Introduction

Polyglutamine (polyQ) tracts are functional features of many conserved transcriptional regulators and allow for complex conformational dynamics and interactions with other polyQ factors [1–9]. A prominent polyQ tract was first identified in the neurogenic gene Notch [10]. The Notch protein is central to a signaling pathway guiding patterning and cell fate decisions during metazoan development [11]. This polyQ tract is embedded in the Notch intracellular domain (NICD), which when cleaved leads to nuclear import and activation via DNA-bound CSL proteins. This polyQ tract is highly polymorphic in Drosophila melanogaster and can generate new alleles that cause developmental and neurogenic defects [7]. All of the identified polymorphic alleles are specific to Drosophila melanogaster because this same tract is uniquely configured in most other Drosophila species as determined by the placement of an intervening histidine residue and the underlying CAX triplet nucleotide repeat pattern [7].

The single polyQ tract of Drosophila Notch proteins is embedded in a much larger intrinsically disordered protein (IDP) region, which is common to many transcriptional activators and co-activators, including NICD, TBP, and many of the Mediator co-activator subunits [12, 13]. It is also common to large scaffolding proteins that serve as signal integration platforms that are regulated by multiple protein-protein interactions. On such example is Huntingtin (Htt), which is conserved in humans, flies [14], and non-metazoan eukaryotes such as social slime molds [15]. Polyglutamine (polyQ) expansions in Htt [1, 8, 9, 16, 17], TBP [18, 19], and other conserved loci in humans underlie various progressive neurodegenerative diseases [1].

IDP regions of regulator and scaffolding proteins provide conformational flexibility and allow the formation of transient regulator complexes under specific conditions [12, 13]. IDPs form random coils or molten globules with very little protein secondary structure and this makes them exceedingly difficult to study biophysically. As such, these regions are typically removed in proteins subjected to structural studies. These regions are also difficult to align with orthologous sequences encoded in other genomes, and are a major impediment to accurate gene annotation in whole genome sequence assemblies. Furthermore, short read assemblies are intractable at loci encoding lengthy polyQ tracts.

Although some eukaryotes, such as the amoebozoan slime mold Dictyostelium, exhibit genome-wide trends in polyQ content [20–22], in general encoded polyQ tracts of conserved regulators expand and contract in a locus-specific manner. The N-terminal polyQ tract in human Htt is highly polymorphic and is expanded relative to early branching primates (e.g., tarsiers), and other mammals (e.g., mouse) (Fig 1A, top). However, the Drosophila Htt ortholog features only a single glutamine residue at this position (Fig 1A, top). Nonetheless, a different region of Drosophila Htt features a prominent polyQ tract, which is absent in vertebrates, including humans, despite the region of its appearance being conserved (Fig 1A, bottom). Both of these polyQ “insertion slots” are preceded by a short leader motif that is predicted to form an amphipathic helix. In general, polyQ tracts evolve in an evolutionary turnover process, the study of which can lead to biophysical insights not immediately accessible via biochemical characterization.

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Fig 1. PolyQ tracts evolve in well-defined slots in Htt and Notch proteins.

(a) Shown is the N-terminal region of Huntingtin (Htt) from humans, tarsier, mouse, and Drosophila. This region evolved a polyglutamine (polyQ) tract at a well-defined location in mammals, which later expanded in the evolution of humans, and is still evolving. While fly Htt is missing the N-terminal polyQ tract, it features a separate tract elsewhere in the protein as shown. Small amino acid residues that are secondary structure breakers are highlighted in blue. Glutamine residues are highlighted in red. Hydrophobic amino acid residues preceding the polyQ tract insertion site are highlighted in cyan. Conserved residues are highlighted in yellow. See Fig 3 for a diagram showing the location of polyQ tracts relative to other NICD domains. (b) Shown is an evolutionary tree of various dipteran genera for which Notch sequences were analyzed in this study. The cladogram is a simplified version of the comprehensive fly tree based on multiple nuclear and mitochondrial genes and morphological markers [42]. The six different families to which these species belong are listed. (c) Shown is the polyQ tract regions of fly Notch proteins. Several sites (lettered slots) have independently evolved a polyQ tract in different fly genera. Most slots are preceded by a leader motif sequence, even when a polyQ tract is not present. (d) Inset shows the high hydrogen bonding potential of carboxamide amino acid side chains.

