Flexible DNA bending in HU–DNA cocrystal structures

HU and IHF are members of a family of prokaryotic proteins that interact with the DNA minor groove in a sequence-specific (IHF) or non-specific (HU) manner to induce and/or stabilize DNA bending. HU plays architectural roles in replication initiation, transcription regulation and site-specific recombination, and is associated with bacterial nucleoids. Cocrystal structures of Anabaena HU bound to DNA (1P71, 1P78, 1P51) reveal that while underlying proline intercalation and asymmetric charge neutralization mechanisms of DNA bending are similar for IHF and HU, HU stabilizes different DNA bend angles (∼105–140°). The two bend angles within a single HU complex are not coplanar, and the resulting dihedral angle is consistent with negative supercoiling. Comparison of HU–DNA and IHF–DNA structures suggests that sharper bending is correlated with longer DNA binding sites and smaller dihedral angles. An HU-induced bend may be better modeled as a hinge, not a rigid bend. The ability to induce or stabilize varying bend angles is consistent with HU’s role as an architectural cofactor in many different systems that may require differing geometries

Azaindole-Based Inhibitors of Cdc7 Kinase: Impact of the Pre-DFG Residue, Val 195

To investigate the role played by the unique pre-DFG residue Val 195 of Cdc7 kinase on the potency of azaindole-chloropyridines (<b>1</b>), a series of novel analogues with various chloro replacements were synthesized and evaluated for their inhibitory activity against Cdc7. X-ray cocrystallization using a surrogate protein, GSK3β, and modeling studies confirmed the azaindole motif as the hinge binder. Weaker hydrophobic interactions with Met 134 and Val 195 by certain chloro replacements (e.g., H, methyl) led to reduced Cdc7 inhibition. Meanwhile, data from other replacements (e.g., F, O) indicated that loss of such hydrophobic interaction could be compensated by enhanced hydrogen bonding to Lys 90. Our findings not only provide an in-depth understanding of the pre-DFG residue as another viable position impacting kinase inhibition, they also expand the existing knowledge of ligand-Cdc7 binding

Identification of a peptide inhibitor for the histone methyltransferase WHSC1

<div><p>WHSC1 is a histone methyltransferase that is responsible for mono- and dimethylation of lysine 36 on histone H3 and has been implicated as a driver in a variety of hematological and solid tumors. Currently, there is a complete lack of validated chemical matter for this important drug discovery target. Herein we report on the first fully validated WHSC1 inhibitor, PTD2, a norleucine-containing peptide derived from the histone H4 sequence. This peptide exhibits micromolar affinity towards WHSC1 in biochemical and biophysical assays. Furthermore, a crystal structure was solved with the peptide in complex with SAM and the SET domain of WHSC1L1. This inhibitor is an important first step in creating potent, selective WHSC1 tool compounds for the purposes of understanding the complex biology in relation to human disease.</p></div

Crystallographic data collection and refinement statistics for WHSC1L1 1054–1285 with PTD2 and SAM.

<p>Crystallographic data collection and refinement statistics for WHSC1L1 1054–1285 with PTD2 and SAM.</p

Representative sensorgram for PTD2 binding to Avi-tagged WHSC1 941–1240 from single-cycle kinetic SPR measurements.

<p>WHSC1 was immobilized on a streptavidin-coated chip and peptide inhibitor was co-injected with SAM utilizing a 3-fold, 5-point dilution series ending at a 20 μM top concentration. Data reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197082#pone.0197082.t002" target="_blank">Table 2</a> is presented as the standard deviation of three independent experiments.</p

WHSC1L1 1054–1285 has a similar overall structure in relation to other NSD family proteins and can form a ternary complex with SAM and PTD2.

<p>(A) Superposition of NSD family proteins (green/yellow = WHSC1L1-PTD2 (PDB code = 6CEN); cyan = WHSC1L1 (PDB code = 5UPD); magenta = WHSC1 (PDB code = 5LSU); purple = NSD1 (PDB code = 3OOI). All protein chains are shown as ribbons; SAM and PTD2 are depicted in stick representation. (B) Structure of WHSC1L1-PTD2-SAM ternary complex. Hydrogen bonds are indicated with dashed lines. (C) Superposition of WHSC1L1-PTD2 and SETD2-H3.3 K36M (grey; PDB code = 5JJY).</p

Representative SPR sensorgrams for PTD2 binding to Avi-tagged WHSC1 941–1240 in the absence or presence of SAM analogs.

<p>WHSC1 was immobilized on a streptavidin-coated chip and peptide inhibitor was either injected in the absence of cofactor (left panel), co-injected with SAH (middle panel), or co-injected with SFG (right panel) utilizing a 2-fold, 10-point dilution series ending at a 100 μM top concentration.</p

Biochemical and biophysical peptide inhibitor potency values for WHSC1 941–1240 and WHSC1L1 1054–1285^a.

<p>Biochemical and biophysical peptide inhibitor potency values for WHSC1 941–1240 and WHSC1L1 1054–1285<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197082#t002fn001" target="_blank">^a</a>.</p

Norleucine-containing peptides can inhibit WHSC1 and WHSC1L1 activity in vitro.

<p>Representative peptide inhibitor biochemical dose-response curves for (A) WHSC1 941–1240 and (B) WHSC1L1 1054–1285. Error bars represent the standard deviation of three independent replicates. Resulting IC₅₀ values are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197082#pone.0197082.t002" target="_blank">Table 2</a>.</p

Isothermal titration calorimetry of PTD2 binding to WHSC1 941–1240.

<p>Upper panel, calorimetric trace for ligand titration; lower panel, binding isotherm from calorimetric trace. WHSC1 concentration in the cell was 10 μM supplemented with 100 μM SAM. PTD2 concentration in the syringe was 100 μM.</p