Structural insights and characterization of human Npas4 protein

Introduction

NPas4 belong to the basic helix loop helix/per-arnt-sim (BHLH/PAS) transcription factor family (Shamloo et al., 2006). This family is responsible for gearing the expression of target genes on either the positive or negative side (Spiegel et al., 2014). Classically, these transcription factors harbor a DNA binding motif and a dimerisation motif called PAS homology domain (Gu, Hogenesch & Bradfield, 2000). The word ‘PAS’ pertains to the first three proteins of the domain; period (per), aryl hydrocarbon receptor translocator (arnt) and single minded (SIM) (Ooe, Saito & Kaneko, 2009). Functionally, Npas4 is categorized as an immediate early gene (IEG), responsible for direct control of a large number of activity dependent genes that are capable of altering their expression input on the basis of sensory stimuli they receive (Madabhushi et al., 2015; Sun & Lin, 2016). Npas4 is also known for development of glutaminergic and GABAergic synapses in neurons suggesting its crucial role in neuro-circuitary homeostasis and memory formation (Spiegel et al., 2014). Deletion of Npas4 may lead to development of several neuronal plasticity disorders and disorganised management of sensory input (Maya-Vetencourt, 2013). Previous studies consolidated its functional role in cerebral ischemia, memory developmental disorders and related pathologies like autism spectrum disorders, and neuropsychiatric disorders (Choy et al., 2015a; Choy et al., 2015b; Ebert & Greenberg, 2013; Shepard, Heslin & Coutellier, 2017). Limited studies are available which clearly delineate Npas4’s role in regulating its discovered function of neuro-protection and hemostasis in synaptic inter connectivity. The tertiary structure of Npas4 protein along with its physico chemical properties is scarcely available. The current study was mainly aimed to provide the structural insight of Npas4 protein and its possible interaction with other proteins.

Materials and Methods

The flow chart of methodology is shown in Fig. 1.

Results

Multiple sequence alignment and phylogenetic analysis

Multiple sequence alignment of human Npas4 sequence with other species is shown in Figure S2. The phylogenetic analysis of the aligned sequences of Npas suggests Npas4’s evolutionary conservativeness. The analysis of taxonomic classification of all organisms (Table S1) revealed human Npas4 to be closely related to other primates including chimpanzees (T.N), monkeys and gibbons. The next closest neighbors were rodents (marmots, elephant-shrews, moles, guinea pigs). Other related organisms to rodents were artiodactyla like camels and alpacas. The farthest neighbors of Npas4 sequences were from rodents including rabbits and pikas (Fig. 2).

Disorder prediction of Npas4

The disorder prediction was carried out using Metadisorder and Mobidb. According to Metadisorder and Mobidb, results indicated Npas4 is an intrinsically disordered protein. According to Metadisorder plot C-terminal half of the protein showed strong disorder while N-terminal of the protein is ordered (position 15–350 a.a). CDF (cumulative distribution function) analysis showed that Npas4 is mixture of ordered and disordered regions as it intersects the boundary (Fig. 3). Primary sequence analysis showed that Npas4 is enriched in disorder promoting amino acids (Pro, Ser, Glu, Gln, Ala and Gly; Table 1). These amino acids have the tendency to prevent the folding of protein (67–70). Pro and Ser have the highest probability. Amino acid sequence analysis of Npas4 protein showed that almost 50% of the protein composed of disorder region. Flexibility analysis of Npas4 was performed using composition profiler and DynaMine server and both servers showed that Npas4 is enriched with flexible amino acids like Pro, Ser and Gln (Fig. 4A). Low hydrophobicity and high hydrophilicity promote disorder in the proteins (Dyson & Wright, 2016). The analysis according to the composition profiler server suggests Npas4 is enriched with hydrophilic amino acids like Gln, Ser, Pro and Thr (Fig. 4B).

