas deregulation of these proteins leads to severe developmental syndromes and cancers. One of a few members of the ETS family that are known to act solely as repressors, ERF, is required for normal osteogenesis and hematopoiesis. Another important function of ERF is acting as a tumor suppressor by antagonizing oncogenic fusions involving other ETS family factors. The structure of ERF and the DNA binding properties specific to this protein have not been elucidated. In this study, we determined two crystal structures of the complexes of the DNA binding domain of ERF with DNA. In one, ERF is in a distinct dimeric form, with Cys72 in a reduced state. In the other, two dimers of ERF are assembled into a tetramer that is additionally locked by two Cys72-Cys72 disulfide bonds across the dimers. In the tetramer, the ERF molecules are bound to a pseudocontinuous DNA on the same DNA face at two GGAA binding sites on opposite strands. Sedimentation velocity analysis showed that this tetrameric assembly forms on continuous DNA containing such tandem sites spaced by 7 bp. Our bioinformatic analysis of three previously reported sets of ERF binding loci across entire genomes showed that these loci were enriched in such 7 bp spaced tandem sites. Taken together, these results strongly suggest that the observed tetrameric assembly is a functional state of ERF in the human cell.

Allosteric substrate activation of SAMHD1 shapes deoxynucleotide triphosphate imbalances by interconnecting the depletion and biosynthesis of different dNTPs.

SAMHD1 is a dNTPase that impedes replication of HIV-1 in myeloid cells and resting T lymphocytes. Here we elucidate the substrate activation mechanism of SAMHD1 that depends on dNTP binding at allosteric sites and the concomitant tetramerization of the enzyme. The study reveals that SAMHD1 activation involves an inactive tetrameric intermediate with partial occupancy of the allosteric sites. The equilibrium between the inactive and active tetrameric states, which is coupled to cooperative binding/dissociation of at least two allosteric dNTP ligands, controls the dNTPase activity of the enzyme, which, in addition, depends on the identity of the dNTPs occupying the four allosteric sites of the active tetramer. We show how such allosteric regulation determines deoxynucleotide triphosphate levels established in the dynamic equilibria between dNTP production and SAMHD1-catalyzed depletion. Notably, the mechanism enables a distinctive functionality of SAMHD1, which we call facilitated dNTP depletion, whereby elevated biosynthesis of some dNTPs results in more efficient depletion of others. The regulatory relationship between the biosynthesis and depletion of different dNTPs sheds light on the emerging role of SAMHD1 in the biology of dNTP homeostasis with implications for HIV/AIDS, innate antiviral immunity, T cell disorders, telomere maintenance and therapeutic efficacy of nucleoside analogs.