NMR Solution Structure of the 32-kDa Platelet Factor 4 ELR-Motif N-Terminal Chimera: A Symmetric Tetramer

Native human platelet factor 4 (PF4) is a homotetrameric protein (70 residues/subunit) known for its anticoagulant heparin binding activity. 2D ¹⁵N-¹H HSQC NMR experiments of native PF4 in solution show the presence of conformational heterogeneity consistent with the formation of asymmetric homotetramers as observed in the X-ray crystal structure of both human and bovine PF4. A chimeric mutant of PF4 (called PF4-M2) which substitutes the first 11 N-terminal residues for the first eight residues from homologous interleukin-8 forms symmetric homotetramers with essentially the same heparin binding activity as native PF4. The solution structure of PF4-M2 has been investigated by using two- and threedimensional ¹H- and ¹⁵N-NMR spectroscopy and NOE-restrained simulated annealing molecular dynamics. As with other members of the CXC chemokine family whose structures are known, the PF4-M2 subunit monomer consists of a mostly hydrophobic, triple-stranded antiparallel β-sheet onto which is folded an amphipathic C-terminal helix and a less periodic N-terminal domain. Although N-terminal substitution with the less acidic interleukin-8 sequence most affects the quartemary structure relative to native PF4 at the AC and AD dimer interfaces, AB dimer stability is weakened as reflected in reduced equilibrium association binding constants.