Part:BBa_K3793004

CDS - Human Aryl hydrocarbon Receptor (Truncated)

This sequence encodes for the truncated version of the human Aryl hydrocarbon receptor (AhR). AhR is a transcription factor that is activated by ligands such as the 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD). Tryptophan metbolites such as kynurenic acid form an important class of ligands to the AhR. Upon Ligand binding, AhR translocates to the nucleus where it forms a heterodimer with the AhR Nuclear Translocator (ARNT). The heterodimer binds to the DNA sequence 5'-TGCGTG-3' within the dioxin response element (DRE) of target gene promoters and activates their transcription.

The part contains the coding sequence for a truncated version of AhR containing the the basic helix-loop-helix, PAS-A and PAS-B domains. The CDS has been codon optimized to remove any type IIS restriction enzyme sites. An N-Terminal 6x HIS tag is added to the CDS.

MilkClear iGEM UCopenhagen 2024

Author: Kate Escobar and Thanh Le, 26-09-2024, MilkClear's Wiki: https://2024.igem.wiki/ucopenhagen/

This part was used in the biosensor devices BBa_K5477041 and BBa_K5477042 to detect polycyclic aromatic hydrocarbons (PAHs), dioxin-like polychlorinated biphenyls (PCBs) and dioxin.

In its inactive state, AhR is located in the cytosol, where it is protected by HSP90 from degradation and nuclear translocation (1). As a part, the CDS for AhR was used without the 6x HIS tag. Upon binding these ligands, AhR undergoes a conformational change that allows it to translocate to the nucleus (2), where it dimerizes with the Aryl hydrocarbon receptor nuclear translocator (ARNT) BBa_K3793005 (1). This complex binds to specific DNA sequences known as xenobiotic response elements (XREs) BBa_K5477008 and therefore allows for transcription of NanoLuc BBa_K16800099 .

Results from Drylab

Aim : To investigate the impact of specific amino acid mutations within the binding pocket of the dioxin receptor (AhR) on ligand-binding affinities, using AutoDock Vina to simulate and predict the interaction energies of various ligands, including Arochlor1260 and FicZ, with wild-type and mutant forms of the receptor.

Objectives :

1. Evaluate the Binding Affinities: Assess the binding affinities of both wild-type and mutated AhR variants for the ligands Arochlor1260 and FicZ using molecular docking simulations.

2. Understand Structural Changes: Analyze the structural effects of specific mutations (e.g., Y336A, F279A, M302W) on the binding pocket and how these changes influence ligand interactions.

3. Correlate Docking Results with Experimental Data: Compare the calculated binding affinities obtained from AutoDock Vina with experimental data to understand how mutations alter ligand binding and potentially receptor function.

4. Investigate Steric Clashes and Conformational Adjustments: Explore how mutations cause steric hindrance or facilitate new interactions within the binding pocket, particularly focusing on large residues (e.g., tryptophan) and how these alter ligand accommodation and affinity.

AhR Mutants Binding Affinity Data

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Mutant	Mutations	Arochlor1260	FicZ
wild type dAhR	none	9.5	13.5
wild type mAhR	none	-3.2	-7
m1	Y336A, F279A	-5.8	-7.5
m2	M331V, F279A	-3.1	-3.1
m3	F279A, L299W, M302W, Y336A	-8	-6.5
m4	F279A, L299W, Y336A	-7.5	-7.1
m5	M302W, L307W, Y336M	7.6	14.5
m6	F279A, L307W, Y336A	5.5	10.3

Figures from AutoDock Vina

Figure 1 (Wild Type dAhR and mAhR Binding Pocket)

This figure shows the binding pockets of the wild-type dAhR (magenta) and mAhR (cyan). The binding sites for the two species differ, as shown by the conformational differences between the two colored meshes.The figure illustrates the structural differences in the binding pockets between dAhR and mAhR. This structural variation could explain the differential binding affinity for Arochlor1260 and FicZ, as reflected in the experimental data. Specifically, wild-type dAhR shows higher binding affinities compared to mAhR.

Figure 2 (dAhR Wildtype and Y336A/F279A Mutant Pocket)

This compares the binding pocket of the wild-type dAhR (white) with the Y336A/F279A mutant (red). The mutations seem to significantly alter the pocket structure, particularly affecting steric and spatial configurations. The mutations Y336A and F279A seem to affect the binding pocket's flexibility and steric hindrance. These changes likely influence ligand binding, potentially lowering affinity or altering specificity, as demonstrated by the binding energy shifts in the table for mutant 1 (m1) and mutant 6 (m6). The Y336A/F279A mutations reduce binding affinity for both Arochlor1260 and FicZ.

Figure 3 (Steric Clashes of Mutant with FicZ)

This figure displays steric clashes caused by mutations (notably involving the indole ring of FicZ). These clashes occur in mutant combinations, particularly in mutants like m3 (F279A, L299W, M302W, Y336A) and m5 (M302W, L307W, Y336M). Mutations that introduce bulky residues, such as tryptophan (W), cause steric clashes with ligands like FicZ, thereby reducing binding affinity. The indole ring of FicZ clashes with the newly introduced residues, contributing to the poor binding affinities observed in mutants m3 and m4. However, in some mutants (like m5), the pocket still accommodates FicZ despite steric clashes, resulting in positive binding affinities.

