A conserved aromatic moiety in the ectodomain is a key determinant for structural integrity and protein trafficking of TNFR superfamily.

Extracellular trafficking of tumor necrosis factor receptor superfamily (TNFRSF) is tightly regulated, disruption of which triggers various autoinflammatory disorders, including TNF receptor-associated periodic syndrome (TRAPS). Here, we provide thus far unraveled molecular basis of noncysteine mutations in TNFR1 ectodomain where loss of an aromatic moiety in cysteine-rich domain (CRD) 2 results in TRAPS disease-associated phenotype. Our study characterized that a missense mutation on phenylalanine residue located in CRD2 (TNFR1F60V ) causes a delay in TNFR1 transport to cell membrane, leading to sustained receptor responsiveness and downstream NF-κB activation, characteristic of clinical manifestation of a prolonged fever. By creating and characterizing identical mutations on structurally conserved ectodomains of osteoprotegerin (OPG) and decoy receptor 3, other two secreted forms of TNFRSF, we further identified that a conserved aromatic residue at the A1 submodule of CRD2 (A1CRD2) confers structural integrity of ectodomain where aromatic sidechain deletion increases thermal instability, interfering with efficient posttranslational modification and subsequent receptor secretion. Interestingly, our functional analyses indicated that this particular noncysteine mutation is not associated with either protein misfolding or loss of function. Finally, by using a synthetic agonist, we demonstrated gain-of-function of the trafficking defect, suggesting the possibility of rescuing affected pathology in related disorders. Given the structural and topological similarities present in the ectodomains of TNFRSF members, our findings provide mechanistic insights of defects in subcellular trafficking of TNF receptors, reported in various TNFRSF-associated diseases.