Structural insights and characterization of human Npas4 protein

Npas4 is an activity dependent transcription factor which is responsible for gearing the expression of target genes involved in neuro-transmission. Despite the importance of Npas4 in many neuronal diseases, the tertiary structure of Npas4 protein along with its physico-chemical properties is limited. In the current study, first we perfomed the phylogenetic analysis of Npas4 and determined the content of hydrophobic, flexible and order-disorder promoting amino acids. The protein binding regions, post-translational modifications and crystallization propensity of Npas4 were predicted through different in-silico methods. The three dimensional model of Npas4 was predicted through LOMET, SPARSKS-X, I-Tasser, RaptorX, MUSTER and Pyhre and the best model was selected on the basis of Ramachandran plot, PROSA, and Qmean scores. The best model was then subjected to further refinement though MODREFINER. Finally the interacting partners of Npas4 were identified through STRING database. The phylogenetic analysis showed the human Npas4 gene to be closely related to other primates such as chimpanzees, monkey, gibbon. The physiochemical properties of Npas4 showed that it is an intrinsically disordered protein with N-terminal ordered region. The post-translational modification analyses indicated absence of acetylation and mannosylation sites. Three potential phosphorylation sites (S108, T130 and T136) were found in PAS A domain whilst a single phosphorylation site (S273) was present in PAS B domain. The predicted tertiary structure of Npas4 showed that bHLH domain and PAS domain possess tertiary structures while the rest of the protein exhibited disorder property. Protein-protein interaction analysis revealed NPas4 interaction with various proteins which are mainly involved in nuclear trafficking of proteins to cytoplasm, activity regulated gene transcription and neurodevelopmental disorders. Moreover the analysis also highlighted the direct relation to proteins involved in promoting neuronal survival, plasticity and cAMP responsive element binding protein proteins. The current study helps in understanding the physicochemical properties and reveals the neuro-modulatory role of Npas4 in crucial pathways involved in neuronal survival and neural signalling hemostasis.