Effect of mutations in Siah1 and Siah2 SBD Chimeras on binding with PHD3.

<p>(<b>a</b>) Pairwise sequence alignment of Siah1 and Siah2 SBD was performed by EMBOSS Needle tool. The 26 amino acids that are unique in Siah1 and Siah2 SBD are highlighted in grey. Dissimilar amino acids are highlighted by ‘*’. Similar amino acids are highlighted by ‘:’ and identical amino acids are highlighted by ‘|’ (top panel). The 10 dissimilar amino acids between Siah1 and Siah2 SBD are shown in diagrammatic representation of the chimeric forms, SBD[S1]^NT[S2]^CT and SBD[S1]^NT[S2]^CT(bottom panel). The original residue numbers are labeled in the respective colors (<b>b</b>) HEK293T cells were transfected with the indicated expression plasmids, followed by FLAG immunoprecipitation (IP) of cell lysates. Immunoprecipitates and lysates were then analyzed by western blotting using the indicated antibodies. The N-terminal mutant chimera, [S1-(E17S/P57S/F98H)]^NT[S2]^CT did not regain binding to PHD3 and the C-terminal mutant chimera, [S2]^NT[S1-(Q121L/T160A]^CT regained complete binding to PHD3 equivalent to WT Siah2 SBD.</p

Effect of additional mutations in the N-terminal region of the SBD on binding with PHD3.

<p>(<b>a</b>) The 10 similar amino acids in the N terminal region (1–00) of Siah1 and Siah2 SBD are highlighted in grey. Mutated residues among the similar amino acids are highlighted within the box. (<b>b</b>) HEK293T cells were transfected with the expression plasmids for the indicated proteins. The cells were lysed and the cell lysates were subjected to FLAG-immunoprecipiation (IP). Immunoprecipitates and lysates were then analyzed by western blotting using the indicated antibodies. The [S1-8Mut]^NT[S2]^CT mutant increased binding compared to [S1-6Mut]^NT[S2]^CT. (<b>c</b>) The amount of PHD3 that coimmunoprecipitated with chimeric and mutated Siah1 and Siah2 SBD was quantified using Gel-pro analyzer software. The binding of the chimeric and mutated SBD to PHD3 was expressed as percentage of the binding of WT Siah2 SBD to PHD3. The data are represented as mean±S.E.M from three independent experiments. Differences in measured variables were assessed with Student's t test. * denotes p<0.05.</p

Docking model of the N-terminal Siah2 SBD and PHD3.

<p>N-terminal region (1–100) of the modeled Siah2 SBD was docked with PHD3 using an automated Cluspro server. The complex was then presented using Pymol as (<b>a</b>) cartoon representation, and (<b>b</b>) space filling representation. (<b>c</b>) The details of the interactions were obtained by PDBsum. The number of H-bond lines between any two residues indicates the number of potential hydrogen bonds between them. For non-bonded contacts, the width of the striped line is proportional to the number of atomic contacts.</p

Interaction of Siah2 with PHD3.

<p>(<b>a</b>) HEK293 cells were transfected in 60-mm cell culture plates for 2 days with the indicated expression plasmids. The cells were lysed, and the lysates were subjected to FLAG immunoprecipitation (IP), as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106547#s2" target="_blank">Materials and Methods</a>”. Aliquots of the cell lysates and immunoprecipitates were analyzed by western blotting with the anti-HA antibody. Both full length Siah2 and Siah2 SBD bind to PHD3 to the same extent. In the IP, the presence of the faint band in the empty vector lane is due to non-specific binding of PHD3. The same membrane was reblotted with FLAG antibody to detect FLAG tagged Siah2 proteins. (<b>b</b>) GST-Siah2 SBD pulldown of HA-PHD3. Cell lysate of HEK293 cells transfected with HA-PHD3 was incubated with GST-Siah2 SBD immobilized on GSH agarose beads and the reaction was performed as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106547#s2" target="_blank">Material and Methods</a>”. The empty expression vector alone was expressed as a GST control for non-specific binding of HA PHD3. After the incubation, the lysate was removed, the GSH-agarose beads were washed, and bound HA-PHD3 was analyzed by Western blotting using anti HA antibody. The pull down assay confirmed the interaction of Siah2 SBD with PHD3.</p

Effect of mutations in the C-terminal region of the SBD on binding with PHD3.

