Asparaginyl β-Hydroxylation of Proteins Containing Ankyrin Repeat Domains Influences Their Stability and Function

doi:10.1016/j.jmb.2009.07.070

Journal of Molecular Biology

Volume 392, Issue 4, 2 October 2009, Pages 994-1006

https://doi.org/10.1016/j.jmb.2009.07.070 Get rights and content

Abstract

Recent reports have provided evidence that the β-hydroxylation of conserved asparaginyl residues in ankyrin repeat domain (ARD) proteins is a common posttranslational modification in animal cells. Here, nuclear magnetic resonance (NMR) and other biophysical techniques are used to study the effect of asparaginyl β-hydroxylation on the structure and stability of ‘consensus’ ARD proteins. The NMR analyses support previous work suggesting that a single β-hydroxylation of asparagine can stabilize the stereotypical ARD fold. A second asparaginyl β-hydroxylation causes further stabilization. In combination with mutation studies, the biophysical analyses reveal that the stabilizing effect of β-hydroxylation is, in part, mediated by a hydrogen bond between the asparaginyl β-hydroxyl group and the side chain of a conserved aspartyl residue, two residues to the N-terminal side of the target asparagine. Removal of this hydrogen bond resulted in reduced stabilization by hydroxylation. Formation of the same hydrogen bond is also shown to be a factor in inhibiting binding of hydroxylated ARDs to factor-inhibiting hypoxia-inducible factor (FIH). The effects of hydroxylation appear to be predominantly localized to the target asparagine and proximal residues, at least in the consensus ARD protein. The results reveal that thermodynamic stability is a factor in determining whether a particular ARD protein is an FIH substrate; a consensus ARD protein with three ankyrin repeats is an FIH substrate, while more stable consensus ARD proteins, with four or five ankyrin repeats, are not. However, NMR studies reveal that the consensus protein with four ankyrin repeats is still able to bind to FIH, suggesting that FIH may interact in cells with natural ankyrin repeats without resulting hydroxylation. Overall, the work provides novel biophysical insights into the mechanism by which asparaginyl β-hydroxylation stabilizes the ARD proteins and reduces their binding to FIH.

Introduction

Recent reports have provided evidence that asparaginyl β-hydroxylation in ankyrin repeat domain (ARD) proteins is a common posttranslational modification in animal cells.1, 2, 3, 4, 5, 6 Following the discovery that factor-inhibiting hypoxia-inducible factor (FIH) catalyzes asparaginyl pro-S β-hydroxylation of ARD proteins from the nuclear factor κβ and nuclear factor κβ inhibitor protein family,² it was found that FIH catalyzes the hydroxylation of Notch receptors,1, 3 the SOCS (suppression of cytokine signaling) box protein 4 (ASB4),⁵ Tankyrase-2, Rabankyrin-5, RNase L,⁴ and MYPT1.⁶

ARDs are ubiquitous and are predicted to be present in greater than 300 human proteins.⁷ ARD proteins are involved in many biological processes including signaling pathways (e.g., Iκβα). Evidence that FIH can catalyze asparaginyl hydroxylation of many ARD proteins comes from work with peptide fragments of ARDs, proteomic analyses,⁴ and the observation that FIH can also catalyze asparaginyl β-hydroxylation in a consensus ARD protein containing three ankyrin repeats.2, 8

Prior to its identification as an ARD asparaginyl hydroxylase, FIH was shown to catalyze the asparaginyl β-hydroxylation of the C-terminal transactivation domain of the α-subunit of hypoxia-inducible factor (HIF-α).9, 10, 11, 12 Asparaginyl β-hydroxylation of HIF-α reduces its interaction with the transcriptional co-activation protein, p300, thus providing a mechanism by which the transcriptional activity of HIF can be regulated in an oxygen-dependent manner.9, 13, 14 Oxygen-dependent regulation of the levels of HIF-α occurs by a separate process involving posttranslational prolyl-4-hydroxlyation, as catalyzed by three or possibly four prolyl hydroxylases (PHDs), in human cells.15, 16, 17 In contrast to the apparent broad substrate specificity of FIH, current evidence suggests that the human PHDs only accept a narrow range of substrates.¹⁸ Both FIH and the PHDs belong to the Fe(II)- and 2-oxoglutarate (2-OG)-dependent superfamily of oxygenases, which are structurally characterized by the presence of a double-stranded β-helix core and conserved ferrous iron and 2-OG binding sites.19, 20

