PDBsum entry: 1p97

Transcription

PDB-id: 1p97

Contents

Description

Header details

Header records

References

PROCHECK

Protein chain

114 a.a. *

* Residue conservation analysis

Tools

Clefts Calculation

PDB id:

1p97

Name:

Transcription

Title:

Nmr structure of thE C-terminal pas domain of hif2a

Structure:

Endothelial pas domain protein 1. Chain: a. Fragment: c-terminal pas domain. Synonym: epas-1, member of pas protein 2, mop2. Engineered: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: epas1. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

UniProt:

Q99814 (EPAS1_HUMAN)

Seq:

Struc:

Seq:

Struc:

Seq:

Struc:

Seq:

870 a.a.

Struc:

114 a.a.*

Key:	PfamA domain
Secondary structure	CATH domain

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

Resolution:

not givenÅ

NMR structure:

20 models

Authors:

P.J.Erbel,P.B.Card,O.Karakuzu,R.K.Bruick,K.H.Gardner

Key ref:

P.J.Erbel et al. (2003). Structural basis for PAS domain heterodimerization in the basic helix--loop--helix-PAS transcription factor hypoxia-inducible factor.. Proc Natl Acad Sci U S A, 100, 15504-15509. [PubMed id: 14668441] [DOI: 10.1073/pnas.2533374100]

Added ref:

J.Yang et al. (2005). Functions of the Per/ARNT/Sim domains of the hypoxia-inducible factor.. J Biol Chem, 280, 36047-36054. [PubMed id: 16129688] [DOI no: 10.1074/jbc.M501755200]

Date:

09-May-03

Release date:

13-Jan-04

Quick_links

Procheck

Clefts

Surface

Key reference

DOI no: 10.1073/pnas.2533374100

Proc Natl Acad Sci U S A 100:15504-15509 (2003)

PubMed id: 14668441

Structural basis for PAS domain heterodimerization in the basic helix--loop--helix-PAS transcription factor hypoxia-inducible factor.

P.J.Erbel, P.B.Card, O.Karakuzu, R.K.Bruick, K.H.Gardner.

ABSTRACT

Biological responses to oxygen availability play important roles in development, physiological homeostasis, and many disease processes. In mammalian cells, this adaptation is mediated in part by a conserved pathway centered on the hypoxia-inducible factor (HIF). HIF is a heterodimeric protein complex composed of two members of the basic helix-loop-helix Per-ARNT-Sim (PAS) (ARNT, aryl hydrocarbon receptor nuclear translocator) domain family of transcriptional activators, HIFalpha and ARNT. Although this complex involves protein-protein interactions mediated by basic helix-loop-helix and PAS domains in both proteins, the role played by the PAS domains is poorly understood. To address this issue, we have studied the structure and interactions of the C-terminal PAS domain of human HIF-2alpha by NMR spectroscopy. We demonstrate that HIF-2alpha PAS-B binds the analogous ARNT domain in vitro, showing that residues involved in this interaction are located on the solvent-exposed side of the HIF-2alpha central beta-sheet. Mutating residues at this surface not only disrupts the interaction between isolated PAS domains in vitro but also interferes with the ability of full-length HIF to respond to hypoxia in living cells. Extending our findings to other PAS domains, we find that this beta-sheet interface is widely used for both intra- and intermolecular interactions, suggesting a basis of specificity and regulation of many types of PAS-containing signaling proteins.

Selected figure(s)

Figure 5.
Fig. 5. Point mutations in the HIF PAS-B central -sheet disrupt the binding of ARNT PAS-B. (a) Superimposed 15N/1H HSQC spectra of 250 µM 15N labeled HIF-2 PAS-B (Left) or triple mutant (Q322E/M338E/Y342T) (Right). Spectra in the presence of 900 µM unlabeled ARNT PAS-B are shown with red contours; those without ARNT are shown in black contours. Similar data for HIF-1 PAS-B are provided in Supporting Methods. (b) PAS-B domain interaction is important to form a biologically active HIF/ARNT complex. A construct expressing a luciferase reporter under the control of an HRE promoter was transfected into Ka-13 (columns 1-5) or CHO (column 6) cells along with various HIF constructs. Values represent the average luciferase activity of three samples, with bars indicating standard error. Luciferase expression was induced by cotransfection of HIF-1 (column 2) or HIF-2 (column 4), particularly under hypoxic conditions. Cotransfection of trHIF-1 (column 3) or trHIF-2 (column 5), full-length HIF proteins containing the three PAS-B mutations, shows a significant drop in luciferase activity compared with wild-type HIF .

