Binding constants of product binding to NUDT16.

<p>All values were determined by Origin from MicroCal Software, Inc., after fitting the experimental data to a one binding-site model, allowing all parameters to evolve freely in the course of the fit.</p

List of enzymes used in this study.

<p>List of enzymes used in this study.</p

Concentration-dependent stabilization of UPP1 (A) in the presence of uridine, vidarabine, idoxuridine, trifluridine and fluorouracil; GDA (B) in the presence of guanine, valaciclovir, aciclovir, thioguanine, penciclovir, ganciclovir and mercaptopurine; and concentration dependent destabilization of RRM1 (C) in the presence of azathioprine.

<p>Concentration-dependent stabilization of UPP1 (A) in the presence of uridine, vidarabine, idoxuridine, trifluridine and fluorouracil; GDA (B) in the presence of guanine, valaciclovir, aciclovir, thioguanine, penciclovir, ganciclovir and mercaptopurine; and concentration dependent destabilization of RRM1 (C) in the presence of azathioprine.</p

Kinetic analysis of UPP1 using Lineweaver-Burk plots.

<p>Uridine was used as substrate (50 to 375 µM) and vidarabine was as inhibitor at 500 µM (?), 250 µM (?), and 100 µM (▴) and 0 µM (⧫).</p

Ligands of GDA and mean ΔT_agg with 1000 µM NA.

<p>Ligands of GDA and mean ΔT_agg with 1000 µM NA.</p

The ΔT_agg values for the 23 enzymes screened against NAL.

<p>The enzymes are numbered: <b>1</b> ADSS2, <b>2</b> ADSL, <b>3</b> BPNT1, <b>4</b> CMPK2, <b>5</b> CTPS2, <b>6</b> DCTD, <b>7</b> DPYS, <b>8</b> GART, <b>9</b> GDA, <b>10</b> GMPR2, <b>11</b> GMPS, <b>12</b> ITPA, <b>13</b> NT5C2, <b>14</b> NT5C3, <b>15</b> NUDT16, <b>16</b> PAICS, <b>17</b> PRTFDC1, <b>18</b> RRM1, <b>19</b> UCK1, <b>20</b> UMPS (1), <b>21</b> UMPS (2), <b>22</b> UPB1, <b>23</b> UPP1, <b>24</b> UPP2. ΔT_agg represents the difference between T_agg of a protein in the presence and absence of a compound. The ΔT_agg values are given as color codes based on ΔT_agg calculated from two values within the same screen. The maximum average mean deviation for ΔT_agg is 0.5°C. However, in some cases, one of the values has been disregarded due to inappropriate curve fitting parameters. In total of 1080 measurements 44 have either one value missing (8) or an average mean deviation greater than 0.5°C (36).</p

Data collection and refinement statistics.

<p>Values in parentheses refer to the highest resolution shell.</p><p>^a</p><p></p><p></p><p></p><p><mi>R</mi></p><p><mi>m</mi><mi>e</mi><mi>r</mi><mi>g</mi><mi>e</mi></p><p></p><mo>=</mo><p></p><p></p><p></p><p><mo>∑</mo></p><p><mi>h</mi><mi>k</mi><mi>l</mi></p><p></p><p></p><p></p><p><mo>∑</mo><mi>i</mi></p><p></p><p><mo stretchy="true">|</mo></p><p></p><p><mi>I</mi><mi>i</mi></p><mo stretchy="false">(</mo><mi>h</mi><mi>k</mi><mi>l</mi><mo stretchy="false">)</mo><mo>−</mo><p><mo stretchy="true">⟨</mo></p><p></p><p><mi>I</mi></p><p><mi>h</mi><mi>k</mi><mi>l</mi></p><p></p><p></p><mo stretchy="true">⟩</mo><p></p><p></p><mo stretchy="true">|</mo><p></p><p></p><p></p><p></p><p></p><p></p><mo stretchy="true">/</mo><p></p><p></p><p><mo>∑</mo></p><p><mi>h</mi><mi>k</mi><mi>l</mi></p><p></p><p></p><p></p><p><mo>∑</mo><mi>i</mi></p><p></p><p><mo stretchy="true">⟨</mo></p><p></p><p><mi>I</mi></p><p><mi>h</mi><mi>k</mi><mi>l</mi></p><p></p><p></p><mo stretchy="true">⟩</mo><p></p><p></p><p></p><p></p><p></p><p></p><p></p><p></p><p></p><p></p><p></p><p>^b</p><p></p><p></p><p></p><p></p><p></p><p><mi>R</mi></p><p><mi>p</mi><mo>.</mo><mi>i</mi><mo>.</mo><mi>m</mi><mo>.</mo></p><p></p><mo>=</mo><p></p><p></p><p></p><p><mo>∑</mo></p><p><mi>h</mi><mi>k</mi><mi>l</mi></p><p></p><p></p><p><mo>[</mo></p><p><mn>1</mn><mo>/</mo></p><p><mo>(</mo></p><p><mi>N</mi><mo>−</mo><mn>1</mn></p><mo>)</mo><p></p><p></p><mo>]</mo><p></p><p></p><p></p><p></p><p><mn>1</mn><mo>/</mo><mn>2</mn></p><p></p><p></p><p><mo>∑</mo><mi>i</mi></p><p></p><p><mo>|</mo></p><p></p><p><mi>I</mi><mi>i</mi></p><p><mo>(</mo></p><p><mi>h</mi><mi>k</mi><mi>l</mi></p><mo>)</mo><p></p><mo>−</mo><p><mo>⟨</mo></p><p></p><p><mi>I</mi></p><p><mi>h</mi><mi>k</mi><mi>l</mi></p><p></p><p></p><mo>⟩</mo><p></p><p></p><mo>|</mo><p></p><p></p><p></p><p></p><mo stretchy="true">/</mo><p></p><p></p><p><mo>∑</mo></p><p><mi>h</mi><mi>k</mi><mi>l</mi></p><p></p><p></p><p></p><p><mo>∑</mo><mi>i</mi></p><p></p><p><mi>I</mi><mi>i</mi></p><p><mo>(</mo></p><p><mi>h</mi><mi>k</mi><mi>l</mi></p><mo>)</mo><p></p><p></p><p></p><p></p><p></p><p></p><p></p><p></p><p></p><p></p><p></p><p>^c</p><p></p><p></p><p></p><p></p><p></p><p><mi>R</mi></p><p><mi>w</mi><mi>o</mi><mi>r</mi><mi>k</mi></p><p></p><mo>=</mo><p><mo>∑</mo></p><p></p><p><mo stretchy="true">|</mo></p><p></p><p><mo>|</mo></p><p></p><p><mi>F</mi></p><p><mi>o</mi><mi>b</mi><mi>s</mi></p><p></p><p></p><mo>|</mo><p></p><mo>−</mo><p><mo>|</mo></p><p></p><p><mi>F</mi></p><p><mi>c</mi><mi>a</mi><mi>l</mi><mi>c</mi></p><p></p><p></p><mo>|</mo><p></p><p></p><mo stretchy="true">|</mo><p></p><p></p><p></p><p></p><mo stretchy="true">/</mo><p></p><p><mo>∑</mo></p><p></p><p><mo>|</mo></p><p></p><p><mi>F</mi></p><p><mi>o</mi><mi>b</mi><mi>s</mi></p><p></p><p></p><mo>|</mo><p></p><p></p><p></p><p></p><p></p><p></p><p></p><p></p> where <i>F</i>_<i>obs</i> and <i>F</i>_<i>calc</i> are observed and calculated structure factors, respectively. R_free correspond to a subset of 5% of reflections randomly selected omitted during refinement.<p></p><p>^d Others refer to the Cl^- ion present in NUDT16 IMP-bound structure.</p><p>^e Values determined by MolProbity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131507#pone.0131507.ref039" target="_blank">39</a>].</p><p>Data collection and refinement statistics.</p