Physico-chemical properties of Npas4

The physico-chemical properties were determined by Protscale server (Fig. 5). The higher score suggested a higher probability of that particular property of Npas4. The ratio of the side chain volume to the length of an amino acid suggested protein bulkiness and may affect the local structure of a protein. The bulkiness values of Npas4 (Fig. 5A) range from 9.713 (position 731aa) to 18.729 (position 295 aa). The hydrophobicity prediction values by Hopp and wood score (Fig. 5B) were between −0.711 (position 630aa) and 0.422 (position 44 aa). The dipole–dipole intermolecular interactions between the positively and negatively charged particles depicted polarity as predicted through Zimmerman score (Fig. 5C) The predicted score lies between 0.272 (position 81 and 82aa) and 34.216 (position 630 aa). This data showed that Npas4 possesses polarity. Mutability, which is the probability that amino acids will bring a change over a particular evolutionary period of time, was determined through the relative mutability score (Fig. 5D) and the values lie between 46.000 (position 295aa) and 102.889 (position 18 aa) which showed that Npas4 has mutability potential.

Table 1:

Disorder promoting amino acids of Npas4.

Disorder promoting amino acids				Order promoting amino acids
Polar/charged amino acids		Small amino acids		Hydrophobic or bulky
Pro	10.3%	Ala	7.8%	Leu	13.0%
Ser	10.2%	Gly	6.6%	Phe	4.9%
Glu	7.2%			Val	3.2%
Gln	5.2%			Tyr	2.6%
Arg	3.4%			Ile	2.5%
Lys	2.4%			Asn	2.1%
				Cys	1.6%
				Trp	0.87%

DOI: 10.7717/peerj.4978/table-1

The signal peptide of Npas4 was computed using SignalP Server. The presence of signal peptide was measured through C- (raw cleavage site score), S- (signal peptide score) and Y-score (combined cleavage site score). S-score is the estimation of possible signal peptide while D-score is the average of mean S and the max Y-score and its discriminate signal peptide from non-signal peptides. In Npas4, the D-score was 0.450 which was less than the cut off value of 0.5 which showed absence of signal peptide (Fig. 6). The secretory nature of Npas4 was predicted through SecretomeP and the NN score which was 0.5.

Post translation modification sites

Attempts were made to predict the protein modification sites that could be occurring in Npas4. The results indicated absence of acetylation and mannosylation sites. However, two potential N-glycosylation sites were found in disorder region of Npas4 at 556 (NPTK) and 671 (NLSL) amino acid positions (Fig. 7).

A total of 80 O’ linked glycosylation sites were present in Npas4. However, only 56 sites were found available for glycosylation according to the GlyCamserver.

According to Netphos 3.1 server predictions, 34 threonine phosphorylation sites, 53 serine phosphorylation sites and four tyrosine specific phosphorylation sites were present in Npas4 (Fig. 8). NetsurfP prediction revealed 43 serine, 28 threonine and three tyrosine residues were exposed for phosphorylation. Table S2 showed the 24 Serine, 13 Threonine and one Tyrosine kinase specific sites in Npas4. Two phosphorylation sites present (S38, S44) in bHLH domain and three sites (S98, S100 and T136) were observed in PASA domain while one site (S273) was seen in PAS B domain. Six Ser and four Thr phosphorylation sites were present in disordered transactivation domain (Table 3).

Table 3:

Phosphorylation sites in disorder region of Npas4.

S.No	Phosphorylation site	Kinases
1	Ser 611	P38MAPK, CDK5
2	Thr 620	PKC
3	Thr 640	CDC2
4	Ser 656	CKII
5	Thr 673	CKI
6	Ser 719	P38MAPK, CKII
7	Ser 728	CKII
8	Ser 759	CKII
9	Ser 777	CKII
10	Thr 785	PKC, ATM

DOI: 10.7717/peerj.4978/table-3

Different physiological parameters of Npas4 were predicted trough Protparam server (Table 4).

Table 4:

Physiological parameters of Npas4.