https://doi.org/10.1371/journal.pone.0174253.g001

To understand the biochemical constraints governing polyQ evolution, I considered the long polyQ tract in the Notch intracellular domain in Drosophila. We previously found that this polyQ tract is highly unstable and variable within the Drosophila melanogaster population [7]. This tract has been continuously evolving since the Drosophila radiation with different species exhibiting distinct polyQ tract configurations within the large IDP region of NICD [7]. Here I analyzed Notch proteins from diverse dipterans (flies) and discovered a previously unknown level of Notch polyQ evolution that is functionally revealing about the Notch intrinsically ordered region and its relation to the adjacent solenoid-forming ankyrin repeats (AR). I show that a polyQ slot insertion model provides a powerful interpretation of the functional interactions between polyQ tracts constrained to a limited number of slots in solenoid-type proteins such as the Notch intracellular domain and Huntingtin. The polyQ insertion model can be informed by use of the fast-evolving dipteran orthologs of human disease-related proteins featuring expanded polyQ tracts.

Results

I define “polyQ insertion slots” as specific positions in a protein where polyQ tracts are favored to occur during evolution. Strong support for a polyQ insertion model of protein evolution could include (i) evidence of a non-random or constrained distribution of polyQ tracts across a set of diverged orthologs, (ii) evidence of a polyQ turnover process whereby polyQ tracts have been lost at one position and gained at another, (iii) identification of structural or peptide sequence motifs serving as polyQ slot leaders or trailers, and (iv) evidence of a functional relationship of polyQ slots to other functional domains within the protein or within a protein interactor.

To identify polyQ insertion slots in the Notch intracellular domain, I analyzed predicted Notch proteins from eight different genera in six different families of Diptera (Fig 1B). I find that polyQ tracts in dipteran Notch proteins have evolved in seven different well-defined polyQ insertion slots (Fig 1C). Each slots is preceded by a conserved polyQ slot leader motif similar to the Htt amphipathic helix, further confirming the utility of the polyQ insertion slot model. Even in the absence of polyQ tracts, slot leaders precede a region enriched in the carboxamide residues, asparagine (N) and glutamine (Q), whose sidechains feature hydrogen bond donor and acceptor groups (Fig 1D). These same slot regions are also enriched in the small amino acid residues that are secondary structure breakers (“g”, “p”, and “s”).

Using the polyQ slot leader organizational model of the IDP region of dipteran NICD and a few reliably homologous slot leader motifs, I identified seven slots in the human Notch1 protein, as shown by an alignment to Drosophila NICD (Fig 2A). As in dipteran Notch proteins, these slots in human Notch1 are also enriched in both carboxamide residues and small secondary structure breakers that lead to IDPs. Thus, carboxamide-enriched IDPs with slot leaders likely represent a deeper organizational principle underlying many polyQ-rich regulators. I demonstrate the utility of this point as follows.

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Fig 2. Notch polyQ slots are conserved IDP interference elements of ankyrin repeats.

