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.
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。