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.
-Based on the data in Figure 2, which statement best supports the formation of a p53-Hdm2 complex in the absence of DNA errors?

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.
-Based on the data in Figure 2, which statement best supports the formation of a p53-Hdm2 complex in the absence of DNA errors?

Quizplus · Accepted Answer

11ecba2d_0e05_8533_a3bf_27d8334e2797_MD0008_00 Proton NMR spectroscopy measures the frequency of hydrogen nuclei spinning in a magnetic field. This frequency, referred to as the chemical shift, is measured in parts per million (ppm) and describes the electron density surrounding a proton within a molecule. When surrounded by high electron density, protons are said to be "shielded" from the magnetic field. However, electronegative atoms draw electron density from nearby protons, causing them to become deshielded. Protons that are shielded appear to the right (upfield) of the NMR spectrum whereas deshielded protons appear to the left (downfield). During protein-protein interactions, hydrophobic residues often associate to avoid contact with water. This interaction increases the electron density around the hydrophobic residues by shielding them from the electronegative oxygen in water. The spectrum in Figure 2 was obtained by analyzing protons on the amino acid residues in the binding region of p53. The passage states that p53 binding to Hdm2 is disrupted by errors in replication. Because DNA replication does not occur in G₀ (and therefore errors are not present), the changes in the chemical shift from G₀ to G₂ are likely related to p53 binding to Hdm2. Protons on the hydrophobic amino acids, F and W, are more shielded (shifted to the right) in G₀ than in G₂, suggesting that hydrophobic interactions are enhanced in G₀, and therefore that p53 binds Hdm2. (Choices A and B) Hydrophilic amino acids on the surface of p53 are more deshielded (shifted to the left) during G₀ than G₂. (Choice D) Hydrophobic amino acids are more shielded during G₀, when p53 is expected to bind Hdm2. Educational objective: High electron densities protect or "shield" protons (right shift) from the magnetic field of the spectrometer whereas electronegative atoms deshield protons (left shift). Hydrophobic residues associate and shield protons at protein-protein interfaces by avoiding water molecules.

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