title: Quantitative investigation of the affinity of human respiratory syncytial virus phosphoprotein C-terminus binding to nucleocapsid protein.
abstract: BACKGROUND
  There are no approved small molecule drug therapies for human respiratory syncytial virus (hRSV), a cause of morbidity and mortality in at-risk newborns, the immunocompromised, and the elderly. We have investigated as a potential novel hRSV drug target the protein-protein interaction between the C-terminus of the viral phosphoprotein (P) and the viral nucleocapsid protein (N), components of the ribonucleoprotein complex that contains, replicates, and transcribes the viral RNA genome. Earlier work by others established that the 9 C-terminal residues of P are necessary and sufficient for binding to N.
  
  
  METHODS
  We used a fluorescence anisotropy assay, surface plasmon resonance and 2-D NMR to quantify the affinities of peptides based on the C terminus of P for RNA-free, monomeric N-terminal-truncated N(13-391). We calculated the contributions to the free energies of binding of P to N(13-391) attributable to the C-terminal 11 residues, phosphorylation of the C-terminal 2 serine residues, the C-terminal Asp-Phe, and the phenyl ring of the C-terminal Phe.
  
  
  RESULTS
  Binding studies confirmed the crucial role of the phosphorylated C-terminal peptide D(pS)DNDL(pS)LEDF for binding of P to RNA-free, monomeric N(13-391), contributing over 90% of the binding free energy at low ionic strength. The phenyl ring of the C-terminal Phe residue contributed an estimated -2.7 kcal/mole of the free energy of binding, the C-terminal Asp-Phe residues contributed -3.8 kcal/mole, the sequence DSDNDLSLE contributed -3.1 kcal/mole, and phosphorylation of the 2 Ser residues contributed -1.8 kcal/mole. Due to the high negative charge of the C-terminal peptide, the affinity of the P C-terminus for N(13-391) decreased as the ionic strength increased.
  
  
  CONCLUSIONS
  The results support the idea that the interaction of the C-terminal residues of P with N constitutes a protein-protein interaction hotspot that may be a suitable target for small-molecule drugs that inhibit viral genome replication and transcription.
authors.affiliation: Biology Department, Infection Innovative Medicines Unit, AstraZeneca R&D Boston, Waltham, MA, USA. adam.shapiro@astrazeneca.com.
authors.forename: Adam B
authors.initials: AB
authors.lastname: Shapiro
authors.forename: Ning
authors.initials: N
authors.lastname: Gao
authors.forename: Nichole
authors.initials: N
authors.lastname: O'Connell
authors.forename: Jun
authors.initials: J
authors.lastname: Hu
authors.forename: Jason
authors.initials: J
authors.lastname: Thresher
authors.forename: Rong-Fang
authors.initials: RF
authors.lastname: Gu
authors.forename: Ross
authors.initials: R
authors.lastname: Overman
authors.forename: Ian M
authors.initials: IM
authors.lastname: Hardern
authors.forename: Graham G
authors.initials: GG
authors.lastname: Sproat
mesh_terms: D020816:Amino Acid Motifs; D001665:Binding Sites; D006801:Humans; D007700:Kinetics; D009698:Nucleoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D018357:Respiratory Syncytial Virus Infections / Q000821:virology*; D018113:Respiratory Syncytial Virus, Human / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D009698:Nucleoproteins; C529603:P protein, human respiratory syncytial virus; D015678:Viral Structural Proteins