Molecular weight	87,116.58
Theoretical PI	4.53
Formula	C3894H5965N1005O1220S23
Total no. of atoms	12,107
Estimated half life	30 h

DOI: 10.7717/peerj.4978/table-4

Tertiary structure of Npas4

The three dimensional structure of Npas4 protein has not been determined to date. Due to the absence of suitable structural template, holmology modeling can not be used (Figure S1). In order to get the high quality structure of Npas4, ab-initio and threading approaches were used. By using these approaches five models were generated through I-Tasser, 10 models through LOMET, one model from Raptor X, 10 models from MUSTER, one model from Phyre and 10 models from SPARSK-X (Table 5). All the 37 models were then subjected to validation through PROSA, Qmean and Ramachandran plot. The models with 96% residues in favoured region were further selected for refinement (Table 6, Fig. 9). All models suggested that Npas4 is a disordered protein with ordered bHLH and PAS domain. Based on Qmean, PROSA Z-score and ramachandran scores, model 9 generated LOMET was the best predicted three dimensional structure of Npas4. The possible phosphorylation sites in PASA and PASB domain of Npas4 is shown in Fig. 10.

Table 5:

Models of Npas4 protein with different methods and their evaluation results.

Model/tool	Method	PROSA	Qmean	Ramachandran plot
				Outlier	Allowed region	Favored region
I-TASSER-1	Multiple-threading alignments	−3.6	−11.55 9.4%	18.6%	72.0%
I-TASSER-2	Multiple-threading alignments	−1.28	−12.31 8.4%	17.4%	74.4%
I-TASSER-3	Multiple-threading alignments	−2.59	−16.61 18.5%	28.0%	53.5%
I-TASSER-4	Multiple-threading alignments	−4.99	−16.83 19.2%	24.6%	56.1%
I-TASSER-5	Multiple-threading alignments	−5.07	−13.55 14.2%	27.8%	58.0%
LEMOT-1	Local meta-threading	−2.89	−3.34 1.6%	2.2%	96.1%
LEMOT-2	Local meta-threading	−3.07	−3.22 1.4%	2.9%	95.8%
LEMOT-3	Local meta-threading	−3.66	−3.08	0.8%	2.4%	96.9%
LEMOT-4	Local meta-threading	−3.72	−3.66 1.6%	2.4%	96.0%
LEMOT-5	Local meta-threading	15.32	−10.17 10.2%	14.2%	75.6%
LEMOT-6	Local meta-threading	−3.11	−3.96	2.0%	5.9%	92.1%
LEMOT-7	Local meta-threading	−2.61	−2.93 1.6%	4.5%	93.9%
LEMOT-8	Local meta-threading	−3.33	−2.75 1.5%	2.9%	95.6%
LEMOT-9	Local meta-threading	−3.63	−2.61 0.6%	3.2%	96.1%
LEMOT-10	Local meta-threading	3.09	−20.65 7.5%	15.9%	76.7%
RaptorX	Threading	−5.42	−5	2.8%	4.7%	92.5%
MUSTER-1	Multi-source threading	−1.57	−12.86	4.5%	8.2%	87.2%
MUSTER-2	Multi-source threading	−1.51	−20.75	6.2%	12.2%	81.5%
MUSTER-3	Multi-source threading	1.09	−2.38	7.2%	11.2%	81.2%
MUSTER-4	Multi-source threading	−3.19	−3.91	1.8%	1.9%	96.4%
MUSTER-5	Multi-source threading	−3.35	−2.88	1.1%	2.6%	96.2%
MUSTER-6	Multi-source threading	−2.87	−20.70	4.8%	10.0%	85.2%
MUSTER-7	Multi-source threading	−0.34	−14.63	4.8%	9.5%	85.8%
MUSTER-8	Multi-source threading	−2.44	−4.14	2.1%	3.4%	94.5%
MUSTER-9	Multi-source threading	0.53	−9.44	7.1%	12.0%	80.9%
MUSTER-10	Multi-source threading	−1.46	−12.05	4.5%	12.9%	82.6%
Phyre	Homology detection method	−4.13	−8.70	7.9%	12.8%	79.4%
SPARSK-1	Fold recognition	−3.9	−9.59	5.5	7.8	86.7%
SPARSK-2	Fold recognition	−4.57	−6.67	2.2	3.9	93.9%
SPARSK-3	Fold recognition	−4.02	−6.12	1.5	3.6	94.9%
SPARSK-4	Fold recognition	−5.58	−6.05	1.6	6.1	92.2%
SPARSK-5	Fold recognition	−5.33	−7.03	1.8	4.4	93.9%
SPARSK-6	Fold recognition	−4.77	−7.39	2.2	4.2	93.5%
SPARSK-7	Fold recognition	−4.05	−6.53	1.4	3.6	95.0%
SPARSK-8	Fold recognition	0.7	−11.0	6.6	7.4	86.0%
SPARSK-9	Fold recognition	−4.3	−7.51	2.4	3.6	94.0%
SPARSK-10	Fold recognition	−4.71	−6.99	1.8	5.0	93.2%

DOI: 10.7717/peerj.4978/table-5

Table 6:

The validated parameters for refined models of Npas4 Protein.

