Lysine 5 and Phenylalanine 9 of the Factor IX -Loop Interact with Phosphatidylserine in a Membrane-Mimetic Environment
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- 作者：Marianne A. Grant ; Roustem F. Baikeev ; Gary E. Gilbert ; and Alan C. Rigby
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：December 14, 2004
- 年：2004
- 卷：43
- 期：49
- 页码：15367 - 15378
- 全文大小：374K
- 年卷期：v.43,no.49(December 14, 2004)
- ISSN：1520-4995
文摘
The binding of factor IX to cell membranes requires a structured N-terminal 
-loopconformation 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 (fIXG4-Q11) andconstrained by an engineered disulfide bond would assume the native factor IX -loop conformation inthe absence of Ca2+. The small size and freedom from aggregation-inducing calcium interactions wouldmake fIXG4-Q11 suitable for structural studies for eliciting details about phospholipid interactions. fIXG4-Q11competes with factor IXa for binding sites on phosphatidylserine-containing membranes with a Ki of 11
M and inhibits the activation of factor X by the factor VIIIa-IXa complex with a Ki of 285 M. TheNMR structure of fIXG4-Q11 reveals an -loop backbone fold and side chain orientation similar to thosefound in the calcium-bound factor IX Gla domain, FIX(1-47)-Ca2+. Dicaproylphosphatidylserine (C6PS) induces HN, H
backbone, and H
chemical shift perturbations at residues Lys5, Leu6, Phe9, andVal10 of fIXG4-Q11, while selectively protecting the NH
side chain resonance of Lys5 from solventexchange. NOEs between the aromatic ring protons of Phe9 and specific acyl chain protons of C6PSindicate that these phosphatidylserine protons reside 3-6 Å from Phe9. Stabilization of the phosphoserineheadgroup and glycerol backbone of C6PS identifies that phosphatidylserine is in a protected environmentthat is spatially juxtaposed with fIXG4-Q11. Together, these data demonstrate that Lys5, Leu6, Phe9, andVal10 preferentially interact with C6PS and allow us to correlate known hemophilia B mutations of factorIX at Lys5 or Phe9 with impaired phosphatidylserine interaction.
-loopconformation 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 (fIXG4-Q11) andconstrained by an engineered disulfide bond would assume the native factor IX -loop conformation inthe absence of Ca2+. The small size and freedom from aggregation-inducing calcium interactions wouldmake fIXG4-Q11 suitable for structural studies for eliciting details about phospholipid interactions. fIXG4-Q11competes with factor IXa for binding sites on phosphatidylserine-containing membranes with a Ki of 11M and inhibits the activation of factor X by the factor VIIIa-IXa complex with a Ki of 285 M. TheNMR structure of fIXG4-Q11 reveals an -loop backbone fold and side chain orientation similar to thosefound in the calcium-bound factor IX Gla domain, FIX(1-47)-Ca2+. Dicaproylphosphatidylserine (C6PS) induces HN, H backbone, and H chemical shift perturbations at residues Lys5, Leu6, Phe9, andVal10 of fIXG4-Q11, while selectively protecting the NH side chain resonance of Lys5 from solventexchange. NOEs between the aromatic ring protons of Phe9 and specific acyl chain protons of C6PSindicate that these phosphatidylserine protons reside 3-6 Å from Phe9. Stabilization of the phosphoserineheadgroup and glycerol backbone of C6PS identifies that phosphatidylserine is in a protected environmentthat is spatially juxtaposed with fIXG4-Q11. Together, these data demonstrate that Lys5, Leu6, Phe9, andVal10 preferentially interact with C6PS and allow us to correlate known hemophilia B mutations of factorIX at Lys5 or Phe9 with impaired phosphatidylserine interaction.