Singleton-Merten syndrome, a critical and rare multifactorial disorder that is closely linked to R516Q mutation in MDA5 protein associated with an enhanced interferon response in the affected individual. In the present study, we provide conclusive key evidence on R516Q mutation and their connectivity towards sequence-structural basis dysfunction of MDA5 protein. Among the various mutations, we found R516Q is the most pathogenic mutation based on mutational signature Q-A-[RE]-G-R-[GA]-R-A-[ED]-[DE]-S-[ST]-Y-[TSAV]-L-V designed from our work. Further, we derived a distant ortholog for this mutational signature from which we identified 343 intra-residue interactions that fall communally in the position required to maintain the structural and functional integration of protein architecture. This identification served us to understand the critical role of hot spots in residual aggregation that holds a native form of folding conformation in the functional region. In addition, the long-range molecular dynamics simulation demarcated the residual dependencies of conformational transition in distinct regions ( L29 360–370 α18 , α19 380–410 L31 , α21 430–480 L33-α22-L35 and α24 510–520 L38 ) occurring upon R516Q mutation. Together, our results emphasise that the dislocation of functional hot spots Pro229, Arg414, Val498, Met510, Ala513, Gly515 and Arg516 in MDA5 protein which is important for interior structural packing and fold arrangements. In a nutshell, our findings are perfectly conceded with other experimental reports and will have potential implications in immune therapeutical advancement for rare singleton-merten syndrome. © 2018, © 2018 Informa UK Limited, trading as Taylor & Francis Group.