Lysine 5 and phenylalanine 9 of the factor IX omega-loop interact with phosphatidylserine in a membrane-mimetic environment.
The binding of factor IX to cell membranes requires a structured N-terminal omega-loop conformation that exposes hydrophobic residues for a highly regulated interaction with a phospholipid. We hypothesized that a peptide comprised of amino acids Gly4-Gln11 of factor IX (fIX(G4)(-)(Q11)) and constrained by an engineered disulfide bond would assume the native factor IX omega-loop conformation in the absence of Ca(2+). The small size and freedom from aggregation-inducing calcium interactions would make fIX(G4)(-)(Q11) suitable for structural studies for eliciting details about phospholipid interactions. fIX(G4)(-)(Q11) competes with factor IXa for binding sites on phosphatidylserine-containing membranes with a K(i) of 11 microM and inhibits the activation of factor X by the factor VIIIa-IXa complex with a K(i) of 285 microM. The NMR structure of fIX(G4)(-)(Q11) reveals an omega-loop backbone fold and side chain orientation similar to those found in the calcium-bound factor IX Gla domain, FIX(1-47)-Ca(2+). Dicaproylphosphatidylserine (C(6)PS) induces HN, Halpha backbone, and Hbeta chemical shift perturbations at residues Lys5, Leu6, Phe9, and Val10 of fIX(G4)(-)(Q11), while selectively protecting the NHzeta side chain resonance of Lys5 from solvent exchange. NOEs between the aromatic ring protons of Phe9 and specific acyl chain protons of C(6)PS indicate that these phosphatidylserine protons reside 3-6 A from Phe9. Stabilization of the phosphoserine headgroup and glycerol backbone of C(6)PS identifies that phosphatidylserine is in a protected environment that is spatially juxtaposed with fIX(G4)(-)(Q11). Together, these data demonstrate that Lys5, Leu6, Phe9, and Val10 preferentially interact with C(6)PS and allow us to correlate known hemophilia B mutations of factor IX at Lys5 or Phe9 with impaired phosphatidylserine interaction.
Grant
Marianne A
MA
Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical Center, Department of Medicine, Harvard Medical School, 330 Brookline Avenue, Boston, Massachusetts 02215, USA.
Baikeev
Roustem F
RF
Gilbert
Gary E
GE
Rigby
Alan C
AC
D002118:Calcium / Q000737:chemistry; D002413:Cations, Divalent / Q000737:chemistry; D002942:Circular Dichroism; D005164:Factor IX / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002836:Hemophilia B / Q000235:genetics; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism*; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D018716:Molecular Mimicry; D019906:Nuclear Magnetic Resonance, Biomolecular; D010456:Peptides, Cyclic / Q000138:chemical synthesis / Q000378:metabolism; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism*; D010718:Phosphatidylserines / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D002413:Cations, Divalent; D008051:Lipid Bilayers; D010456:Peptides, Cyclic; D010718:Phosphatidylserines; D010649:Phenylalanine; D005164:Factor IX; D008239:Lysine; D002118:Calcium