title: 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.
abstract: 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.
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
authors.forename: M R
authors.initials: MR
authors.lastname: Jagannath
authors.forename: M M
authors.initials: MM
authors.lastname: Kesavulu
authors.forename: R
authors.initials: R
authors.lastname: Deepa
authors.forename: P Narayan
authors.initials: PN
authors.lastname: Sastri
authors.forename: S Senthil
authors.initials: SS
authors.lastname: Kumar
authors.forename: K
authors.initials: K
authors.lastname: Suguna
authors.forename: C Durga
authors.initials: CD
authors.lastname: Rao
mesh_terms: D002460:Cell Line; D004768:Enterotoxins / Q000737:chemistry* / Q000502:physiology; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012401:Rotavirus / Q000737:chemistry*; D014118:Toxins, Biological / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D004768:Enterotoxins; D006023:Glycoproteins; C081369:NS28 protein, rotavirus; D014118:Toxins, Biological; D017361:Viral Nonstructural Proteins