<p>HEK293T cells were transfected with the expression plasmids for the indicated proteins. The cells were lysed followed by FLAG immunoprecipitation (IP) of cell lysates. Immunoprecipitates and lysates were then analyzed by western blotting using the indicated antibodies. The [S2]^NT[S1-Q121L]^CT chimera regained binding equivalent to Siah2 SBD wild type. In contrast, [S2]^NT[S1-T160A]^CT showed only a small increase in PHD3 binding. FLAG-SBD Siah in the IP was masked by the IgG light chain.</p

Siah1 exhibits weak binding compared to Siah2 with PHD3.

<p>HEK293T cells were transfected in 60-mm cell culture plates for 2 days with expression plasmids for the proteins indicated at the top of each panel. (<b>a</b>) Cell lysates were subjected to HA-IP and aliquots of the cell lysates and immunoprecipitates were analyzed by western blotting with the anti-FLAG antibody. Both the Full length and Siah1 SBD did not show binding to PHD3 (<b>b</b>) The lysates were subjected to reciprocal FLAG-IP. Immunoprecipitates and aliquots of the cell lysates were analyzed by Western blotting with anti-HA and anti-FLAG antibodies. In the IP, FLAG-SBD overlaps with the IgG light chain. Compared to Siah2 SBD, only weak binding of Siah1 SBD to PHD3 was observed.</p

Effect of mutations in the N-terminal region of the SBD on binding with PHD3.

<p>HEK293T cells were transfected with the expression plasmids for the proteins indicated at the top of each panel. The cells were lysed and the cell lysates were subjected to FLAG-immunoprecipiation (IP). Immunoprecipitates and lysates were then analyzed by western blotting using the indicated antibodies. (<b>a</b>) Both [S1-(P21H/A26P/Q62A)]^NT[S2]^CT and [S1-6Mut]^NT[S2]^CT chimeras only partially regained binding to PHD3, compared to binding of wild type Siah2 SBD to PHD3. (<b>b</b>) Only the chimera with the P21H/A26P mutations regained partial binding with PHD3. In contrast, mutation of Q62A did not increase PHD3 binding. FLAG-SBD Siah in the IP was masked by the IgG light chain.</p

Kinetic monitoring of ADC activity carried out using 1 mM L-aspartate and 3 µM enzyme.

<p>The different points correspond to conversion percentages of the individual ¹H NMR spectra taken at increasing reaction times after initiation of the reaction in D₂O at 25°C. Percentage of product formation and substrate depletion is represented by filled and empty circles, respectively. The percentage of product and substrate after 30 min of the reaction in the presence of D-tartrate is represented by filled and empty squares, respectively.</p

Relative inhibitory effects of selected known and newly tested compounds against ADC.^[a]

[a]<p>The measurements were performed using 1 mM L-aspartate, 3 µM ADC, and 1 mM compound (potential inhibitor) in D₂O at 25°C.</p>[b]<p>The conversion percentage corresponds to the product formed by integration of the ¹H NMR signals corresponding to substrate and product of the enzymatic reaction after ca. 30 min upon addition of the enzyme. The time was adjusted to correspond to 50% conversion in the <i>absence</i> of inhibitor (reference). The absolute values were averaged from at least two independent assays.</p>[c]<p>The relative inhibitory effect, <i>k</i>_rel, was calculated as the ratio of the conversion percentages in the presence and absence of compound.</p>[d]<p>While full inhibition was also observed when using double the enzyme concentration, i.e., 6 µM, only a small nhibitory effect could be detected (<i>k</i>_rel = 0.9) when the assay was performed with 100 µM oxaloacetate, i.e., at a 10-fold lower inhibitor concentration.</p>[e]<p>A smaller <i>k</i>_rel value of 0.74, suggesting moderate inhibition, was observed upon preincubation with ADC for 1 h at ambient temperature.</p

Chemical structures of known inhibitors against ADC (<i>K1–K7</i>) and computationally identified potential inhibitors obtained <i>via</i> virtual screening (<i>I1–I7</i>).

<p>Chemical structures of known inhibitors against ADC (<i>K1–K7</i>) and computationally identified potential inhibitors obtained <i>via</i> virtual screening (<i>I1–I7</i>).</p