Nutlin shows reduced inhibition of the direct interaction between p53 and HDM2 N-terminal domain.

<p>F2H assay to investigate the interaction of p53 (bait) with wild-type (WT) HDM2, mutant Q24R and M62A (preys). The F2H assay measures the interaction between two proteins as ratio of cells showing co-localization of bait and prey at the nuclear F2H interaction platform, to cells not showing this co-localization. Titration of Nutlin on to BHK cells co-transfected with GFP-p53 and RFP-HDM2 (wt) immediately shows declined percentage of co-localization. In contrast, p53 interaction with HDM2 mutants Q24R and M62A upon Nutlin treatment is clearly less reduced, indicating Nutlin resistance. Graph bars show means of normalized interaction values (in %) ± s.e.m. from three to five independent experiments. n.s. no significance, *p<0.05, **p<0.01.</p

<i>In vitro</i> detection of HDM2-p53-DNA complex and disruption by Nutlin.

<p>A, Schematic depicting complex formed between HA-tagged HDM2, p53 and DNA captured on beads coated with anti-HA antibody. The HDM2 expression construct (purple bar) comprises HA-tag encoding sequence (black) and p53 RE (green). Arrows depict PCR primers to quantify captured DNA. B, Real-time PCR assay to measure complex formation (shown in A) in the presence of Nutlin (0,10,100 µM). Values indicate fold increase over HDM2 gene control without any p53 response element (2CONA) appended. Values represent mean ± SD (n = 2), *p<0.05. C, Western blot of p53 captured by immobilised HDM2 and effect of Nutlin (10 µM).</p

The M62A mutant in the HDM2 p53-binding domain selectively results in loss of Nutlin binding.

<p>A, Simulations (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062564#s2" target="_blank">Materials and Methods</a>) indicate loss of significant packing interactions with Nutlin (left structure, circled) when M62 is mutated to alanine (right structure, circled). Nutlin respectively depicted in purple and orange. B, Packing interactions with p53 (left structure, circled) are minimally disrupted by M62A mutation (right structure, circled). p53 (amino acids 17–29) respectively depicted in purple and orange.</p

Selection of Nutlin-resistant HDM2 by <i>in vitro</i> compartmentalisation.

<p>1, HDM2 expression constructs (blue and purple bars) appended with 2CONA p53 response element (green) and HA-tag coding sequence (black) and p53 expression construct (red bar) are segregated into aqueous emulsion compartments along with Nutlin (yellow orb). Protein expression occurs within compartments. Nutlin inhibition of HDM2 results in no HDM2-p53-DNA complex formation (left bubble), whereas resistant HDM2 can form the complex (right bubble). 2–3, The emulsion is broken and complexes captured with anti-HA antibody. DNA encoding resistant HDM2 variants is amplified by PCR. 4, Selectants further evaluated by secondary pull-down assay or subjected to further rounds of selection.</p

Selected HDM2 variants display <i>in vitro</i> Nutlin-resistance phenotype.

<p>A,B <i>In vitro</i> pull-down assay showing reduced inhibition by Nutlin (10 µM) to binding of p53 for indicated parental HDM2 variants and point mutants derived from parental clones (5.9 and 5.14 respectively). 50% of p53 protein pulled down in absence of Nutlin treatment loaded. C, Analysis of L82P mutation indicates reduced inhibition by Nutlin (100 µM). Control indicates background p53 binding in absence of HDM2. * Indicates no Nutlin treatment.</p

Nutlin shows reduced inhibition of selected variants in p53-null H1299 cells.

<p>A, H1299 cells transfected with either p53 alone or p53 and indicated HDM2 variants. P53 function measured by reporter gene activity in presence of Nutlin (0,2,5 µM). Values represent mean ± SD, n = 2. B, Same as in A, with p53 activity shown as fold-increase over the base-line value of inhibition in the absence of drug treatment (set to 1). Values represent mean ± SD, n = 2. *p<0.5, **p<0.05. C, Western blot indicating expression levels of HDM2 variants and p53 in H1299 cells.</p

The Q24R mutation in the HDM2 lid region is predicted to enhance affinity for p53 but not Nutlin.

<p>A, Molecular simulations (detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062564#s2" target="_blank">Materials and Methods</a>) indicate mutation of Q24 to arginine (right structure) leads to repulsion of proximal K51 and stabilization of E28 in p53 (amino acids 17–29, blue) through charge-charge interaction. This additional stabilization is not seen in wild-type HDM2 bound to p53 (left structure, p53 shown in purple). B, Q24 makes no significant contacts with Nutlin (left structure) and mutation to arginine (right structure) makes no additional difference.</p

Mutation V280A in acidic domain results in reduced interaction with p53 DNA binding domain.

<p>A, Models of the native (left) and V280A mutant (right) peptides docked to the p53 DNA binding domain. Residues set as “active” in the Haddock server run (details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062564#s2" target="_blank">Materials and Methods</a>) are shown as sticks (red and yellow for p53 and blue for peptide). Residues of p53 known to make direct contact with DNA are colored in yellow. V280 and A280 are shown in green in the respective structures. In the native case V280 appears to make more contacts with p53 than A280 in the mutated peptide. B, <i>In vitro</i> pulldown assay shows reduced interaction between HDM2-V280A and N-terminally truncated p53 (Δ133, comprising secondary HDM2 interaction site only). Interaction with full-length p53 (comprising both N-terminal and secondary HDM2 interaction sites) is unaffected.</p

Apparent Kds (nM) of indicated ligands for HDM2 mutants determined by competitive fluorescence anisotropy titrations.

<p>Apparent Kds (nM) of indicated ligands for HDM2 mutants determined by competitive fluorescence anisotropy titrations.</p

Molecular simulations show negative impact of the P20L and Q24R mutations on the docking of Nutlin to the HDM2 N-terminal domain.

<p>A, Space-filling (left) and ribbon (right) depictions of Nutlin (orange) binding the main (arrowed) and secondary (circled) binding sites of HDM2. The p53-peptide (cyan) binding to the main site is overlaid. B, Space-filling model of the HDM2 N-terminal domain showing migration of the lid region when P20 (left, orange sphere,) is mutated to leucine (right, green sphere). Arrow depicts main Nutlin binding site. C, Mutation of Q24 (left) to arginine (right) results in extensive hydrogen bond network with E23 and Y100 and likely occlusion of the secondary Nutlin binding site.</p