Model/tool	Method	PROSA	Qmean	Ramachandran plot
				Outlier	Allowed region	Favored region
MUSTER-4	Multi-source threading	−3.19	−3.91	1.8%	1.9%	96.4%
MUSTER-5	Multi-source threading	−3.35	−2.88	1.1%	2.6%	96.2%
LEMOT-1	Local meta-threading	−2.89	−3.34	1.6%	2.2%	96.1%
LEMOT-3	Local meta-threading	−3.66	−3.08	0.8%	2.4%	96.9%
LEMOT-4	Local meta-threading	−3.72	−3.66	1.6%	2.4%	96.0%
LEMOT-9	Local meta-threading	−3.63	−2.61	0.6%	3.2%	96.1%

DOI: 10.7717/peerj.4978/table-6

Protein-protein interaction analysis of Npas4

To study interaction of Npas4 with other proteins, STRING database v10 (Szklarczyk et al., 2015) was used (Fig. 11). The results suggested Npas4 interaction with ARNT (Aryl hydrocarbon receptor nuclear translocator); NXF2B (nuclear RNA export factor 2B); NXF2 (nuclear export factor 2); NXF3 (nuclear export factor 3); NDNL2 (necdin-like 2); MAGEF1 (melanoma antigen family F, 1); C3orf58 (chromosome 3 open reading frame 58); NXF1 (nuclear RNA export factor 1); NUP107 (nucleoporin 107KDa); CSAG1 (Chondrosarcoma associated gene 1); MAGEF1 (Melanoma antigen family F, 1); SLC9A6 (Solute carrier family 9, subfamily A (NHE6, cation proton antiporter 6), member 6); MAGED2 (Melanoma antigen family D, 2); SLC9A9 (Solute carrier family 9, subfamily A (NHE9, cation proton antiporter 9), member 9); MAGEL2 (MAGE-like 2); SSX2B (Synovial sarcoma, X breakpoint 2B) SLC9A1 (Solute carrier family 9, subfamily A (NHE1, cation proton antiporter 1), member 1); CREB1 (cAMP responsive element binding protein 1); MAGEA1 (Melanoma antigen family A, 1) & EPAS1 (Endothelial PAS domain-containing protein 1 (also known as hypoxia-inducible factor-2alpha (HIF-2alpha).

Discussion

The Npas4 gene has been in debate for more than a decade, however its structural information is scarcely available. Npas4 expression is primarily traced to neural cells, where it has been reported to be an important contributor of dendritic growth in phases of neuronal development and for modulating limbic patterning and function (Moser et al., 2004). Recently, it has been reported to be also expressed in pancreatic cells (Speckmann et al., 2016). Functionally, Npas4 is an immediate early gene, which under activation, triggers the activation of battery of genes involved in regulating brain plasticity and cognition, attributed mainly to its interaction with number of transcription factors. Npas4 belongs to the bHLH-PAS family of transcription factors and the other Npas members (1 and 3) are also linked to numerous psychiatric disorders namely autism, bipolar disorders, schizophrenia and depressive disorders (Adachi et al., 2014; Kamnasaran et al., 2003). A simulated dimerised structure of Npas4 with ARNT was reported before to discuss the potential implication of gene variants (Bersten et al., 2014). Current findings address physicochemical properties on human Npas4 protein, with a plausible 3D model, providing useful information about amino acid characteristics and possible identification of interactable proteins.

