Heparin dodecasaccharide binding to platelet factor-4 and growth-related protein-alpha. Induction of a partially folded state and implications for heparin-induced thrombocytopenia.
alpha-Chemokines are known heparin-binding proteins. Here, a heparin dodecasaccharide (H12) was purified and used in NMR studies to investigate binding to growth-related protein-alpha (Gro-alpha) and to platelet factor-4-M2 (PF4-M2), an N-terminal chimera of PF4. Pulsed field gradient NMR was used to derive diffusion coefficients as the protein (monomer):H12 ratio was varied. In the absence of H12, both PF4-M2 and Gro-alpha give diffusion coefficients consistent with the presence of mostly dimers. As the PF4-M2:H12 ratio is increased from 1:6 to 2:1, the diffusion coefficient increases, indicating dissociation to the monomer state. On addition of H12 to either protein, (15)N/(1)H heteronuclear single quantum coherence NMR data demonstrate loss of (1)H resonance dispersion and intensity, particularly at protein:H12 ratios of 2:1 to 4:1, indicating significant perturbation to native structures. For Gro-alpha in particular, (1)H resonance dispersion appears random coil-like. At these same ratios, circular dichroism (CD) data show general retention of secondary structure elements with a slight shift to additional helix formation. Random coil NMR resonance dispersion suggests a shift to a less compact, partially folded, and/or more flexible state. Further addition of H12 causes resonance intensity and dispersion to return making NMR spectra appear native-like. At low PF4-M2:H12 ratios, loss of resonance intensity for residues proximal to Arg-20 and Arg-22 in three-dimensional NMR HCCH-TOCSY spectra suggests that the Arg-20-Arg-22 loop either interacts most strongly with H12 and/or that binding at this site is heterogeneous. This domain was previously shown to be crucial to heparin binding. Of particular interest to the biology of PF4-heparin complex formation, heparin-induced thrombocytopenia antibody binding occurs at about the same PF4-M2:H12 ratio as does this transition to a partially folded PF4-M2 state, strongly suggesting that heparin-induced thrombocytopenia antibody recognizes a less folded, lower aggregate state of the protein.
Mikhailov
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Department of Biochemistry, Molecular Biology & Biophysics, Biomedical Engineering Center, University of Minnesota Health Science Center, Minneapolis, Minnesota 55455, USA.
Young
H C
HC
Linhardt
R J
RJ
Mayo
K H
KH
D054360:Chemokine CXCL1; D019743:Chemokines, CXC / Q000737:chemistry / Q000378:metabolism; D002630:Chemotactic Factors / Q000737:chemistry / Q000378:metabolism*; D004187:Disaccharides / Q000737:chemistry / Q000378:metabolism; D006133:Growth Substances / Q000737:chemistry / Q000378:metabolism*; D006493:Heparin / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D010978:Platelet Factor 4 / Q000737:chemistry / Q000378:metabolism*; D017510:Protein Folding; D013921:Thrombocytopenia / Q000097:blood*
C475829:CXCL1 protein, human; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors; D004187:Disaccharides; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D010978:Platelet Factor 4; D006493:Heparin