Mean ΔTagg (°C) for UPP1.

<p>The average deviation from mean value is less than ±0.5°C.</p>*<p>Indicates that deviation is greater than ±0.5°C. T_agg of UPP1 in the absence of NA is listed. The concentration of NA was 500 µM and R1P (ribose-1-phosphate) was 1 mM.</p

Interactions between NUDT16 and IMP.

<p>(A) Overview of the IMP molecules bound to NUDT16 dimer. Both the view and the color codes correspond to the ones used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131507#pone.0131507.g001" target="_blank">Fig 1A</a>. IMP and Mg²⁺ ions are shown as sticks and van der Waals spheres, respectively. (B) Electrostatic surface representation of NUDT16 bound to IMP. Molecular surface of NUDT16 is colored according to its electrostatic potential as calculated by the APBS program [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131507#pone.0131507.ref024" target="_blank">24</a>]. The color ramp varies from blue to red corresponding to +5<i>k</i>T/e and to -5<i>k</i>T/e, respectively. (C) Interactions between NUDT16, IMP and Mg²⁺ around the ribose and the base. NUDT16 is shown in the cartoon representation and colored either in violet (NUDIX motifs) or blue-grey (other region of the protein). IMP, IMP-interacting residues and Val22 are shown as sticks with their carbon atoms colored either in violet (residues belonging to the NUDIX motif), blue-grey (other NUDT16 residues) or green (IMP). NUDT16 residues are labeled using the single-letter code. Mg²⁺ ions are displayed as orange van der Waals spheres. Water molecules involved in hydrogen-bond network between NUDT16 and IMP are represented as small red spheres and labeled in red according to their numbering in the PDB file. H-bonds are symbolized by dashed lines. (D) Interactions between NUDT16, IMP and Mg²⁺ around the phosphate. Same color code as in (C) except that H-bonds and salt-bridges are represented by purple and yellow dashed lines respectively. (E) Ligplot [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131507#pone.0131507.ref025" target="_blank">25</a>] diagram of the interactions between NUDT16, IMP and Mg²⁺. Water molecules and Mg²⁺ ions are show as cyan and green spheres respectively. Covalent bonds in the IMP molecule are colored in violet and in orange when they belong to NUDT16. Hydrogen bonds and salt-bridges are represented by green dashed lines, hydrophobic interactions by red dashed lines. NUDT16 residues are labeled in black and red when they are involved in hydrophobic and polar interactions, respectively. Heteroatoms on the hypoxanthine ring of IMP are labelled in violet. Bonds between the Mg²⁺ ions and other atoms are depicted by violet plain lines. Water molecules (when they are shown in Fig 3C) are labeled in cyan according to their numbering in the PDB file.</p

Mean ΔTagg (°C) for dCK.

<p>The Mean ΔT_agg was based on two samples within the screen. The average deviation from mean value was less than ±0.5°C. The NAs were listed according to the largest increase in thermal shifts (ΔT_agg) in the presence of 500 µM NA and 1 mM ATP. T_agg for dCK in each screening is presented. * Indicate that the result is based on one value.</p