Example 2 - TP53 from Structure to Function

The objective of this exercise is to explore active site residues and structural motifs to gain a better understanding of TP53 functional pathways. Participants can use a handful of features within iCn3D’s Sequences and Annotation window to visualize structure-function relationships and render an informative visualization. Follow Links are provided for guidance on time consuming steps. 

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Background on TP53 Structure and Cellular Processes

The TP53 protein contains functional domains critical for binding DNA and interacting with other proteins to coordinate various cellular responses. TP53 plays a critical role in maintaining cellular health by regulating cell cycle progression, DNA repair, and apoptosis, preventing the uncontrolled growth that can lead to disease. TP53 is known to form higher-order structures, including dimers and tetramers, which are important for high-affinity DNA binding.

Part 1: Background and Secondary Structure

1. Go to iCn3D and load PDB structure "1TUP"

   Note: You can reuse a previous rendering or start fresh

2. Open "Sequence & Annotations" window (Analysis > Seq. & Annotations), toggle on “Details”, and scroll down to view independent structural units within the protein.

   • Refamiliarize yourself with the different molecular components in this structure.

   • Review secondary structure information, shown above the primary sequence- strands (green arrow), helices (pink coil), and loops (blue line) within the chains. Dashed lines may indicate gaps or unstructured regions.

   • To better visualize the overall folding pattern, select your protein chains (e.g., 1TUP_A +Ctrl 1TUP_B +Ctrl 1TUP_C), then go to Color > Secondary and choose Sheet in Green
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3. Review the 1TUP Structure Summary page to provide a more complete understanding of the structure. Navigate here from iCn3D by clicking on “1TUP” in the title.

   Answer the following question:

   • Considering TP53's functions, how might secondary structure elements (strands, helices) suggest potential functional regions? For instance, are there regions rich in alpha helices (often involved in protein-protein interactions) that might be important for dimerization?

Part 2: Explore Interactions and Functional Sites

1. Go back to iCn3D and load PDB structure "1TUP" and open "Sequence & Annotations" window (Analysis > Seq. & Annotations)

2. To understand potential interactions between TP53 and other molecules, like DNA or protein partners, check "Interactions", toggle on “Details”, select "Show All Chains" to ensure annotations include all protein chains, and scroll down to view independent structural units within the protein. Adjust Style to better understand which residues are involved in binding.

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   Answer the following question:

   • Consider what you know about DNA- and protein-protein interactions. What are common amino acid properties that can help coordinate binding?

     Hint: for this question, ignore "Interact Misc (water)" and remember that charged and polar amino acids in TP53 play a crucial role in interactions with other molecules like DNA or protein partners by forming hydrogen bonds and ionic interactions, allowing for specific recognition and binding that governs TP53's function.

3. To identify key amino acid residues critical for specific TP53 functions like DNA binding or dimerization, check "Functional Sites", toggle on “Details”, select “Show All Chains”. Adjust style to better understand which residues are involved in binding.

   Answer the following questions:

   • For DNA binding, what properties are common for the interacting amino acids involved in DNA binding?

     Hint: protein-DNA interactions often feature positively charged amino acids that interact with the negatively charged phosphate groups in DNA.

   • For the zinc binding site, how does this compare to our findings in iCn3D Fundamentals with a 5Å ion-residue cutoff?
     

   • You may notice that there is an overlap in the zinc binding and dimerization functional sites. What might this mean for the relationship between ion binding and P53's ability to form quaternary structure?
     Hint: Studies suggest that zinc binding might induce or stabilize the p53 dimer conformation and mutations in the zinc-binding site can also affect p53 dimerization, potentially leading to loss of function. So, the overlap in the zinc binding and dimerization functional sites of p53 suggests a potentially interdependent relationship between these two processes.
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4. Using what you've learned about TP53's secondary structures, active site residues, and functional domains, render an informative iCn3D visualization(s) on the structure-function relationship in TP53.