CRY2 is one of the four central proteins of the cell-autonomous molecular clock in mammals. Numerous missense SNPs have been reported in the cry2 gene which results in missense variants of CRY2. These were correlated with diverse metabolic diseases as well as autism spectrum disorders. Thus, we performed in silico analysis of the human CRY2 (hCRY2) protein, assessing the structural stability and interaction of the protein with the FBXL3 and PER2. Multiple computational tools were used in each phase of the analysis assuring the reliability of the data. The computational study suggests that among 436 missense variants of hCRY2, variants: L74P, L274P, L309P, F315V and Y485H were the most destabilizing missense mutants. These variants were found to alter hCRY2 structure and FAD binding pocket that likely affects the binding of its cofactor FAD and interfering the binding mode of other CRY activating compounds. Structural alterations also reduce the binding affinity to regulatory proteins FBXL3 and PER2, which may cause imbalance in the circadian period length in cells as well as develop multiple abnormalities. These five missense variants warrant detailed in vitro and in vivo investigations to solidify their roles in damaging the protein structure, stability, interaction with protein partners.

Background Pirin, a member of the cupin superfamily, is an iron-binding non-heme protein. It acts as a coregulator of several transcription factors, especially the members of NFκB transcription factor family. Based on the redox state of its iron cofactor, it can assume two different conformations and thereby act as a redox sensor inside the nucleus. Previous studies suggested that pirin may be associated with cancer, inflammatory diseases as well as COVID-19 severities. Hence, it is important to explore the pathogenicity of its missense variants. In this study, we used a number of in silico tools to investigate the effects of missense variants of pirin on its structure, stability, metal cofactor binding affinity and interactions with partner proteins. In addition, we used protein dynamics simulation to elucidate the effects of selected variants on its dynamics. Furthermore, we calculated the frequencies of haplotypes containing pirin missense variants across five major super-populations (African, Admixed American, East Asian, European and South Asian). Results Among a total of 153 missense variants of pirin, 45 were uniformly predicted to be pathogenic. Of these, seven variants can be considered for further experimental studies. Variants R59P and L116P were predicted to significantly destabilize and damage pirin structure, substantially reduce its affinity to its binding partners and alter pirin residue fluctuation profile via changing the flexibility of several key residues. Additionally, variants R59Q, F78V, G98D, V151D and L220P were found to impact pirin structure and function in multiple ways. As no haplotype was identified to be harboring more than one missense variant, further interrogation of the individual effects of these seven missense variants is highly recommended. Conclusions Pirin is involved in the transcriptional regulation of several genes and can play an important role in inflammatory responses. The variants predicted to be pathogenic in this study may thus contribute to a better understanding of the underlying molecular mechanisms of various inflammatory diseases. Future studies should be focused on clarifying if any of these variants can be used as disease biomarkers.

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