Hypoxia-inducible Factor 2 Alpha in Complex with Aryl Hydrocarbon Receptor Nuclear Translocator and Hypoxia Response Element DNA

Fielding Fischer '21 and Liana Valin '21


Contents:


Model View:


I. Introduction

Hypoxia inducible factors (HIFs) are transcription factors that respond to low availability of oxygen (hypoxia) within a cell. HIF-2-alpha can only function efficiently as a transcription factor when in complex with the aryl hydrocarbon receptor nuclear translocator (ARNT). The HIF-2-alpha and ARNT proteins belong to the PER-ARNT-SIM (PAS) subfamily of transcription factors, which is part of the mammalian basic helix-loop-helix (bHLH) family.1 HIF-2-alpha-ARNT heterodimers bind in a sequence specific manner to a hypoxia response element (HRE) sequence on DNA, targeting the expression of hundreds of genes, including those that regulate angiogenesis, erythropoiesis, and glucose uptake and metabolism.2

Under normoxia, enzymatic hydroxylation of conserved residues within the transactivation domains of HIF-2-alpha ultimately results in proteasomal degradation of the complex, and inhibited recruitment of cofactors, both of which decrease transcriptional activity and target gene-expression.3 The von Hippel-Lindau tumor suppressor protein (pVHL) is linked with this proteasomal degradation pathway, but cannot function in certain cancers, allowing HIF to escape degradation in hypoxic settings, such as tumors.2 Subsequent intratumoral accumulation of HIF-2-alpha allows for heterodimerization with ARNT and tumor growth associated with the expression of target genes. Disruption of heterodimerization by small molecule binding is therefore being researched as a direct therapy for certain cancers.


II. General Structure

The HIF-2-alpha-ARNT consists of HIF-2 alpha and ARNT. HIF-2-alpha and ARNT are comprised of the same including bHLH, PAS-A, and PAS-B.

The bHLH domains of HIF-2-alpha and ARNT consists of each, which are important for DNA binding. The PAS-B domains include , which are important for heterodimerization.1

There are no intramolecular interactions between the three domains of ARNT , allowing for adaptability in heterodimerization with other proteins in the bHLH-PAS family. Most of the heterodimer interactions between HIF-2 alpha and ARNT occur at the interfaces of PAS-B and PAS-B. This includes the formation of and a salt bridge . Two other important heterodimer interfaces occur between PAS-B and PAS-A, and bHLH and bHLH . These specific interfaces are important because they stabilize the molecule as a heterodimer; without them the HIF-2-alpha and ARNT subunits cannot form a stable complex.1


III. Binding with HRE DNA

The DNA-reading of the HIF-2-alpha-ARNT complex is composed of the two respective bHLH domains of HIF-2-alpha and ARNT. The alpha helices of the bHLH domains insert into the major groove of hypoxia response element (HRE) Specifically, the alpha helices interact with the HRE recognition (5’-TACGTG-3’) and its pseudo-symmetric (5’-CACGTA-3’). Two residues from bHLH form contacts with the pseudo-symmetric complement of the HRE recognition sequence: forms hydrophobic contact with the eighth nucleotide (thymine) and forms a hydrogen bond with the fourth nt (guanine). Three residues from bHLH, R-102, H-94, and E-98, form contacts with both the HRE recognition sequence and its complement . 1

Residues N184 and K186 of PAS-A interact with the DNA downstream from the HRE recognition sequence. Both residues form hydrogen bonds with the DNA backbone of a downstream cytosine. . 1


IV. Small Molecule Binding

Within the HIF-2-alpha-ARNT complex there are five known distinct small-molecule binding cavities. Certain molecules that inhibit heterodimerization of HIF-2-alpha-ARNT have been found to bind in distinct cavities.1,4,5

An artificial molecule, 0X3, binds within a cavity of the PAS-B domain through van der Waals and electrostatic interactions.5 Specifically, the nitro group of 0X3 interacts with the adjacent residue. Binding is also facilitated by a pi-hydrogen bond between the A-ring of 0X3 and the of Y281.6 A preclinical inhibitor of HIF-2 heterodimerization named PT2399 has been found to bind in the same cavity as 0X3, but with higher affinity.5 Both molecules share interactions with the following .

