Structural Features of a Tiny Viral Protein, ORF7b of SARS-CoV-2.

Accessory proteins of SARS-CoV-2 play crucial roles in viral pathogenesis, yet their structural properties remain elusive. ORF7b, a small accessory protein comprising only 43 amino acids, is widely assumed to parallel the structure-function relationships of its SARS-CoV ortholog based solely on sequence homology. In this study, we challenge this paradigm through direct physicochemical and structural characterization. Sequence analysis and electrostatic profiling reveal that the SARS-CoV-2 protein is a macromolecular polyanion with a net charge of -4 at neutral pH, featuring a diffuse negative surface that is highly responsive to pH changes. Complete 3D structures generated via ab initio modeling display a helical core flanked by two highly fluctuating, disordered termini. Residue Interaction Network (RIN) topology and Normal Mode Analysis (NMA) identified specific hinges governing these flexible extremities. Furthermore, the calculated dipole moment vector is tilted outward by 24°, misaligning with the central axis. Molecular dynamics simulations suggest that while the soluble structure is highly stable in water, it undergoes severe distortions and insufficient solvation within a membrane-mimetic environment. Thermodynamic association profiles and verified interactomic data from BioGRID reveal a strong propensity for ORF7b to participate in liquid-liquid phase transitions alongside human and viral partners. Taken together, these unique properties suggest that ORF7b operates as a dynamic peripheral membrane protein rather than a sedentary transmembrane component, providing a fresh framework for future therapeutic targeting. Overall, these in silico findings shift the current paradigm on ORF7b2 topology and provide a robust, physically grounded framework that identifies specific molecular priorities for future in vitro and in vivo validation.