Figure 6.
Fig. 6. Versatility of protein interactions involving PAS domain -sheets. HIF2 is shown in the same orientation as Fig. 4b and colored by residues experiencing significant 15N/1H chemical shifts on complex formation (red) and those used to generate the complex-disrupting trHIF-2 (blue). Phototropin (AsLOV2) (36) and photoactive yellow protein (33) highlight the -helices external to the PAS core (magenta) and any atoms located within 5 Å of those helices (pink). HERG (42) shows functionally important, solvent-exposed residues (dark blue) and residues present in a surface hydrophobic patch suggested to be important for channel function (light blue) (42).

Figures were selected by the author.

Author's comment

See also PDB structures 1x0o and 2a24.
Kevin Gardner

Added reference

DOI no: 10.1074/jbc.M501755200

J Biol Chem 280:36047-36054 (2005)

PubMed id: 16129688

Functions of the Per/ARNT/Sim domains of the hypoxia-inducible factor.

J.Yang, L.Zhang, P.J.Erbel, K.H.Gardner, K.Ding, J.A.Garcia, R.K.Bruick.

ABSTRACT

Biological responses to oxygen availability play important roles in development, physiological homeostasis, and many disease processes. In mammalian cells, this adaptation is mediated in part by a conserved pathway centered on the hypoxia-inducible factor (HIF). HIF is a heterodimeric protein complex composed of two members of the basic helix-loop-helix Per-ARNT-Sim (PAS) (ARNT, aryl hydrocarbon receptor nuclear translocator) domain family of transcriptional activators, HIFalpha and ARNT. Although this complex involves protein-protein interactions mediated by basic helix-loop-helix and PAS domains in both proteins, the role played by the PAS domains is poorly understood. To address this issue, we have studied the structure and interactions of the C-terminal PAS domain of human HIF-2alpha by NMR spectroscopy. We demonstrate that HIF-2alpha PAS-B binds the analogous ARNT domain in vitro, showing that residues involved in this interaction are located on the solvent-exposed side of the HIF-2alpha central beta-sheet. Mutating residues at this surface not only disrupts the interaction between isolated PAS domains in vitro but also interferes with the ability of full-length HIF to respond to hypoxia in living cells. Extending our findings to other PAS domains, we find that this beta-sheet interface is widely used for both intra- and intermolecular interactions, suggesting a basis of specificity and regulation of many types of PAS-containing signaling proteins.

Selected figure(s)

Figure 2.
FIGURE 2. HIF-2 PAS domains are required for HIF activity. Cell lines were transiently transfected with the 3xHRE-tk-luc HIF reporter gene and incubated under normoxic or hypoxic (1% O[2]) conditions for 12 h. The values represent the average luciferase activity of three replicates with the bars indicating standard error.

Figure 5.
FIGURE 5. Both HIF-2 PAS domains are required for heterodimerization with ARNT. A, heterodimerization of HIF-2 and ARNT requires both HIF-2 PAS domains. ARNT was immunoprecipitated (IP) from nuclear extracts prepared from each stable cell line (top left panel). The levels of HIF-2 protein that coimmunoprecipitated with ARNT were assessed by Western blot analysis (bottom left panel). Input levels are indicated in the panels on the right. B, the DNA binding activity of HIF-2 from nuclear lysates is dependent upon both HIF-2 PAS domains. The ability of HIF-2 to bind a DNA fragment derived from the HIF-responsive vascular endothelial growth factor promoter was assessed by an electrophoretic mobility shift assay. A HIF-2-containing complex previously verified by supershift experiments using an antibody to HIF-2 is indicated.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2005, 280, 36047-36054) copyright 2005.

Figures were selected by the author.