Heparin binding to platelet factor-4. An NMR and site-directed mutagenesis study: arginine residues are crucial for binding.
Native platelet factor-4 (PF4) is an asymmetrically associated, homo-tetrameric protein (70 residues/subunit) known for binding polysulphated 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 native 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-heparin binding models which centre around C-terminal alpha-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 binding. Site-directed mutagenesis and heparin binding data support these NMR findings by indicating that arginines more than C-terminal lysines, are crucial to the heparin binding process.
Department of Biochemistry, University of Minnesota, Minneapolis 55455, USA.
K H
KH
Mayo
E
E
Ilyina
V
V
Roongta
M
M
Dundas
J
J
Joseph
C K
CK
Lai
T
T
Maione
T J
TJ
Daly
D000595:Amino Acid Sequence; D001120:Arginine; D001665:Binding Sites; D005813:Genes, Synthetic; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010978:Platelet Factor 4 / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism
D011994:Recombinant Proteins; D010978:Platelet Factor 4; D006493:Heparin; D001120:Arginine