NMR SOLUTION STRUCTURE OF THE 32-KDA PLATELET FACTOR-4 ELR-MOTIF N-TERMINAL CHIMERA - A SYMMETRICAL TETRAMER

Native human platelet factor 4 (PF4) is a homotetrameric protein (70 r esidues/subunit) known for its anticoagulant heparin binding activity. 2D N-15-H-1 HSQC NMR experiments of native PF4 in solution show the p resence of conformational heterogeneity consistent with the formation of asymmetric homo-tetramers as observed in the X-ray crystal structur e of both human and bovine PF4. A chimeric mutant of PF4 (called PF4-M 2) which substitutes the first 11 N-terminal residues for the first ei ght residues from homologous interleukin-8 forms symmetric homotetrame rs with essentially the same heparin binding activity as native PF4. T he solution structure of PF4-M2 has been investigated by using two- an d three-dimensional H-1- and N-15-NMR spectroscopy and NOE-restrained simulated annealing molecular dynamics. As with other members of the C XC chemokine family whose structures are known, the PF4-M2 subunit mon omer consists of a mostly hydrophobic, triple-stranded antiparallel be ta-sheet onto which is folded an amphipathic C-terminal helix and a le ss periodic N-terminal domain. Although N-terminal substitution with t he less acidic interleukin-8 sequence most affects the quarternary str ucture relative to native PF4 at the AC and AD dimer interfaces, AB di mer stability is weakened as reflected in reduced equilibrium associat ion binding constants.