Figure 4 (Steric Clashes of Y336 with BPA)

This figure highlights the steric clashes between Y336 and BPA. The clashes are shown via distance measurements indicating close contact points between the aromatic ring of BPA and Y336. The close proximity and steric clashes between BPA and Y336 explain why Y336 mutations drastically impact ligand binding. Specifically, in mutant m1 (Y336A, F279A), the removal of the bulky tyrosine alleviates steric hindrance, resulting in altered binding dynamics for both FicZ and Arochlor1260. The absence of the Y336 side chain enables the ligand to bind more favorably, explaining the observed experimental data for these mutants.

Experimental Results Analysis

Wild Type dAhR: Exhibits favorable binding to both Arochlor1260 (9.5) and FicZ (13.5), consistent with the structural integrity of its binding pocket.

Wild Type mAhR: Has poor binding affinity for both Arochlor1260 (-3.2) and FicZ (-7), suggesting significant structural or conformational differences in its binding pocket compared to dAhR.

Mutants (m1–m6)

m1 (Y336A, F279A): The double mutation significantly reduces binding affinity compared to wild-type mAhR, with Arochlor1260 (-5.8) and FicZ (-7.5). This suggests that the removal of Y336 and F279 reduces overall pocket stability and ligand compatibility.

m2 (M331V, F279A): The effects of this mutation on binding are less severe, with binding affinities of -3.1 for both Arochlor1260 and FicZ, suggesting that these mutations cause a moderate disruption.

m3 (F279A, L299W, M302W, Y336A): This combination leads to a significant reduction in binding affinity (-8 for Arochlor1260 and -6.5 for FicZ). The bulky substitutions (W) lead to steric clashes, severely impacting ligand interactions.

m4 (F279A, L299W, Y336A): Shows similarly reduced binding, with Arochlor1260 (-7.5) and FicZ (-7.1), indicating that the combination of these mutations disrupts the pocket, but to a lesser extent than m3.

m5 (M302W, L307W, Y336M): Uniquely, this mutant exhibits improved binding to both Arochlor1260 (7.6) and FicZ (14.5). This suggests that, despite the steric bulk of tryptophan (W), the mutation enhances hydrophobic interactions or stabilizes the ligand in the pocket.

m6 (F279A, L307W, Y336A): Exhibits moderate binding with Arochlor1260 (5.5) and FicZ (10.3). The reduced steric hindrance from Y336A might allow more favorable interactions, but the presence of bulky tryptophan (L307W) still causes some spatial constraints.

Conclusion

The mutations in dAhR generally impact binding affinity due to changes in steric hindrance and pocket flexibility. Mutants like m5 exhibit improved binding, likely due to favorable hydrophobic interactions despite steric hindrance, while others (e.g., m3, m4) demonstrate poor binding due to steric clashes. The experimental results from AutoDock Vina align well with the structural changes observed in the figures, where specific mutations like Y336A and bulky tryptophan substitutions (W) lead to steric conflicts, significantly influencing ligand binding.

Mutant sequences

>7VNA|Y336A|F279A| GSIPHKENMFKSKHKLDASLVSMDQRGKHILGYADAELVNMGGYDLVHYDDLAYVASAHQ ELLKTGASGMIAYRAQKKDGEWQWLQTSSRLVYKNSKPDFVICTHRQLMDEEGHDLLGKR

>7VNA|M331V|F279A| GSIPHKENMFKSKHKLDASLVSMDQRGKHILGYADAELVNMGGYDLVHYDDLAYVASAHQ ELLKTGASGVIAYRYQKKDGEWQWLQTSSRLVYKNSKPDFVICTHRQLMDEEGHDLLGKR

>7VNA|F279A|L299W|M302W|Y336A| GSIPHKENMFKSKHKLDASLVSMDQRGKHILGYADAEWVNWGGYDLVHYDDLAYVASAHQ ELLKTGASGMIAYRAQKKDGEWQWLQTSSRLVYKNSKPDFVICTHRQLMDEEGHDLLGKR

>7VNA|F279A|L299W|Y336A| GSIPHKENMFKSKHKLDASLVSMDQRGKHILGYADAEWVNMGGYDLVHYDDLAYVASAHQ ELLKTGASGMIAYRAQKKDGEWQWLQTSSRLVYKNSKPDFVICTHRQLMDEEGHDLLGKR

>7VNA|F279A|L307W|Y336A| GSIPHKENMFKSKHKLDASLVSMDQRGKHILGYADAELVNMGGYDWVHYDDLAYVASAHQ ELLKTGASGMIAYRAQKKDGEWQWLQTSSRLVYKNSKPDFVICTHRQLMDEEGHDLLGKR

>7VNA|Y336A|M302W|L307W| GSIPHKENMFKSKHKLDFSLVSMDQRGKHILGYADAELVNWGGYDWVHYDDLAYVASAHQ ELLKTGASGMIAYRAQKKDGEWQWLQTSSRLVYKNSKPDFVICTHRQLMDEEGHDLLGKR

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 364
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 364
  Illegal NheI site found at 169
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 364
  Illegal BglII site found at 1179
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 364
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 364
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. Carambia, A., Schuran, F.A. The aryl hydrocarbon receptor in liver inflammation. Semin Immunopathol 43, 563–575 (2021). https://doi.org/10.1007/s00281-021-00867-8

2. Mandal A, Biswas N, Alam MN. Implications of xenobiotic-response element(s) and aryl hydrocarbon receptor in health and diseases. Hum Cell. 2023 Sep;36(5):1638-1655. doi: 10.1007/s13577-023-00931-5. Epub 2023 Jun 17. PMID: 37329424.