PDBsum entry 2k7s

Transcription

PDB id: 2k7s

Contents

Protein chain

119 a.a.

References listed in PDB file

Key reference

Title

Arnt pas-B has a fragile native state structure with an alternative beta-Sheet register nearby in sequence space.

Authors

M.R.Evans, P.B.Card, K.H.Gardner.

Ref.

Proc Natl Acad Sci U S A, 2009, 106, 2617-2622. [DOI no: 10.1073/pnas.0808270106]

PubMed id

19196990

Abstract

The aryl hydrocarbon receptor nuclear translocator (ARNT) is a basic helix-loop-helix Period/ARNT/Single-minded (bHLH-PAS) protein that controls various biological pathways as part of dimeric transcriptional regulator complexes with other bHLH-PAS proteins. The two PAS domains within ARNT, PAS-A and PAS-B, are essential for the formation of these complexes because they mediate protein-protein interactions via residues located on their beta-sheet surfaces. While investigating the importance of residues in ARNT PAS-B involved in these interactions, we uncovered a point mutation (Y456T) on the solvent-exposed beta-sheet surface that allowed this domain to interconvert with a second, stable conformation. Although both conformations are present in equivalent quantities in the Y456T mutant, this can be shifted almost completely to either end point by additional mutations. A high-resolution solution structure of a mutant ARNT PAS-B domain stabilized in the new conformation revealed a 3-residue slip in register and accompanying inversion of the central Ibeta-strand. We have demonstrated that the new conformation has >100-fold lower in vitro affinity for its heterodimerization partner, hypoxia-inducible factor 2alpha PAS-B. We speculate that the pliability in beta-strand register is related to the flexibility required of ARNT to bind to several partners and, more broadly, to the abilities of some PAS domains to regulate their activities in response to small-molecule cofactors.

Figure 2.
ARNT PAS-B can exist in two distinct conformations. (A) Widespread peak doubling is observed throughout ^15N-^1H HSQC spectra of Y456T ARNT PAS-B, as seen by comparison with spectra from the wild-type protein. Several doubled sites are highlighted with blue circles in the Y456T spectrum. (B) The E403 resonance from the Y456T ARNT mutant indicates two slowly exchanging conformations, but the relative populations of these can be perturbed by additional mutations. (C) Demonstration that conformations 1 and 2 are in equilibrium is provided by Mono Q separation of conformation 2 from a Y456T sample and monitoring the reestablishment of the equilibrium with extended incubation.

Figure 5.
Residues adopt a similar conformation in both structures. (A and B) Sequence (A) and structure (B) alignment of both mutant and wild-type Iβ strands. Shifted side chains adopt conformations of wild-type residues despite changes in identity or sequence. This is clearly seen by residue C459, where in the wild-type conformation it is buried in the core but in the mutant conformation, it slips 3 residues, becoming solvent-exposed and filling the space occupied by T462.

Secondary reference #1

Title

Structural basis of arnt pas-B dimerization: use of a common beta-Sheet interface for hetero- And homodimerization.

Authors

P.B.Card, P.J.Erbel, K.H.Gardner.

Ref.

J Mol Biol, 2005, 353, 664-677. [DOI no: 10.1016/j.jmb.2005.08.043]

PubMed id

16181639

Figure 1.
Figure 1. Regulation of the hypoxia-inducible factor (HIF) by intracellular oxygen levels. Under normoxia, O[2]-dependent modifications target HIF-α subunits (green) for proteosomal degradation and interfere with their ability to interact with CBP/p300 transcriptional coactivators. Under the low O[2] levels of hypoxic conditions, HIF-α monomers are stabilized and transported to the nucleus where they form functional heterodimers with ARNT (blue) on hypoxia-responsive elements (HREs), upregulating gene transcription via the activity of their transcriptional activation domains (TADs, including ARNT TAD and HIF-α N and C-terminal TADs (NTAD and CTAD, respectively)).

Figure 4.
Figure 4. HADDOCK-derived structure of the HIF-2a/ARNT PAS-B domain complex. (a) Superposition of 20 lowest energy structures of the HIF-2a/ARNT PAS-B complex, with HIF-2a shown in green and ARNT in blue. The structure closest to the mean is highlighted with a tube representation for the backbone, and yellow sticks indicate the locations of the side-chains of three HIF-2a PAS-B residues previously shown to be critical for heterodimer formation and HIF-driven transcriptional activation.15^ and 16 (b) Ribbon diagram of the lowest energy complex structure (backbone r.m.s.d.=1.05(±0.31) Å for ordered residues 242-342 for HIF-2a and 361-445, 456-463 for ARNT PAS-B, relative to other members of ensemble). Backbone amide sites that display significant changes in chemical shift or intensity upon complex formation (shown as spheres) define the primary interface used for the HADDOCK calculations.

The above figures are reproduced from the cited reference with permission from Elsevier