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This Sandbox is Reserved from 06/12/2018, through 30/06/2019 for use in the course "Structural Biology" taught by Bruno Kieffer at the University of Strasbourg, ESBS. This reservation includes Sandbox Reserved 1480 through Sandbox Reserved 1543.

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PDX-1 Homeodomain

spinqualitylabelsall models PDB ID 2h1k Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image
2h1k, resolution 2.42Å ()
Gene:	IPF1, PDX1 (Mesocricetus auratus)
Structural annotation: Resources: InterPro : Ipr012287, Ipr009057, Ipr001356, Ipr001827, Ipr000047 Pfam : PF00046 UniProt : P70118
Functional annotation: Cellular component: nucleus Biological process: regulation of transcription regulation of transcription, DNA-dependent transcription Molecular function: DNA binding transcription factor activity sequence-specific DNA binding
Evolutionary conservation: Toggle Conservation Colors: Rows = identical sequences: Further Information: Caveats, Explanation, Complete Results at ConSurfDB
Resources:	FirstGlance, OCA, RCSB, PDBsum
Coordinates:	save as pdb, mmCIF, xml

IntroductionIntroduction

The cytogenetic location of the PDX-1 gene (Ipf1) is on the chromosome 13q12.1

Pancreatic and Duodenal homeoboX 1, called PDX-1, is a transcription factor which is encoded by the gene Ipf1. This one is located at the human chromosome 13q12.1.

Human PDX-1 is a protein of 283 amino acids with a molecular weight of 30,64 kDa. This transcription factor belongs to the ParaHox transcription factor family, which diverged from the Hox subfamily through a duplication event. Moreover, it is important to note that PDX-1 is located in the nucleus of cells.

Schematic representation of functional domains and phosphorylation sites of PDX-1

PDX-1 contains, at the N-terminus, a transactivation domain (from 1 to 79 amino acids) and the middle region of the protein is composed of a homeodomain (from 146 to 206 amino acids) which is essential for DNA binding and protein-protein interactions.

The homeodomain plays a key role in the pancreatic development, the β-cell maturation and survival. Indeed, transcription factors play an important role in gene regulation by recognizing specific DNA control regions associated with each gene. That is why a modification in the structure of the transcription factor can affect the regulation of the gene and so cause different diseases. In our case, PDX-1 modifications can lead to a type II diabetes.

The DNA binding domains of transcription factors belong to evolutionarily conserved families including homeodomain family.

Structure of PDX-1 homeodomainStructure of PDX-1 homeodomain

General structure of PDX-1 homeodomain

GeneralityGenerality

The homeodomain is one of several small DNA binding motifs with DNA binding specificity which is present into approximately 235 transcription factors. The homeodomain protein folds into three (Wikipédia), with helices 1 and 2 antiparallel to each other and perpendicular to helix 3, and a flexible .

Helices 2 and 3 form a helix-turn-helix type motif (Protéopédia, Wikipedia). Indeed this tertiary structure motif consists of two α-helices and a short extended amino acids chain between them. The more carboxyl-terminal helix can fit into the major groove of DNA. That is why, helix 3 (also known as the recognition helix) interacts with the the major groove of the DNA.

The N-terminal arm binds with a specific DNA sequence through the minor groove.

Motifs of the PDX-1 homeodomain [1]Motifs of the PDX-1 homeodomain [1]

The homeodomain contains a Protein Transduction Domain (PTD : from 188 to 203 amino acids) and a Nuclear Localization Signal motif (NLS : from 197 to 203 amino acids), which allow PDX-1 to permeate into cells.

Nuclear translocation of transcription factors is a crucial requirement for their action and stimulus-dependent nuclear translocation can serve as a mechanism to regulate gene expression at the level of transcription initiation. NLS is composed of several basic amino acids such as arginine (R) and lysine (K), as we can see in the NLS sequence RRMKWKK (basic amino acids) of PDX-1. This motif is sufficient for the nuclear import of PDX-1.

DNA binding properties of homeodomainsDNA binding properties of homeodomains

All recognition helices (also, called helices 3) of Hox factors (such as PDX-1) are able to recognize the TAAT core of the DNA through van der Waals contacts made by Ile 47 with Ade 3 and Thy 4, and through two hydrogen bonds by with Ade 3. Asn 51 also forms a hydrogen bond with Ade 2. Finally, bases Cyt 5*, Thy 6*, and Cyt 7* are recognized through van der Waals contacts with Gln 50 and Met 54. [[2] Part B]

The N-terminal arm sequence is less well conserved than the recognition helix, but typically includes positively charged . The arm sequence contributes to DNA binding specificity. The N-terminal arm facilitates searching the DNA for binding sites through electro-static attraction by a sliding mechanism or transferring between DNA strands by a ‘‘fly catching’’ mechanism.

Two different stable conformations of PDX-1 homeodomainTwo different stable conformations of PDX-1 homeodomain

There are two complexes of the PDX-1 homeodomain with differences in the conformation of the N- terminal arm and helix 3. Indeed, the homeodomain of PDX-1 binds DNA in two different conformations named conformations A and B. Moreover, the two conformations of the Pdx1/DNA complex contained invariant contacts found in both conformations A and B, and variant contacts specific to each conformation.

Differences between conformation A and B

* Helix 3

Helix 3 of PDX-1 forms specific interactions in the major groove with the bases Ade 2, Ade 3, and Thy 4 of the TAAT core of the DNA, and the bases Cyt 5*, Thy 6*, and Cyt 7* of the DNA complementary strand. Two residues form direct hydrogen bonds with DNA bases in both conformations: Asn 51 with Ade 3 in the major groove, and Arg 5 with Thy 1 and Gua -1* in the minor groove. Majors differences between conformations A and B are in the major groove. Three phosphate contacts are specific to conformation A: Asn 51 with Ade 2, and Arg 31 and Lys 46 with Ade 8*. However, conformation B is more specific than conformation A.

