title: Novel pentameric structure of the diarrhea-inducing region of the rotavirus enterotoxigenic protein NSP4.
abstract: A novel pentameric structure which differs from the previously reported tetrameric form of the diarrhea-inducing region of the rotavirus enterotoxin NSP4 is reported here. A significant feature of this pentameric form is the absence of the calcium ion located in the core region of the tetrameric structures. The lysis of cells, the crystallization of the region spanning residues 95 to 146 of NSP4 (NSP4(95-146)) of strain ST3 (ST3:NSP4(95-146)) at acidic pH, and comparative studies of the recombinant purified peptide under different conditions by size-exclusion chromatography (SEC) and of the crystal structures suggested pH-, Ca(2+)-, and protein concentration-dependent oligomeric transitions in the peptide. Since the NSP4(95-146) mutant lacks the N-terminal amphipathic domain (AD) and most of the C-terminal flexible region (FR), to demonstrate that the pentameric transition is not a consequence of the lack of the N- and C-terminal regions, glutaraldehyde cross-linking of the ΔN72 and ΔN94 mutant proteins, which contain or lack the AD, respectively, but possess the complete C-terminal FR, was carried out. The results indicate the presence of pentamers in preparations of these longer mutants. Detailed SEC analyses of ΔN94 prepared under different conditions, however, revealed protein concentration-dependent but metal ion- and pH-independent pentamer accumulation at high concentrations which dissociated into tetramers and lower oligomers at low protein concentrations. While calcium appeared to stabilize the tetramer, magnesium in particular stabilized the dimer. ΔN72 existed primarily in the multimeric form under all conditions. These findings of a calcium-free NSP4 pentamer and its concentration-dependent and largely calcium-independent oligomeric transitions open up a new dimension in an understanding of the structural basis of its multitude of functions.
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India.
authors.forename: Anita R
authors.initials: AR
authors.lastname: Chacko
authors.forename: Mohammed
authors.initials: M
authors.lastname: Arifullah
authors.forename: Narayan P
authors.initials: NP
authors.lastname: Sastri
authors.forename: Jeyaraman
authors.initials: J
authors.lastname: Jeyakanthan
authors.forename: Go
authors.initials: G
authors.lastname: Ueno
authors.forename: Kanagaraj
authors.initials: K
authors.lastname: Sekar
authors.forename: Randy J
authors.initials: RJ
authors.lastname: Read
authors.forename: Eleanor J
authors.initials: EJ
authors.lastname: Dodson
authors.forename: Durga C
authors.initials: DC
authors.lastname: Rao
authors.forename: Kaza
authors.initials: K
authors.lastname: Suguna
mesh_terms: D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D003460:Crystallization; D018360:Crystallography, X-Ray; D003967:Diarrhea / Q000821:virology*; D004768:Enterotoxins; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000378:metabolism; D055503:Protein Multimerization; D012401:Rotavirus / Q000472:pathogenicity*; D012400:Rotavirus Infections / Q000235:genetics / Q000821:virology*; 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; D010446:Peptide Fragments; D014118:Toxins, Biological; D017361:Viral Nonstructural Proteins; D008274:Magnesium; D002118:Calcium