title: Structural plasticity of the coiled-coil domain of rotavirus NSP4.
abstract: Rotavirus (RV) nonstructural protein 4 (NSP4) is a virulence factor that disrupts cellular Ca(2+) homeostasis and plays multiple roles regulating RV replication and the pathophysiology of RV-induced diarrhea. Although its native oligomeric state is unclear, crystallographic studies of the coiled-coil domain (CCD) of NSP4 from two different strains suggest that it functions as a tetramer or a pentamer. While the CCD of simian strain SA11 NSP4 forms a tetramer that binds Ca(2+) at its core, the CCD of human strain ST3 forms a pentamer lacking the bound Ca(2+) despite the residues (E120 and Q123) that coordinate Ca(2+) binding being conserved. In these previous studies, while the tetramer crystallized at neutral pH, the pentamer crystallized at low pH, suggesting that preference for a particular oligomeric state is pH dependent and that pH could influence Ca(2+) binding. Here, we sought to examine if the CCD of NSP4 from a single RV strain can exist in two oligomeric states regulated by Ca(2+) or pH. Biochemical, biophysical, and crystallographic studies show that while the CCD of SA11 NSP4 exhibits high-affinity binding to Ca(2+) at neutral pH and forms a tetramer, it does not bind Ca(2+) at low pH and forms a pentamer, and the transition from tetramer to pentamer is reversible with pH. Mutational analysis shows that Ca(2+) binding is necessary for the tetramer formation, as an E120A mutant forms a pentamer. We propose that the structural plasticity of NSP4 regulated by pH and Ca(2+) may form a basis for its pleiotropic functions during RV replication.
  
  
  OBJECTIVE
  The nonstructural protein NSP4 of rotavirus is a multifunctional protein that plays an important role in virus replication, morphogenesis, and pathogenesis. Previous crystallography studies of the coiled-coil domain (CCD) of NSP4 from two different rotavirus strains showed two distinct oligomeric states, a Ca(2+)-bound tetrameric state and a Ca(2+)-free pentameric state. Whether NSP4 CCD from the same strain can exist in different oligomeric states and what factors might regulate its oligomeric preferences are not known. This study used a combination of biochemical, biophysical, and crystallography techniques and found that the NSP4 CCD can undergo a reversible transition from a Ca(2+)-bound tetramer to a Ca(2+)-free pentamer in response to changes in pH. From these studies, we hypothesize that this remarkable structural adaptability of the CCD forms a basis for the pleiotropic functional properties of NSP4.
authors.affiliation: Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USA.
authors.forename: Narayan P
authors.initials: NP
authors.lastname: Sastri
authors.affiliation: Verna Marrs Mclean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, USA.
authors.forename: Maria
authors.initials: M
authors.lastname: Viskovska
authors.affiliation: Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USA.
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Hyser
authors.affiliation: Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USA.
authors.forename: Mark R
authors.initials: MR
authors.lastname: Tanner
authors.affiliation: Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USA.
authors.forename: Lori B
authors.initials: LB
authors.lastname: Horton
authors.affiliation: Berkeley Center for Structural Biology, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
authors.forename: Banumathi
authors.initials: B
authors.lastname: Sankaran
authors.affiliation: Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USA Verna Marrs Mclean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, USA mestes@bcm.edu vprasad@bcm.edu.
authors.forename: B V Venkataram
authors.initials: BV
authors.lastname: Prasad
authors.affiliation: Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USA mestes@bcm.edu vprasad@bcm.edu.
authors.forename: Mary K
authors.initials: MK
authors.lastname: Estes
mesh_terms: D055592:Biophysical Phenomena; D002118:Calcium / Q000378:metabolism; D004252:DNA Mutational Analysis; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D055503:Protein Multimerization; D012401:Rotavirus / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D014118:Toxins, Biological / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D006023:Glycoproteins; C081369:NS28 protein, rotavirus; D014118:Toxins, Biological; D017361:Viral Nonstructural Proteins; D002118:Calcium