Paramyxovirus fusion protein: Characterization of the core trimer, a rod-shaped complex with helices in anti-parallel orientation

The fusion (F) protein of the paramyxovirus SV5 contains two heptad repeat regions, HRA adjacent to the fusion peptide and HRB proximal to the transmembrane domain. Peptides, N-1 and C-1, respectively, corresponding to these heptad repeat regions form a thermostable, α-helical trimer of heterodimers (S. B. Joshi, R. E. Dutch, and R. A. Lamb (1998), Virology 248, 20-34). Further characterization of the N-1/C-1 complex indicated that the C- 1 peptides, which are predicted to residue on the outside of the complex, are resistant to digestion by several proteases when present in the complex. Only proteinase K digested most of the C-1 peptide, though the small remaining protease protected fragment of C-1 confers extreme thermostability on the proteinase-K-resistant N-1 trimeric coiled-coil. Carboxypeptidase Y digestion of the N-1/C-1 complex indicates that the C-1 peptides associate in an antiparallel orientation relative to the N-1 peptides. Electron microscopy of the N-1/C-1 complex showed a rod-shaped complex with an average length of 9.7 nm, consistent with all of N-1 existing as an α helix. Mutations at heptad repeat a and d residues of N-1, positions that are predicted to point inward to the center of the N-1 trimeric coiled-coil, were found to have varying effects as-analyzed by circular dichroism measurements. The mutation I₁₃₇M did not affect the helical structure of the isolated N-1 peptide but did affect the thermostability of the N-1/C-1 complex. Mutations L₁₄₀M and L₁₆₁M perturbed the helical structure formed by N-1 in isolation but did not affect formation of a thermostable N-1/C-1 complex. Finally, a peptide, SV5 F 255-293, corresponding to a proposed leucine zipper region, was analyzed for effects on N-1, C-1, or the N-1/C-1 complex. Circular dichroism analysis demonstrated that while the presence of peptide 255-293 increased the helical signal from either N-1 or the N-1/C-1 complex, no change in thermostability was observed, indicating that this region is not a component of the final, most stable core of the F protein.