Abstract：
High-molecular weight kininogen (HK) circulates in plasma as a complex with zymogen prekallikrein (PK). HK is both a substrate and a cofactor for activated plasma kallikrein, and the principal exosite interactions occur between PK N-terminal apple domains and the C-terminal D6 domain of HK.
To determine the structure of the complex formed between PK apple domains and an HKD6 fragment and compare this with the coagulation factor XI (FXI)-HK complex.
We produced recombinant FXI and PK heavy chains (HCs) spanning all 4 apple domains. We cocrystallized PKHC (and subsequently FXIHC) with a 31-amino acid synthetic peptide spanning HK residues Ser565-Lys595 and determined the crystal structure. We also analyzed the full-length FXI-HK complex in solution using hydrogen deuterium exchange mass spectrometry.
The 2.3Å PKHC-HK peptide crystal structure revealed that the HKD6 sequence WIPDIQ (Trp569-Gln574) binds to the apple 1 domain and HK FNPISDFPDT (Phe582-Thr591) binds to the apple 2 domain with a flexible intervening sequence resulting in a bent double conformation. A second 3.2Å FXIHC-HK peptide crystal structure revealed a similar interaction with the apple 2 domain but an alternate, straightened conformation of the HK peptide where residues LSFN (Leu579-Asn583) interacts with a unique pocket formed between the apple 2 and 3 domains. HDX-MS of full length FXI-HK complex in solution confirmed interactions with both apple 2 and apple 3.
The alternate conformations and exosite binding of the HKD6 peptide likely reflects the diverging relationship of HK to the functions of PK and FXI.
To determine the structure of the complex formed between PK apple domains and an HKD6 fragment and compare this with the coagulation factor XI (FXI)-HK complex.
We produced recombinant FXI and PK heavy chains (HCs) spanning all 4 apple domains. We cocrystallized PKHC (and subsequently FXIHC) with a 31-amino acid synthetic peptide spanning HK residues Ser565-Lys595 and determined the crystal structure. We also analyzed the full-length FXI-HK complex in solution using hydrogen deuterium exchange mass spectrometry.
The 2.3Å PKHC-HK peptide crystal structure revealed that the HKD6 sequence WIPDIQ (Trp569-Gln574) binds to the apple 1 domain and HK FNPISDFPDT (Phe582-Thr591) binds to the apple 2 domain with a flexible intervening sequence resulting in a bent double conformation. A second 3.2Å FXIHC-HK peptide crystal structure revealed a similar interaction with the apple 2 domain but an alternate, straightened conformation of the HK peptide where residues LSFN (Leu579-Asn583) interacts with a unique pocket formed between the apple 2 and 3 domains. HDX-MS of full length FXI-HK complex in solution confirmed interactions with both apple 2 and apple 3.
The alternate conformations and exosite binding of the HKD6 peptide likely reflects the diverging relationship of HK to the functions of PK and FXI.
摘要：
背景：高分子量激肽原（HK）作为与酶原激肽释放酶（PK）的复合物在血浆中循环。HK既是活化血浆激肽释放酶的底物又是辅因子，主要的外部位相互作用发生在PKN端苹果结构域和HK的C端D6结构域之间。
目的：确定PK苹果结构域与HKD6片段之间形成的复合物的结构，并将其与凝血因子XI（FXI）-HK复合物进行比较。
方法：我们生产了跨越所有4个苹果结构域的重组FXI和PK重链（HC）。我们将PKHC（以及随后的FXIHC）与跨越HK残基Ser565-Lys595的31个氨基酸的合成肽共结晶，并确定了晶体结构。我们还使用氢氘交换质谱法分析了溶液中的全长FXI-HK复合物。
结果：2.3µPKHC-HK肽晶体结构显示，HKD6序列WIPDIQ（Trp569-Gln574）与苹果1结构域结合，HKFNPISDFPDT（Phe582-Thr591）与苹果2结构域结合，具有柔性插入序列，导致弯曲的双构象。第二个3.2µFXIHC-HK肽晶体结构揭示了与苹果2结构域的相似相互作用，HK肽的拉直构象,其中残基LSFN(Leu579-Asn583)与苹果2和3结构域之间形成的独特口袋相互作用。溶液中全长FXI-HK复合物的HDX-MS证实了与苹果2和苹果3的相互作用。
结论：HKD6肽的交替构象和外位点结合可能反映了HK与PK和FXI功能的不同关系。
目的：确定PK苹果结构域与HKD6片段之间形成的复合物的结构，并将其与凝血因子XI（FXI）-HK复合物进行比较。
方法：我们生产了跨越所有4个苹果结构域的重组FXI和PK重链（HC）。我们将PKHC（以及随后的FXIHC）与跨越HK残基Ser565-Lys595的31个氨基酸的合成肽共结晶，并确定了晶体结构。我们还使用氢氘交换质谱法分析了溶液中的全长FXI-HK复合物。
结果：2.3µPKHC-HK肽晶体结构显示，HKD6序列WIPDIQ（Trp569-Gln574）与苹果1结构域结合，HKFNPISDFPDT（Phe582-Thr591）与苹果2结构域结合，具有柔性插入序列，导致弯曲的双构象。第二个3.2µFXIHC-HK肽晶体结构揭示了与苹果2结构域的相似相互作用，HK肽的拉直构象,其中残基LSFN(Leu579-Asn583)与苹果2和3结构域之间形成的独特口袋相互作用。溶液中全长FXI-HK复合物的HDX-MS证实了与苹果2和苹果3的相互作用。
结论：HKD6肽的交替构象和外位点结合可能反映了HK与PK和FXI功能的不同关系。