Human Npas4 is located on human chromosome 11 reference genomic contig NC000011.10, mapping to the chromosomal position 11q13.2. It possesses 11 exons that encode 2406 bp mRNA which translates into 802 amino acid long protein with 87.1 KDa molecular weight (https://www.ncbi.nlm.nih.gov/gene/266743).

In the current study the functional characterization of Npas4 has been done using in silico approaches. The MSA and phylogentic analysis showed that Npas4 is evolutionarily conserved, reflecting the highest homology to primates such as chimpanzees and monkeys.

Npas4 does not have any signal peptide hence it may not be classically secreted extracellularly. The NN score of SecretomeP also verifes Npas4 to be a non-classically secreted protein.

The physicochemical and fuctional properties of proteins are affected by post-translational modifications. Phosphorylation, acetylation and glycosylation are the most common type of post-translational modifications of proteins (Koh et al., 2012).

Acetylation is one of the major post-translational modifications (PTMs) and is important in determining the cellular localization of proteins (Qin, Pang & Zhou, 2011). There is no acetylation site present in Npas4 protein.

Glycosylation, the addition of glycosyl moiety to protein, is the most common post-translational modification in eukaryotes. N-linked and O-linked glycosylations are common while C-linked (mannosylation) is rare (Koh et al., 2012). No C-linked glycosylation sites were present in Npas4. In Npas4, two N-linked glycosylation sites were present but these sites were not available for glycosylation due to the absence of signal peptide.

Phosphorylation is another important feature contributing in PTM. Phosphorylation changes the conformation of protein making it active, inactive or modifying its function (Raghava et al., 2014). In Npas4 there were 90 potential phosphorylation sites which include 34 Threonine, 53 Serine and four Tyrosine phosphorylation sites. But not all the sites were available for phosphorylation. There were 24 Serine, 13 Theronine and one Tyrosine site available for phosphorylation.The important phosphorylation sites present in PAS A and PAS B domain were found at position 130, 136 and 273 respectively. Further experimental studies are needed to explore the role of these sites in Npas4 function.

Prediction analysis in the current study suggested presence of intrinsically disordered protein residues (IDRs) in Npas4 sequence at the C terminal, that can have profound effect on its functional versatility. Conventionally, proteins at physiological temperature exhibit a particular conformational ensemble based on optimal thermal accessibility of the ensemble to various molecular crowders and interacting proteins (Wright & Dyson, 1999). However, intrinsically disordered proteins or intrinsically disordered protein residues (IDRs) in a particular protein enables the protein to interconvert among various topological conformations (Dyson & Wright, 2005). These IDRs therefore, can provide Npas4 ease of flexibility to engage multiple targets. Moreover, IDRs can also aid in interactions by efficient utilization of less residues. This will enhance spontaneous disassociation or displacement in neuronal pathways. Previously it has been reported that structural homogeneity exhibited by IDRs is deficient which results from varied composition of Gly and Pro charged residues (Wright & Dyson, 2015), as is the case of Npas4, which may subsequently result in decreased protein folding. This can further be implicated to enhance conformational plasticity of Npas4 which may help in behaving differently to various transcription factors and enhancing its functional repertoire. The presence of IDRs C-terminal regulatory domain have also been reported in other Bhlh proteins (Fribourgh & Partch, 2017).

Moreover, previous reported behavior of IDRs suggest that the presence of more charged residues enhances electrostatic interactions which augments the propensity of post-translational modifications (Hofmann et al., 2012; Mao et al., 2010). This subsequently resulted in altered binding affinities and increased range of structural heterogeneity derived from disorder to order transitions altering protein compactness. The simultaneous consideration of Npas4 functioning is as an ‘immediate early gene’, along with all the predicted and implied consequences of harboring IDRs in its sequence. The PAS domains carrying multi-ligand binding properties can aid Npas4 to impart its role as a ‘hub protein’ in forming various complexes and key mediator in neuronal signaling.

The presence of IDR within Npas4 sequence can pose difficulty in sculpting protein structure through X-ray crystallography which may be attributable to hindrances caused by the crystal packing forces from promiscuous conformations of disordered regions (Wells et al., 2008). This could be one of the reasons that determination of Npas4 crystallization propensity was reported to be on very low scores from all servers used.

