Passage
The tumor suppressor p53 is a homotetrameric protein with an N-terminal transactivation domain (TAD) , a proline-rich domain, a DNA-binding domain (DBD) , a tetramerization domain (TD) , and a C-terminal regulatory domain (REG) on each subunit (Figure 1) .
Figure 1 Organization of the p53 domains (1-393 amino acids) Human double minute 2 (Hdm2) is a lysine-specific ubiquitin protein ligase that can change the half-life of some proteins, including p53, by signaling for their proteolytic cleavage. Under normal circumstances, Hdm2 binds p53 at the AD1 region within the TAD and attaches ubiquitin to a lysine residue. However, errors in DNA replication induce the phosphorylation of residue T18 on p53, which inhibits p53's association with Hdm2. The overexpression of Hdm2 has been correlated with the downregulation of tumor suppressor p53 in certain types of cancers. Changes in the NMR chemical shift (ppm) of amino acids in the AD1 region shown in Figure 2 provide evidence for the formation of a p53-Hdm2 complex.
Figure 2 ¹H NMR spectrum of the AD1 region in ¹⁵N-labeled p53 during G₀ and G₂ phases of the cell cycleHdm2 also binds to Nedd8 ultimate buster 1 (NUB1) , which enhances signaling for the degradation of Nedd8 proteins. Preliminary data suggest that Hdm2 interacts differently with wild-type NUB1 (WTNUB1) than with mutant K159RNUB1. To assess the effect of Hdm2 on NUB1 and p53, the half-lives of p53 and Nedd8 protein variants were compared in cells that overexpress Hdm2 (Figure 3) .
Figure 3 Half-lives of p53, phosphorylated p53 (p53-PO₄) , and Nedd-8 proteins in cells with wild-type or mutant NUB1 over time
Adapted from Ferreon JC, Lee CW, Arai M, Martinez-yamout MA, Dyson HJ, Wright PE. Cooperative regulation of p53 by modulation of ternary complex formation with CBP/p300 and HDM2. Proc Natl Acad Sci USA. 2009;106(16) :6591-6.
-The data in Figure 3 support which of the following conclusions?

Question

Passage
The tumor suppressor p53 is a homotetrameric protein with an N-terminal transactivation domain (TAD) , a proline-rich domain, a DNA-binding domain (DBD) , a tetramerization domain (TD) , and a C-terminal regulatory domain (REG) on each subunit (Figure 1) .

Figure 1 Organization of the p53 domains (1-393 amino acids) Human double minute 2 (Hdm2) is a lysine-specific ubiquitin protein ligase that can change the half-life of some proteins, including p53, by signaling for their proteolytic cleavage. Under normal circumstances, Hdm2 binds p53 at the AD1 region within the TAD and attaches ubiquitin to a lysine residue. However, errors in DNA replication induce the phosphorylation of residue T18 on p53, which inhibits p53's association with Hdm2. The overexpression of Hdm2 has been correlated with the downregulation of tumor suppressor p53 in certain types of cancers. Changes in the NMR chemical shift (ppm) of amino acids in the AD1 region shown in Figure 2 provide evidence for the formation of a p53-Hdm2 complex.

Figure 2 ¹H NMR spectrum of the AD1 region in ¹⁵N-labeled p53 during G₀ and G₂ phases of the cell cycleHdm2 also binds to Nedd8 ultimate buster 1 (NUB1) , which enhances signaling for the degradation of Nedd8 proteins. Preliminary data suggest that Hdm2 interacts differently with wild-type NUB1 (WTNUB1) than with mutant K159RNUB1. To assess the effect of Hdm2 on NUB1 and p53, the half-lives of p53 and Nedd8 protein variants were compared in cells that overexpress Hdm2 (Figure 3) .

Figure 3 Half-lives of p53, phosphorylated p53 (p53-PO₄) , and Nedd-8 proteins in cells with wild-type or mutant NUB1 over time
Adapted from Ferreon JC, Lee CW, Arai M, Martinez-yamout MA, Dyson HJ, Wright PE. Cooperative regulation of p53 by modulation of ternary complex formation with CBP/p300 and HDM2. Proc Natl Acad Sci USA. 2009;106(16) :6591-6.
-The data in Figure 3 support which of the following conclusions?

Quizplus · Accepted Answer

11ecba2d_0e01_dbb0_a3bf_af33571c802d_MD0008_00 Binding affinity describes the strength of noncovalent binding interactions between two molecules. The equilibrium dissociation constant K_d measures the tendency of a protein-ligand complex to separate into free protein and free ligand. A low K_d value is indicative of a high tendency for the complex to remain bound, and therefore indicates a high binding affinity. Alternately, a high K_d value suggests a low binding affinity. In other words, the dissociation constant of a protein-ligand complex is inversely proportional to its binding affinity. Regulatory proteins such as Hdm2 can negatively or positively affect the activity, half-life, or expression of the proteins to which they bind. Based on the data in Figure 3, Hdm2 can be classified as a negative regulator of p53 because it binds and signals for the cleavage or degradation of unphosphorylated p53. The half-life graph in Figure 3 illustrates that unphosphorylated p53 proteins have a much shorter half-life than their phosphorylated counterparts. This difference in half-lives reflects the gap between the dissociation constants of phosphorylated and unphosphorylated p53. The passage suggests that phosphorylation of p53 at residue T18 reduces the binding affinity of p53 for Hdm2. Consequently, phosphorylated p53 is degraded at a slower rate because it has a higher K_d than unphosphorylated p53. (Choice B) Figure 3 shows that unphosphorylated p53 is degraded rapidly. The passage states that Hdm2 binds p53, indicating a low K_d. (Choice C) Dephosphorylation of p53 increases the rate of p53 degradation by decreasing K_d (raising the binding affinity) of the Hdm2-p53-PO₄ complex. (Choice D) Phosphorylated p53 is cleaved less readily because it has lower affinity (higher K_d) for Hdm2. Educational objective: Binding affinity describes the strength of noncovalent interactions between two molecules and is measured using equilibrium dissociation constants (K_d). The dissociation constant of a protein for its substrate is inversely proportional to the binding affinity. Negative regulators decrease the expression, activity, or half-life of their target proteins.

Passage the Tumor Suppressor P53 Is a Homotetrameric Protein with an an N-Terminal