N- and C-terminal cooperation in rotavirus enterotoxin: novel mechanism of modulation of the properties of a multifunctional protein by a structurally and functionally overlapping conformational domain.

Rotavirus NSP4 is a multifunctional endoplasmic reticulum (ER)-resident nonstructural protein with the N terminus anchored in the ER and about 131 amino acids (aa) of the C-terminal tail (CT) oriented in the cytoplasm. Previous studies showed a peptide spanning aa 114 to 135 to induce diarrhea in newborn mouse pups with the 50% diarrheal dose approximately 100-fold higher than that for the full-length protein, suggesting a role for other regions in the protein in potentiating its diarrhea-inducing ability. In this report, employing a large number of methods and deletion and amino acid substitution mutants, we provide evidence for the cooperation between the extreme C terminus and a putative amphipathic alpha-helix located between aa 73 and 85 (AAH73-85) at the N terminus of DeltaN72, a mutant that lacked the N-terminal 72 aa of nonstructural protein 4 (NSP4) from Hg18 and SA11. Cooperation between the two termini appears to generate a unique conformational state, specifically recognized by thioflavin T, that promoted efficient multimerization of the oligomer into high-molecular-mass soluble complexes and dramatically enhanced resistance against trypsin digestion, enterotoxin activity of the diarrhea-inducing region (DIR), and double-layered particle-binding activity of the protein. Mutations in either the C terminus, AAH73-85, or the DIR resulted in severely compromised biological functions, suggesting that the properties of NSP4 are subject to modulation by a single and/or overlapping highly sensitive conformational domain that appears to encompass the entire CT. Our results provide for the first time, in the absence of a three-dimensional structure, a unique conformation-dependent mechanism for understanding the NSP4-mediated pleiotropic properties including virus virulence and morphogenesis.