The function of protein can be best predicted if we have insight about the protein tertiary structure. The Npas4 sequence has less than 25% homology with the crystal structure of PAS domains of Npas3 and Npas1. In the absence of any structural template, ab-initio modeling approach for Npas4 structure prediction was opted. The modeled structure was composed of alpha helices and B sheets in the bHLH domain and PAS A and B domains, while the transactivation domain does not passess any tertiary structure due to the presence of IDRs. Due to the presence of proper tertiary structure of PAS domain it was involved in heterodimarization with its partner (ARNT). Current study of amino acid properties suggested preponderance of hydrophobic amino acids in both PAS A and B domains. These findings are in accordance with other bHLH proteins like Npas1, Npas3, BMAL1 and CLOCK where PAS domains are harboring internal hydrophobic cavities (Wu et al., 2016).

The Npas4 was also subjected to prediction of interacting proteins and it reflected its interaction with ARNT, which is its main partner for dimerization. The STRING analysis also showed some important interactions. The analysis showed that Npas4 functional interaction can be divided into four clusters or groups. Cluster 1 involves proteins which are responsible for forming a large protein complex (the SMC5-SMC6 complex). This protein complex is responsible for inducing double stranded DNA breaks (DSB) under neuronal stimulation, which is crucial for the expression downstream promoters such as BDNF (Madabhushi et al., 2015). Through its interaction with CDK5, which has a key role in BDNF induced dendrite development, it implicates the crucial role of Npas4 in memory plasticity through regulating the balance of inhibition in neuronal synapses. This interaction may also be related to Rett Syndrome, a disorder in which activity dependent BDNF transcription is hampered (Liang et al., 2015). Cluster 2 (Fig. 8) suggested Npas4 interact with Nuclear RNA export factor (NXF) proteins, which are RNA binding proteins, with various paralogs reported to be found in dendritic granules (Mamon et al., 2017), suggesting Npas4’s active involvement in the nuclear export of different mRNA and translational control over different proteins. Cluster 3 proteins reported to have altered expression in autism spectrum disorders (ASD), that may consequently result from knockdown of activity regulated gene transcription mediated by Npas4 (Morrow et al., 2008). Cluster 4 represented Npas4’s interaction with cyclic AMP responsive element binding (CREB) proteins. These proteins under phosphorylated state can alter chromatin structure by histone acetylation which speeds up the RNA polymerase II recruitment, triggering the gene transcriptional programs involved in synapse development (Cohen & Greenberg, 2008; Greer & Greenberg, 2008). The CREB along with Npas4 is also involved in regulating inhibitory synapses of excitatory neurons by activating BDNF transcription (Hong, McCord & Greenberg, 2008; Lin et al., 2008). Interstingly, we found three binding sites in the transactivation domain which fall in the region of IDR. This information provides a plausible rationale that CREB may be interacting with any of these residues from the transactvation domain through interaction with transcriptional coactivator, CBP which is known to cause such interaction through IDRs (Dyson & Wright, 2016).

Conclusion

Npas4 is an intrinsically disordered protein with ordered bHLH and PAS domain. The amino acid sequence analysis showed that Npas4 has a high proportion of flexible and hydrophobic amino acids that promote the disorder properties of proteins. The model 9 predicted through LOMET is the best structure of Npas4 and can be used for further analysis. This protein is difficult to crystalize, so in order to determine its tertiary structure we can use NMR and other related techniques. It has strong interactions with NXF 2B and NXF 3 proteins which implicate its potential role in cytoplasmic export of proteins from nucleus, thus influencing protein translation. This Current study also elucidates Npas4 activation with CREB proteins, suggesting Npas4’s reliance on Ca²⁺ dependent kinases (CDKs). The information compiled in this research can serve as useful information for identifying new drug targets, which can modulate synaptic hemostasis in neuropshychiatric and neuro-developmental disorders. Moreover, possible interaction with Nuclear RNA export factor (NXF) protein family role identification needs further elaboration. The results reported by our study are more structural and theoretical in nature, however, they may help in biophysical studies, NMR and crystallographic studies directed towards Npas4. Moreover, the considerations of results may help in future studies designed to understand binding interactions of Npas4.

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