Of the recombinant ARD proteins examined as FIH substrates to date, hydroxylation has been shown to occur on the ankyrin repeats that closely resemble consensus ARD sequences.² Individual ankyrin repeats typically contain approximately 33 residues arranged in two α-helices composed of around 8 and 10 residues, respectively. Ankyrin repeats are linked by β-hairpin loops that project perpendicularly away from the helices leading to a characteristic L shape.²¹ ARDs composed of 5/6 ankyrin repeats are common, although ARDs with many more ankyrin repeats have been reported.²² The asparaginyl residues of ankyrin repeats that are known to be hydroxylated are conserved and located on the β-hairpin loops.

Comparisons of ankyrin repeat sequences identified as FIH substrates (Fig. 1a) reveal high levels of similarity outside the conserved DXN motif, including a leucine residue at − 8 (i.e., to the N-terminal side) relative to the target asparagine, a glycine at the − 4-position, and an alanine at − 3. However, because these residues are highly (but not totally) conserved within ARD proteins, in general, they would be expected to be present in sequences that are and are not FIH substrates. Indeed, at present, it appears that the minimum requirement for ARD asparaginyl β-hydroxylation is an asparagine appropriately positioned within an ankyrin repeat. However, the conservation of a leucinyl (or similar) residue at the − 8-position may be significant (despite being conserved in greater than 80% of ankyrin repeats²³) because a leucine is also present at an analogous position in HIF-α (Leu795 in human HIF-1α). Crystal structures of FIH complexed with HIF-1α and ankyrin repeat peptides reveal distinct binding interactions between the − 8 leucinyl residue and a hydrophobic pocket on FIH.¹ However, not all appropriately positioned asparaginyl residues are hydroxylated either in vivo or in vitro; for example, Notch-4 seems to contain an appropriately positioned asparaginyl residue (N1686), which has not yet been observed to be hydroxylated within the limits of detection.¹

In contrast to the apparent direct role of asparaginyl and prolyl hydroxylation in HIF-α regulation, the biological function of asparaginyl β-hydroxylation of ARD proteins is uncertain. Because asparaginyl β-hydroxylation decreases the affinity of ARD proteins for FIH, it has been proposed that the hydroxylation status of the ‘pool’ of FIH accessible ARD proteins regulates the amount of free FIH available to catalyze HIF-α hydroxylation.⁴ Asparaginyl β-hydroxylation does not alter the conformation of ARD proteins in the crystalline state.1, 8 However, in solution, it was found that β-hydroxylation stabilizes the fold of a consensus ankyrin repeat protein containing three repeats.⁸ To date, there is no evidence that such stabilization occurs when a fragment of human HIF-1α (which does not adopt a well-defined structure while free in solution) is hydroxylated at Asn803.

The findings concerning ankyrin hydroxylation have raised the question of how hydroxylation stabilizes the ARD fold and the factors that govern the effect of asparaginyl hydroxylation on binding of the protein to FIH. Here, we provide biophysical evidence that asparaginyl hydroxylation enables an additional hydrogen bond between hydroxyl-asparaginyl and the aspartyl residue of the conserved DXN(OH) motif. The results also reveal that the thermodynamic stability of an ARD fold is a factor in deciding whether a specific ARD is an FIH substrate; FIH can also bind ARDs that are not substrates for hydroxylation.

Section snippets

Is the stability of ankyrin repeats a factor in determining if an ARD is an FIH substrate?