The binding of these two molecules causes conformational changes in the A and B beta sheets of PAS-B, altering residue-residue interactions on the PAS-B: PAS-B interface. Specifically, interactions crucial for heterodimerization along the interface, including the hydrogen bonds between Asp-240 and Arg-366 , the salt bridge between Arg-247 and Glu-362 , and other hydrophobic interactions, are disrupted. 5

Proflavine, a component of acriflavine, is another small molecule that binds to the complex by intercalating between the PAS-B and PAS-A domains.1 Proflavine binds to of ARNT, both crucial residues in the PAS-B: PAS-A interface, subsequently disrupting heterodimerization.4


V. Discussion

Although HIF factors are not ligand binding proteins with active sites for substrate binding, certain molecules can bind and disrupt complex formation with ARNT. The inability of HIF-2-alpha to form this complex inhibits its ability to upregulate target genes in hypoxic environments. This lack of target gene expression stems from the inability of HIF-2-alpha and ARNT to form functional structures needed for transcriptional activity, including a DNA-reading head.

Because many cancers contain intratumoral hypoxia, and chemotherapy and radiation therapy only target proliferating and well-oxygenated cancer cells, recent cancer research has focused on small molecule binding that disrupts formation of HIF-2-alpha-ARNT.7 Both acriflavine and PT2399 have been found to inhibit complex formation, downregulating expression of tumor-growth associated genes. Despite the fact that these molecules bind in distinct cavities within the complex, both have been found to reduce tumor vascularization and growth.4,8 The discovery of heterodimer-inhibiting molecules are the most direct therapy for certain cancers as of now,8 and an important area of ongoing and future oncological research.


VI. References

1. Wu, D., Potluri, N., Lu, J., Kim, Y., Rastinejad, F. 2015. Structural integration in hypoxia-inducible factors. Nature.524:303–308.

2. Kaelin, W.G. 2007. von Hippel-Lindau Disease. Annual Review of Pathology: Mechanisms of Disease.2:145-173.

3. Kaelin, W.G. 2005. Proline hydroxylation and gene expression. Annual Review of Biochemistry. 74: 115-128.

4. Lee, K., Zhang, H., Qian, D. Z., Rey, S., Liu, J. O., and Semenza, G. L. 2009. Acriflavine inhibits HIF-1 dimerization, tumor growth, and vascularization.Proceedings of the National Academy of Sciences of the United States of America. 106(42): 17910-17915.

5. Sun, D-R, Wang, ZJ, Zheng, QC, Zhang, HX. 2018. Exploring the inhibition mechanism on HIF 2 by inhibitor PT2399 and 0X3 using molecular dynamics simulations. Journal of Molecular Recognition. 31:e2730.

6. Scheuermann, T. H., Li, Q., Ma, H. W., Key, J., Zhang, L., Chen, R., Garcia, J. A., Naidoo, J., Longgood, J., Frantz, D. E., Tambar, U. K., Gardner, K. H., Bruick, R. K. 2013. Allosteric inhibition of hypoxia inducible factor-2 with small molecules. Nature chemical biology. 9(4): 271-276

7. Schito, L., Semenza, G.L. 2016. Hypoxia-inducible factors: master regulators of cancer progression. Trends Cancer. 2:758-770.

8. Martínez-Sáez, O., Borau, P.G., Alonso-Gordoa, T., Molina-Cerrillo, J., Grande, E. 2017. Targeting HIF-2 alpha in clear cell renal cell carcinoma: A promising therapeutic strategy. Critical Reviews in Oncology/Hematology. 111: 117-123

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