(a) Shown is an alignment of Drosophila Notch and human Notch1 intracellular domains (NICD) beginning at the S3 cleavage site (caret). The CSL/RBPJ-associated molecule (RAM) region is located at the N-terminus of NICD. Two nuclear localization signals (green with wavy underlining) flank the seven ankyrin repeats (ARs). The 33 residues of each AR features a helix-turn-helix peptide motif (helical regions in double underlining). This is followed by a conserved neddylation site and the six or seven polyQ insertion slots each starting with a polyQ slot leader sequence motif. A conserved domain of unknown function (DUF3454) follows the polyQ insertion slots. The polyQ slots fill up the region that is intrinsically disordered, but some slots are more conserved in sequence than others, such as slot-B, slot-D, and slot-G. See Fig 3 for a diagram showing the location of polyQ slots relative to other NICD domains. (b) The crystal structure of the human AR2–AR7 region is annotated to show the carboxamide residues that are conserved between fly Notch and human Notch1. These conserved Q/N residues and others not conserved are featured in the terminal loop and strand elements of each ankyrin repeat. (c) Shown is a close-up of ankyrin repeat 5 and the conserved double asparagines in the turn element.

https://doi.org/10.1371/journal.pone.0174253.g002

The seven N-terminal ankyrin repeats (ARs) in NICD form a solenoid-like domain, which fold by delicate intra-repeat interactions. Given the approximate 1-to-1 conservation of ARs to the IDP repeats with polyQ slots, it is possible that the IDP array is a kinetically active ensemble on one side of the linear AR solenoid. To investigate whether the polyQ IDP slots could be proteodynamic modulators of ARs, I analyzed the AR structural elements for carboxamide residues that could be hydrogen bonding with the carboxamide and polyQ rich IDPs. I find that the terminal halves of ARs are >10-fold enriched (13-to-1) in conserved carboxamide residues relative to the first half (see boxed residues in Fig 2A, and labeled residues in Fig 2B). Additional non-conserved carboxamide residues are also enriched in the terminal halves of ARs of both species, particularly in the terminal loop and strand elements (red Q’s and N’s in Fig 2A).

The terminal β-strand of each AR forms a hairpin with the starting β-strand of the next AR, and is important for induced propagation of solenoid formation [23]. I thus propose that the conserved array of seven polyQ insertion slots, which feature polyQ and/or carboxamide enriched IDPs, constitute AR interference elements (ARIEs). A pair of adjacent asparagines are also conserved in AR-5, which could be available for hydrogen bonding interactions in unfolded Notch AR solenoids (Fig 2C). Furthermore, the approximate 1-to-1 conservation of ARs to ARIEs in Notch suggest that ARIEs are proteodynamic modulators can be associated with specific solenoid repeats or pairs of repeats. The proteodynamic ARIEs array would attenuate AR solenoid formation via kinetic coupling and constitute a critical aspect of NICD regulation at multiple steps in the Notch signaling pathway.

Analogous to Notch and other AR-based solenoids, the proteins Htt, EF3, the regulatory A subunit of PP2A, and TOR1 (target of rapamycin) are solenoids based on HEAT repeats with Htt having three HEAT repeats [24]. Thus, I further suggest that solenoid interference arrays featuring polyQ turnover dynamics are functionally-coupled components of solenoids. Solenoid folding is a delicate regulatory-prone process and is distinct from the folding seen in stable globular structures driven by hydrophobic packing and long-distance interactions. Thus, a folding funnel landscape view of NICD and Htt might be described by bi-stable minima involving a lower moat ARIEs-ARs interference state and a higher dimple solenoid state [25]. Alternatively, or possibly occurring only in special contexts, the AR solenoid of NICD may “organize” the folding of the intrinsically disordered region contains the ARIEs array.

The conserved carboxamide residues of NICD occupy the inner side of the AR solenoid (Fig 2B, numbered residues on inner side). This side of the AR solenoid corresponds to the interaction surface in transcriptional complexes with Mastermind and CSL [23, 26]. Possibly, the polyQ richness of Mastermind (Mam/Maml) [4, 5, 27], a dedicated Notch-coactivator, functions as an AR solenoid chaperone by competing with the same solenoid surface entangled with the ARIEs array. Similarly, the Notch interacting protein Deltex features polyQ tracts in Drosophila, which has a longer polyQ tract than human Notch proteins, but not in human Deltex homologs (not shown). As such these functional intermolecular interactions would stem directly from AR-ARIE intramolecular coupling. Intermolecular ARIE-ARIE interactions in transcriptional NICD multimers [28, 29] may also relieve intramolecular inhibition of AR solenoid formation by the ARIEs array. Thus, polyQ expansions and contractions are likely to be under complex selection for their regulatory interactions with the adjacent solenoid repeats and their regulatory modulators.

