Crystal Structure and Interactions of the PAS Repeat Region of the Drosophila Clock Protein PERIOD

250

333

The hypoxia-inducible factor (HIF) basic helix-loop-helix Per-aryl hydrocarbon receptor nuclear translocator (ARNT)-Sim (bHLH-PAS) transcription factors are master regulators of the conserved molecular mechanism by which metazoans sense and respond to reductions in local oxygen concentrations. In humans, HIF is critically important for the sustained growth and metastasis of solid tumors. Here, we describe crystal structures of the heterodimer formed by the C-terminal PAS domains from the HIF2␣ and ARNT subunits of the HIF2 transcription factor, both in the absence and presence of an artificial ligand. Unexpectedly, the HIF2␣ PAS-B domain contains a large internal cavity that accommodates ligands identified from a small-molecule screen. Binding one of these ligands to HIF2␣ PAS-B modulates the affinity of the HIF2␣:ARNT PAS-B heterodimer in vitro. Given the essential role of PAS domains in forming active HIF heterodimers, these results suggest a presently uncharacterized ligand-mediated mechanism for regulating HIF2 activity in endogenous and clinical settings.internal cavity ͉ NMR ͉ X-ray crystallography ͉ hypoxia ͉ protein-ligand interactions T he hypoxia-inducible factor (HIF) transcription factors are present in multicellular organisms and adopt conserved roles in maintaining cellular oxygen homeostasis. In humans, HIF misregulation correlates with aggressive solid tumor growth and poor clinical outcomes (1, 2). Transcriptionally active HIF proteins are heterodimers of the HIF␣ and aryl hydrocarbon receptor nuclear translocator (ARNT, also known as HIF␤) subunits (3, 4), each containing an N-terminal basic helix-loophelix (bHLH) domain for specific DNA binding, two tandem Per-ARNT-Sim (PAS) domains to facilitate heterodimerization and C-terminal regulatory regions (5-7). Three known human HIF␣ subunit isoforms share ARNT as their bHLH-PAS protein binding partner. HIF1␣ and HIF2␣ are similarly regulated, but show cell line-specific differences in expression and gene regulation patterns (8). HIF3␣ and its splicing isoforms (9, 10) lack C-terminal sequences that recruit transcriptional coactivator proteins, suggesting that these proteins act as dominant negative pathway regulators by forming regulatory-incompetent heterodimers with ARNT.Regulation of this pathway is governed in large part by posttranslational modifications that down-regulate HIF activity under adequate cellular oxygenation levels (normoxia). The best characterized of these modifications are hydroxylations of key proline and asparagine residues in the HIF␣ C-terminal region (11,12). These hydroxylated prolines recruit the von Hippel Lindau (pVHL) E3 ubiquitin ligase, which ultimately downregulates HIF␣ protein levels through proteasomal degradation, whereas the hydroxylated asparagines block HIF␣-coactivator interactions. Oxygen-insufficient conditions (hypoxia) inactivate the hydroxylases, allowing HIF␣ subunits to escape degradation, heterodimerize with ARNT, and ultimately control the levels of Ͼ100 proteins (13). Misregulation of the HIF path...

Section: Discussionmentioning

confidence: 99%

Artificial ligand binding within the HIF2α PAS-B domain of the HIF2 transcription factor

Scheuermann

Tomchick²,

Machius³

et al. 2009

250

333

“…Interestingly, this tyrosine is replaced by an alanine (Ala287 dPER ) in the Drosophila PERIOD (dPER) homologue. This substitution enables the insertion of Trp482 dPER (corresponding to Trp448 mPER1 ∕ Trp419 mPER2 ∕Trp359 mPER3 ) into the PAS-A domain binding pocket of the dimerizing molecule and hence the formation of a completely different dPER homodimer (27,31). Gly264 and Gly268 of mPER1, which allow for a close approach of the dimerizing αC helices and hence the formation of a tighter PAS-A/αC interface than in mPER3, are conserved in mammalian PER1 homologues but not in other PER proteins or bHLH-PAS transcription factors.…”