Actually, in conformation B, Gln 50 formes a water-mediated contact with Gua 5 and Thy 6* , and binds Ade 2 in addition to Ade 3.

PDX-1 homedomain/DNA interactions. A) Structure of the PDX-1 homedomain/DNA complex. B) Hydrogen bond contacts with the DNA differ between Conformation A and B.

* The N-terminal arm

The (residues 1 to 9) of PDX-1 homeodomain, first contacts the core TAAT bases of the DNA through the minor groove and contributes to the binding specificity. In PDX-1, the N-terminal sequence contains  : Lys 2, Arg 3 and Arg 5.

In both PDX-1 conformations, Arg 5 forms hydrogen bonds with the bases of Thy 1 and Gua -1* through the minor groove, and van der Waals contact with Ade 2.

In conformation B, the N-terminal arm is more ordered with Lys 2 hydrogen bonded with the bases Ade 3 and Thy 2* in the minor groove, whereas in the conformation A, the N-terminal arm is mostly disordered. Scientists attributed the different contacts between the two conformations, to differences in DNA bending.

Arg 3 and Arg 43 help the stabilization of the N-terminal arm. In fact, the contact by from the major groove with the phosphate backbone correlates with stabilizing the N-terminal arm. Moreover, these residues are more mobile in conformation A than in conformation B.

So, the most stable configuration for the N-terminal arm of Pdx1 consists of Lys 2 inserted in the minor groove and Arg 3 outside of the minor grove contacting the phosphate backbone and Arg 43.

* The DNA curvature

Furthermore, the DNA curvature in conformation A or B differs from each other. The curvature of the DNA in conformation B is around 34° while the conformation A enable a curvature around 16°.

In summary, conformation A represents a specific DNA bound configuration with a single base contact by Arg 5 in the minor groove whereas the conformation B represents another specific PDX-1 conformation. Indeed, this one is able to form additional water-mediated contacts with DNA bases by Asn 51 and Gln 50 in the major groove, and by Lys 2 in the minor groove. This difference of conformations allows the flexibility of homeodomains in binding DNA which may be important for the functioning of PDX-1.

Functions of the PDX-1 homeodomainFunctions of the PDX-1 homeodomain

Functions with DNAFunctions with DNA

This flexibility of homeodomains in binding DNA may facilitate diverses functions of the PDX-1 transcription factor. Indeed, the PDX-1 protein has multiple functional roles in the developing of the pancreas, in mature pancreatic β and δ-cells, and neural cells. As a transcription factor, PDX-1 is able to regulate a lot of different genes.

It regulates differentiation of the duodenum and stomach and is involved as a key regulator of pancreas development. Indeed, PDX-1 evolves during the life from a differenciation factor during embryogenesis to a regulation factor of gene transcription of proteins which regulate the rate of glucose of the islets of Langerhans in adults. In the mature pancreas Pdx1 is expressed in β and δ-cells that secrete the endocrine hormones called respectively insulin and somatostatin.

Protein-Protein interactionsProtein-Protein interactions

In order to regulate all of these genes, PDX1 interacts with different cofactors.

In pancreatic δ-cells, PDX-1 interacts with PBX1 and PREP1 corresponding respectively to PBC and MEIS proteins. These proteins belong to the three amino acids loop extension (TALE) family of homeodomains. By interacting with these proteins, PDX-1 can regulate the somatostatin promoter. Interaction with the TALE proteins is mediated through a conserved pentapeptide motif, FPWMK, located in the N-terminal region of the protein and separated from the homeodomain by around 10 residues.

In neural cells, PDX-1 utilizes a different binding site on the somatostatin promoter than in δ-cells, suggesting a distinct protein complex. PDX-1 interacts also with the basic helix-loop-helix (bHLH: Wikipedia) factor E47/NeuroD. In pancreatic acinar cells, PDX-1 interacts with Pbx1b and Mrg1 to regulate the elastase I gene.

Disordered sequences outside of the homeodomain can influence DNA binding specificity. Moreover, phosphorylation or sumoylation are important for nuclear localization and may affect activity. The activity of PDX-1 is regulated by recruiting different coactivators and corepressors to non-conserved regions outside of the homeodomain.

Consequences of the modification of PDX-1 homeodomainConsequences of the modification of PDX-1 homeodomain

Diabete is a metabolic disorder characterized by a hyperglycemia resulting from defects in insulin secretion or insulin action in function of the type of the diabetes (type I or II).

Five different proteins with functional defects, cause a form of diabetes termed MODY ( included in type II diabetes). Those include the enzyme GK and four transcription factors, including PDX-1 (MODY-4). Mutations of PDX-1 have also effects on the development of the pancreas and on the function of insulin-secreting cells. It could also be a pharmacological target for beta-cell defects in Type II diabetes, since it has a role as regulator of islet of Langerhans stem cell activity.

External Ressources External Ressources

http://atlasgeneticsoncology.org/Genes/PDX1ID43921ch13q12.html

http://ghr.nlm.nih.gov/gene/PDX1

http://diabetes.diabetesjournals.org/content/51/suppl_3/S320.long

Protreopedia Page Contributors and EditorsProtreopedia Page Contributors and Editors

Mégane DENU and Claire HUBRECHT

Student 1A ESBS (Promo 2016)