Analysis of the evolutionary turnover of polyQ tracts in dipteran NICD moieties provides a novel perspective of the IDP region of NICD, and possibly completes the functional domain inventory of a key developmental signaling molecule (Fig 3A). ARIEs are evident by evolutionary polyQ slot turnover and by the subsequently identified common leader motif resembling the amphipathic leader in Huntingtin. To see if additional peculiarities pertain to the NICD slot leaders, I derived an NICD-specific polyQ slot leader motif by taking the nine residue peptide leader sequences from Drosophila and Stomoxys slot leaders except for those from the hypothesized polyQ slot-C, for which polyQ insertions have not yet been seen (Fig 3B). Not only is this a short amphipathic helix with hydrophobic residues on one side but there is typically at least one glutamine residue adjacent to the hydrophobic side (Fig 3B). Thus, the leader motif itself may function to both display and interact with polyQ epitopes or carboxamide-rich IDPs in slots, or with the carboxamide rich terminal elements of associated ankyrin repeats.

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Fig 3. Notch AR interference elements (ARIEs) are present as a distinct array within NICD.

(a) The polyQ slot leader motif defines 7 possible insertion sites, for which six have independently evolved polyQ tracts in distinct lineages of flies. The number of slots is also suggestive of an interference with the seven ankyrin repeats or the six pairs of adjacent repeats. (b) The leader sequences from Drosophila and Stomoxys polyQ insertion slots (except for slot C) were used to produce the motif logo shown. Also shown is an amphipathic helix starting with residue number four and showing how the hydrophobic amino acids at positions 6, 9, and 10 of the slot leader motif are segregated to one side of the helix (yellow circles). Interestingly, position 7 frequently features a single glutamine residue, which could interact with an adjacent polyQ tract. (c) Drosophila Notch and vertebrate Notch1 feature a polyQ tract in slot-G, although it is much more expanded in Drosophila than humans. (d, e) Unlike Drosophila Notch, the Notch proteins from several other fly genera, including Musca, Stomoxys, Glossina, Lucilia, Bactrocera, and Ceratitis, feature a more prominent polyQ tract in slot-F. These flies also have a much smaller polyQ tract in slot-G. The tephritid genera also feature a tract in slot-A, demonstrating that N-terminal slots can also accept polyQ tracts. (f) The Notch protein from Anopheles darlingii features polyQ tracts in slot-B and slot-E in addition to a very long tract in slot-G.

https://doi.org/10.1371/journal.pone.0174253.g003

Discussion

Altogether, the dipteran Notch proteins reveal the evolutionary appearance of lengthy polyQ tracts in five of the seven ARIEs and constitute an evolutionary Rosetta Stone for understanding NICD and its disordered carboxamide interference elements (Fig 3C–3F). The single polyQ tract of Drosophila melanogaster is polymorphic and homologous to the single polyQ tract that is configured differently in other Drosophila species. However, order-wide patterns across multiple dipteran genera are needed to reveal the entire expanse within which polyQ turnover happens. Similar deep evolutionary profiling of polyQ turnover in other conserved regulators may reveal additional solenoid-modulating interference arrays. Thus fast evolving dipteran genera may provide an ideal evolutionary system for understanding mutant human proteins associated with proteotoxicity diseases.

