HEPARIN-BINDING TO PLATELET FACTOR-IV - AN NMR AND SITE-DIRECTED MUTAGENESIS STUDY - ARGININE RESIDUES ARE CRUCIAL FOR BINDING

Native platelet factor-4 (PF4) is an asymmetrically associated, homo-t etrameric protein (70 residues/subunit) known for binding polysulphate d glycosaminoglycans like heparin, PF4 N-terminal chimeric mutant M2 ( PF4-M2), on the other hand, forms symmetric tetramers [Mayo, Roongta, Ilyina, Milius, Barker, Quinlan, La Rosa and Daly (1995) Biochemistry 34, 11399-11409] making NMR studies with this 32 kDa protein tractable . PF4-M2, moreover, binds heparin with a similar affinity to that of n ative PF4. NMR data presented here indicate that heparin (9000 Da cut- off) binding to PF4-M2, while not perturbing the overall structure of the protein, does perturb specific side-chain proton resonances which map to spatially related residues within a ring of positively charged side chains on the surface of tetrameric PF4-M2. Contrary to PF4-hepar in binding models which centre around C-terminal a-helix lysines, this study indicates that a loop containing Arg-20, Arg-22, His-23 and Thr -25, as well as Lys-46 and Arg-49, are even more affected by heparin b inding. Site-directed mutagenesis and heparin binding data support the se NMR findings by indicating that arginines more than C-terminal lysi nes, are crucial to the heparin binding process.