Background Retinoic acid inducible gene 1 (RIG-1), multi-domain protein has a role-play in detecting viral nucleic acids and stimulates the antiviral response. Dysfunction of this protein due to mutations makes the route vulnerable to viral diseases. Aim Identification of functional hotspots that maintains conformational stability in RIG-1 domains. Methods In this study, we employed a systematic in silico strategy on RIG-1 protein to understand the mechanism of structural changes upon mutation. We computationally investigated the protein sequence signature for all the three domains of RIG-1 protein that encloses the mutation within the motif. Further, we carried out a structural comparison between RIG-1 domains with their respective distant orthologs which revealed the minimal number of interactions required to maintain its structural fold. This intra-protein network paved the way to infer hotspot residues crucial for the maintenance of the structural architecture and folding pattern. Key findings Our analysis revealed about 40 hotspot residues that determine the folding pattern of the RIG-1 domains. Also, conventional molecular dynamic simulation coupled with essential dynamics provides conformational transitions of hot spot residues among native and mutant structures. Structural variations owing to hotspot residues in mutants again confirm the significance of these residues in structural characterization of RIG-1 domains. We believe our results will help the researchers to better comprehend towards regulatory regions and target-binding sites for therapeutic design within the pattern recognition receptor proteins. Significance Our protocol employed in this work describes a novel approach in identifying signature residues that would provide structural insights in protein folding. © 2017 Elsevier Inc.