Section: Discussionmentioning

confidence: 99%

Unwinding the differences of the mammalian PERIOD clock proteins from crystal structure to cellular function

Kucera

Schmalen

Hennig

et al. 2012

Self Cite

The three PERIOD homologues mPER1, mPER2, and mPER3 constitute central components of the mammalian circadian clock. They contain two PAS (PER-ARNT-SIM) domains (PAS-A and PAS-B), which mediate homo-and heterodimeric mPER-mPER interactions as well as interactions with transcription factors and kinases. Here we present crystal structures of PAS domain fragments of mPER1 and mPER3 and compare them with the previously reported mPER2 structure. The structures reveal homodimers, which are mediated by interactions of the PAS-B β-sheet surface including a highly conserved tryptophan (Trp448 mPER1 , Trp419 mPER2 , Trp359 mPER3 ). mPER1 homodimers are additionally stabilized by interactions between the PAS-A domains and mPER3 homodimers by an N-terminal region including a predicted helix-loop-helix motive. We have verified the existence of these homodimer interfaces in solution and inside cells using analytical gel filtration and luciferase complementation assays and quantified their contributions to homodimer stability by analytical ultracentrifugation. We also show by fluorescence recovery after photobleaching analyses that destabilization of the PAS-B/tryptophan dimer interface leads to a faster mobility of mPER2 containing complexes in human U2OS cells. Our study reveals structural and quantitative differences between the homodimeric interactions of the three mouse PERIOD homologues, which are likely to contribute to their distinct clock functions.circadian clock | PAS domains | PERIOD proteins | protein interactions

“…The Ala scan data indicate that a significant fraction of the side chains in PYP is dedicated to tuning the interaction between the N-terminal region of PYP and its PAS domain core, and thus regulate τ pB (Table S3). The C-terminal helical extensions of a PAS domain from the plant photoreceptor phototropin (40) and the Drosophila clock protein Period (41) and the N-terminal extension of a PAS domain in the fungal photoreceptor Vivid (42) all pack against the PAS core of the protein in a position similar to that of the N-terminal region of PYP. Functionally important conformational changes and partial unfolding occur in all of these extensions (30,31,(40)(41)(42).…”

Section: Discussionmentioning

confidence: 99%

Robustness and evolvability in the functional anatomy of a PER-ARNT-SIM (PAS) domain

Philip

Kumauchi

Hoff

2010

The robustness of proteins against point mutations implies that only a small subset of residues determines functional properties. We test this prediction using photoactive yellow protein (PYP), a 125-residue prototype of the PER-ARNT-SIM (PAS) domain superfamily of signaling proteins. PAS domains are defined by a small number of conserved residues of unknown function. We report high-throughput biophysical measurements on a complete Ala scan set of purified PYP mutants. The dataset of 1,193 values on active site properties, functional kinetics, stability, and production level reveals that 124 mutants retain the characteristic photocycle of PYP, but that the majority of substitutions significantly alter functional properties. Only 35% of substitutions that strongly affect function are located at the active site. Unexpectedly, most PAS-conserved residues are required for maintaining protein production. PAS domain activation often involves conformational changes in α-helices linked to the PAS core. However, the mechanism of transmission and kinetic regulation of allosteric structural changes from the PAS domain to these helices is not clear. The Ala scan data reveal interactions governing allosteric switching in PYP. The photocycle kinetics is significantly altered by substitutions at 58 positions and spans a 3,000-fold range. Nine residues that dock the N-terminal α-helices of PYP to its PAS core regulate signaling kinetics. Ile39 and Asn43 are identified as part of a mechanism for regulating allosteric switching that is conserved among PAS domains. These results show that PYP combines robustness with a high degree of evolvability and imply production level as an important factor in protein evolution.allosteric signal transmission | molecular evolution | protein structure-function relationship