Kelly et al.⁸ have reported that a three-repeat consensus ankyrin protein (3CA_1A2N), containing a single appropriately positioned asparaginyl residue, is a substrate for FIH. However, a four-repeat consensus ankyrin protein (4CA_1A2A3N), also containing a single potential target asparaginyl residue, was not an FIH substrate.⁸ The conformational stability of proteins with consensus ARDs is reported to increase as the number of ankyrin repeats increases.24, 25 We therefore compared the activity of

Discussion

There is increasing evidence that ARD asparaginyl hydroxylation is a common intracellular posttranslational modification. As the list of confirmed sites of FIH-mediated ARD hydroxylations has grown, it has become apparent that the sequence requirements for this reaction are relatively lenient.⁴ At present, it seems that the residue targeted for hydroxylation itself is the only residue absolutely conserved among FIH substrates, with the only requirement being that this residue resides at a

Design of genes for common ARDs

The nomenclature of Kelly et al.⁸ is used to describe the number of ankyrin repeats and potential hydroxylation sites in the consensus ARDs used in these studies, that is, 2CA_N, 3CA_1A2N, 4CA_1A2A3N, and 5CA_1A2A3A4N. The regular font number defines the total number of ankyrin repeats in the construct. The subscript (A or N) refers to the presence of an alaninyl or asparaginyl residue at the site of the residue ‘targeted’ for hydroxylation. The associated number defines the ankyrin repeat in which

Acknowledgements

This work was funded by the Biotechnology and Biological Sciences Research Council (UK), the European Union, and the Wellcome Trust. We are grateful to I. Vakonakis for providing the rhamnose binding lectin domain from mouse latrophilin 1 as a control, M. Nutley for contributions to the DSC experiments and P. J. Ratcliffe, M. L. Coleman and M. A. McDonough for discussions.

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The ASPPs were shown to compete with a HIF-1α fragment and a previously reported consensus ankyrin substrate (19) for FIH-catalyzed hydroxylation (Figs. S10–S12), raising the possibility that they might, along with other FIH substrates, be involved in regulating the role of FIH in the hypoxic response. Consensus ARD-derived peptides (19, 20) containing VNVN, ANVN, and VNAN motifs were also shown to be FIH substrates undergoing single hydroxylations (Fig. S13). The combined turnover and structural studies imply that in order for the second FIH-catalyzed hydroxylation to occur, interactions in addition to those at the active site (Figs 1 and S6) and which do not manifest in the FIH:peptide fragment crystal structures are involved in the second hydroxylation.
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In human cells oxygen levels are ‘sensed’ by a set of ferrous iron and 2-oxoglutarate dependent dioxygenases. These enzymes regulate a broad range of cellular and systemic responses to hypoxia by catalysing the post-translational hydroxylation of specific residues in the alpha subunits of hypoxia inducible factor (HIF) transcriptional complexes. The HIF hydroxylases are now the subject of pharmaceutical targeting by small molecule inhibitors that aim to activate or augment the endogenous HIF transcriptional response for the treatment of anaemia and other hypoxic human diseases. Here we consider the rationale for this therapeutic strategy from the biochemical, biological and medical perspectives. We outline structural and mechanistic considerations that are relevant to the design of HIF hydroxylase inhibitors, including likely determinants of specificity, and review published reports on their activity in pre-clinical models and clinical trials.
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Citation Excerpt :
The natural ARD proteins, Notch and IκBα, contain more than six ARDs and are known to be relatively efficient substrates for FIH (43, 62), including in cells (18). Thus, the number of contiguous ARDs in a protein does not necessarily correlate with the hydroxylation efficiency, which is also related to local stability and sequence of the hydroxylated region (21, 22). ( Note also that FIH probably interacts with ARDs that it does not hydroxylate (22).)
The hypoxia-inducible factor (HIF) hydroxylases regulate hypoxia sensing in animals. In humans, they comprise three prolyl hydroxylases (PHD1–3 or EGLN1–3) and factor inhibiting HIF (FIH). FIH is an asparaginyl hydroxylase catalyzing post-translational modification of HIF-α, resulting in reduction of HIF-mediated transcription. Like the PHDs, FIH is proposed to have a hypoxia-sensing role in cells, enabling responses to changes in cellular O₂ availability. PHD2, the most important human PHD isoform, is proposed to be biochemically/kinetically suited as a hypoxia sensor due to its relatively high sensitivity to changes in O₂ concentration and slow reaction with O₂. To ascertain whether these parameters are conserved among the HIF hydroxylases, we compared the reactions of FIH and PHD2 with O₂. Consistent with previous reports, we found lower K_m^app(O₂) values for FIH than for PHD2 with all HIF-derived substrates. Under pre-steady-state conditions, the O₂-initiated FIH reaction is significantly faster than that of PHD2. We then investigated the kinetics with respect to O₂ of the FIH reaction with ankyrin repeat domain (ARD) substrates. FIH has lower K_m^app(O₂) values for the tested ARDs than HIF-α substrates, and pre-steady-state O₂-initiated reactions were faster with ARDs than with HIF-α substrates. The results correlate with cellular studies showing that FIH is active at lower O₂ concentrations than the PHDs and suggest that competition between HIF-α and ARDs for FIH is likely to be biologically relevant, particularly in hypoxic conditions. The overall results are consistent with the proposal that the kinetic properties of individual oxygenases reflect their biological capacity to act as hypoxia sensors.
Factor inhibiting HIF (FIH) recognizes distinct molecular features within hypoxia-inducible factor-α (HIF-α) versus ankyrin repeat substrates
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Factor Inhibiting HIF (FIH) catalyzes the β-hydroxylation of asparagine residues in HIF-α transcription factors as well as ankyrin repeat domain (ARD) proteins such as Notch and Gankyrin. Although FIH-mediated hydroxylation of HIF-α is well characterized, ARDs were only recently identified as substrates, and less is known about their recognition and hydroxylation by FIH. We investigated the molecular determinants of FIH substrate recognition, with a focus on differences between HIF and ARD substrates. We show that for ARD proteins, structural context is an important determinant of FIH-recognition, but analyses of chimeric substrate proteins indicate that the ankyrin fold alone is not sufficient to explain the distinct substrate properties of the ARDs compared with HIF. For both substrates the kinetic parameters of hydroxylation are influenced by the amino acids proximal to the target asparagine. Although FIH tolerates a variety of chemically disparate residues proximal to the asparagine, we demonstrate that certain combinations of amino acids are not permissive to hydroxylation. Finally, we characterize a conserved RLL motif in HIF and demonstrate that it mediates a high affinity interaction with FIH in the presence of cell lysate or macromolecular crowding agents. Collectively, our data highlight the importance of residues proximal to the asparagine in determining hydroxylation, and identify additional substrate-specific elements that contribute to distinct properties of HIF and ARD proteins as substrates for FIH. These distinct features are likely to influence FIH substrate choice in vivo and, therefore, have important consequences for HIF regulation.