These results also raise the likelihood of blind spots in our understanding of solenoids formed by ankyrin repeats in Notch and other AR solenoids, HEAT repeats in Htt and other HEAT solenoids, and others. Much, much more work will be needed to understand how these delicate structures are coupled in turn to intrinsically disordered modules. Biophysical studies that necessarily start with truncated solenoid domains are unlikely to approximate the more dynamic lives these domains have in their full-length proteins and within the cell. Furthermore, studies focused on the biochemistry of Q/N-rich and polyQ-rich polypeptides are providing evidence of complex conformational dynamics involving multiple structural states including coiled-coils, α-helices, and β-sheets, and distinct properties promoting or disallowing multimerization and/or aggregation [30–32]. Theoretical modeling has also provided some support for the ability of these conformational states to be mechanically coupled to the cell cytoskeleton [33]. In this regard, a recent study has tested the complex dynamics bound to emerge in a full-length Huntingtin protein and has found evidence for the proposed intra-repeat modulatory effect on solenoid formation by its N-terminal polyQ tract [34]. Additional studies in which these expanded polyQ tracts are moved either to polyQ insertion slots identified by evolutionary comparisons or to negative control slots could highlight the extent to which the concept of polyQ insertion slots is productive. If so, the identification of such slots in fast-evolving dipteran orthologs of key human proteins should be prioritized.

The identification of satisfying multiple sequence alignments (MSA) for fast-evolving proteins enriched in intrinsically-disordered peptide regions is an important but difficult problem. The absence of protein-folding domains in these regions exacerbates the underlying problem. Furthermore, such regions are highly tolerant of insertions and deletions (indels), particularly for homopolymeric runs. To this end, newer advanced MSA methods, such as Bayesian Markov chain Monte Carlo samplers that can infer variable gap penalties (HMM transition probabilities for indels) from sequence data, could be extremely useful [35]. Such methods could also be applied to fast-evolving cis-regulatory sequences that tend to become enriched in microsatellite repeats [36, 37]. In the case of the NICD ARIEs region, polyQ slot leaders, if well conserved, can serve as ungapped alignment blocks separated by gap-tolerant linkers. Similarly, in the case of cis-regulatory DNAs, transcription factor binding motifs can serve as ungapped alignment blocks separated by gap-tolerant and MSR-enriched linkers. Thus, examples such as the NICD ARIEs can serve as a valuable data set for the development of newer MSA methods that can learn complex patterns of variable gap tolerance.

Methods

Protein structures were analyzed and annotated using NCBI’s Cn3D 4.3.1 software (http://www.ncbi.nih.gov/Structure) and high resolution structures of the ankyrin repeats of human Notch1 (PDB ID: 2F8Y, MMDB: 38239) [26]. The motif logo of Fig 3B was constructed using WebLogo (http://weblogo.berkeley.edu/logo.cgi). The source sequences for dipteran Notch proteins are from the following accessions and additional analyses: AAC36153.1, AAC36151.1 (Lucilia cuprina), XP_011292982.1 (Musca domestica), XP_013109509.1 (Stomoxys calcitrans), XP_004535280.1 (Ceratitis capitata), and XP_014087559.1 (Bactrocera oleae). The NICD sequence of Glossina morsitans (Tsetse fly) is a conceptual translation of CCAG010014983. The NICD sequence of Anopheles darlingii is a combination of ETN64594 and a splice corrected sequence from ADMH02000947. Alignments of Notch intrinsically disordered protein regions were explored by extensive variation of parameter space using both CLUSTALW [38] and MUSCLE [39, 40] on MEGA7 [41], but these were incongruent with each other and frequently subject to polyQ alignment artifact. Computed alignments improved when the sequences were anchored and or trimmed to include only the N-terminal ARIEs slot leaders to the highly conserved C-terminal DUF3454 sequence. Alignments in their final form shown in the figures were constructed manually and represent a maximization of the number of optimal local alignments. S1 and S2 Files contain text editable versions of the alignments of Figs 1 and 2.

Supporting information

Acknowledgments

I thank members of the Erives Laboratory for feedback at lab meetings and for asking good questions. I thank members of the Kreinitz Laboratory for the same. I thank Dr. Jan Fassler for providing critical feedback on an earlier version of this manuscript.