Example 1 - TP53 Mutation Analysis

The objective of this exercise is to investigate mutations in TP53 (PDB 1TUP) using iCn3D’s mutation analysis and the "ClinVar" feature in "Sequences & Annotations". Together, these analysis tools will help you better understand how disease-associated mutation within a functional domain might affect DNA binding. Follow Links are provided for guidance on time consuming steps.

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Background on P53 and DNA Binding

P53, a tumor suppressor protein, regulates cell cycle and apoptosis by binding to DNA. This binding is facilitated by interactions between P53's positively charged residues (like Lysine) and the negatively charged phosphate groups in DNA's backbone and major groove.

Part 1: iCn3D's Mutation analysis  

1. Go to iCn3D and load PDB structure "1TUP"
   Note: You can reuse a previous rendering or start fresh

2. To analyze DNA Interactions, use the select by distance function and pinpoint residues in the protein that are interacting with the DNA. Keep in mind, charged residues often interact with the negatively charged DNA backbone (usually at the major groove).

   • Select > By Distance, choose nucleotides, set sphere radius to 3.5 Å, choose 1TUP_B, and click Display. For this step, focus on Chain B (1TUP_B) as it has the most interactions with DNA.

   • Style > Side Chains > Stick for better residue visualization

   • Select > Save Selection and name "resi_3.5_dna"

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3. View Interacting Residues:

   • Defined Sets: Select nucleotides +Ctrl resi_3.5_dna.

   • View > View Selection to visualize DNA and interacting residues. Adjust the Style and Color as needed.

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4. Focus on Lys120 and answer the following question:

   • Considering the properties and position of K120, how might a mutation at this site affect TP53's interaction with DNA? 
     Hint: View residue Lys120 (positively charged, pointing into major DNA groove). This mutation (K120A) affects DNA binding in mouse models (reference publication)

5. To perform Mutation Analysis select Analysis > Mutation, follow the noted format, and input 1TUP_B_120_A to perform mutation analysis on Lys120 to Ala120. Choose Interactions.

   • Type "A" to alternate between K120 and A120. Visually compare K120 and A120.

     Answer the following question:

   • Are there any missing interactions around the mutation site when comparing wild type and mutant structures?

6. Review Key and interaction maps (salt bridge/ionic and pi-cation interactions lost with mutation).

   • Explore Defined Sets (Analysis > Defined Sets) for closer inspection of interactions

     Answer the following questions:

   • Focusing on the "Different interactions" subgraph in the interaction maps, what types of interactions are lost upon mutation based on the color key?

   • Considering the missing interactions you identified (like salt bridges or pi-cation interactions), how might this loss affect TP53's ability to interact with DNA?
     Hint: Lost ionic/aromatic interactions often weaken protein-DNA binding.

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Part 2: ClinVar Feature in Sequences & Annotations 

Now, we will use the ClinVar feature within iCn3D to explore documented mutations in P53 and understand how they might be linked to human diseases. NCBI's ClinVar aggregates information about genomic variation and its relationship to human health.

ClinVar Color Codes

Red mutations: Pathogenic or likely pathogenic (stronger association with disease).
Green mutations: Variants of unknown significance (VUS) - require further investigation.

1. Open "Sequence & Annotations" window (Analysis > Seq. & Annotations)

2. Check "ClinVar", toggle on “Details”, select "Show All Chains" to ensure annotations include all protein chains and scroll down to view mutations

   • Highlighting a residue with a ClinVar mutation in this window can display a label on the 3D structure indicating the associated disease

   • Focus on residue 120 again. These mutations (K>*, K>E, K>Q) are all pathogenic (Li-Fraumeni-like syndrome). What might this mean about the importance of K120 in biological function?

3. Review the ClinVar entries to learn more about genetic variation, including its location, and its potential health impact

4. Review "Interactions" presets for these mutations

   • Describe any structural variations and consider how these changes may impact protein function

   • Time permitting, explore other mutations of interest to you, the associated ClinVar page, and/or create a rendering highlighting the mutation