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¹: Present address: I. Prokes, Department of Chemistry, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK.

^†: I.P. and L.K. contributed equally to this work.

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Journal of Molecular Biology

Asparaginyl β-Hydroxylation of Proteins Containing Ankyrin Repeat Domains Influences Their Stability and Function

Abstract

Introduction

Section snippets

Is the stability of ankyrin repeats a factor in determining if an ARD is an FIH substrate?

Discussion

Design of genes for common ARDs

Acknowledgements

J. Biol. Chem.

Mol. Cell. Proteomics

J. Biol. Chem.

Cell

J. Biol. Chem.

J. Biol. Chem.

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

J. Inorg. Biochem.

Structure

J. Mol. Biol.

J. Mol. Biol.

Structure

J. Biol. Chem.

Posttranslational hydroxylation of ankyrin repeats in IκB proteins by the hypoxia-inducible factor (HIF) asparaginyl hydroxylase, factor inhibiting HIF (FIH)

Proc. Natl Acad. Sci. USA

Interaction with factor inhibiting HIF-1 defines an additional mode of cross-coupling between the Notch and hypoxia signaling pathways

Proc. Natl Acad. Sci. USA

ASB4 is a hydroxylation substrate of FIH and promotes vascular differentiation via an oxygen-dependent mechanism

Mol. Cell. Biol.

MYPT1, the targeting subunit of smooth muscle myosin phosphatase, is a substrate for the asparaginyl hydroxylase factor inhibiting hypoxia inducible factor (FIH)

Biochem. J.