Characterizing the interaction between F12-ATP and GST-HEPN.

<p>A. Overlay of ¹⁵N,¹H-HSQC NMR spectra of 200 μM ¹⁵N-HEPN alone (<i>red</i>) and following the addition of GTP (50 μM GTP in <i>green</i> and 100 μM in <i>blue</i>). Residues that showed chemical shift perturbations in the presence of GTP were identified from the assigned HEPN spectrum and labeled. B. Residues involved in GTP-binding are mapped onto the HEPN crystal structure (PDB: 3O10) and labeled in <i>blue</i>. Malonate ions present in the crystal structure are presented in stick representation, with their oxygen atoms labeled in <i>red</i>. C. Overlay of ¹⁵N,¹H-HSQC NMR spectra of 110 μM ¹⁵N-HEPN alone (<i>red</i>) and following the addition of F12-ATP (55 μM in <i>green</i> and 110 μM in <i>blue</i>). D. Residues involved in F12-ATP-binding are mapped onto the HEPN crystal structure. Residues involved in both F12-ATP and GTP-binding are labeled in <i>blue</i> and additional residues that shifted during the F12-ATP titration are labeled in <i>red</i>.</p

Effects of MgCl₂ and EDTA on the interaction between F12-ATP and GST-HEPN.

<p>A. F12-ATP vs. GST-HEPN FP titration was carried out in the presence of 1 mM MgCl₂ and increasing concentrations of EDTA. B. F12-ATP vs. GST-HEPN FP titration with increasing concentrations of EDTA alone. C. EDTA-interacting residues are labeled in <i>blue</i> on the HEPN crystal structure. D. G-tetra-P-binding residues are mapped onto HEPN. G-tetra-P-binding residues that are also involved in GTP-binding are labeled in <i>blue</i> and additional residues only involved in G-tetra-P-binding are labeled in <i>red</i>.</p

Fluorescence polarization assay development and validation.

<p>A. Chemical structure of 8[(4-amino)butyl]-amino-ATP-MNT (MABA-ATP). B. Chemical structure of fluorescein-12-ATP (F12-ATP). C. 0.5 μM MABA-ATP and 5 nM F12-ATP were titrated against various concentrations of GST-HEPN. D. 0.5 μM MABA-ATP and 5 nM F12-ATP and 120 nM / 7 μM GST-HEPN were titrated against various concentrations of GTP to verify that the probes bind to HEPN at its nucleotide-binding site.</p

Residues involved in binding mapped onto HEPN crystal structure.

<p>Residues that produced chemical shift changes during the NMR titrations with DMSO (A), AN670 (B), AK968 (C) and AN652 (D) were mapped onto the HEPN crystal structure. In each structure, DMSO-binding residues are labeled in <i>blue</i> and additional residues binding to the hit candidates are labeled in <i>red</i>.</p

Affinities of nucleotides and nucleotide analogs for HEPN estimated from FP competition assays.

<p>The affinities of the compounds were summarized along with their chemical structures.</p

Confirmation of HTS candidates by FP competition assay.

<p>A and B. 5 nM F12-ATP and 200 nM GST-HEPN were titrated with the hits to confirm their binding to GST-HEPN. C. Affinities of the compounds for HEPN were estimated from the competition assays and summarized along with their chemical structures.</p