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Analysis of the factor XI variant Arg184Gly suggests a structural basis for factor IX binding to factor XIa.
机构信息
Department of Pathology, Microbiology and Immunology, Vanderbilt University, Nashville, TN, USA.
出版信息
J Thromb Haemost. 2013 Jul;11(7):1374-84. doi: 10.1111/jth.12275.
BACKGROUND
A patient with factor XI (FXI) deficiency was reported with an Arg184Gly substitution in the FXI A3 domain. The A3 domain contains an exosite required for binding of FIX to activated FXI (FXIa).
OBJECTIVE
To test the effects of the Arg184Gly substitution on FIX activation, and to characterize the FIX-binding site on FXIa.
METHODS
Recombinant FXIa and FIX variants were used to identify residues involved in FIX activation by FXIa. Analysis of the FXI structure was used to identify potential FIX-binding sites.
RESULTS
The Km for FIX activation by FXIa-Gly184 was approximately three-fold higher than for FXIa, suggesting that Arg184 is part of the exosite. Arg184 and the adjacent residues, Ile183 and Asp185, contribute to charged and hydrophobic areas that are not present in the FXI homolog prekallikrein (PK). Replacing residues 183-185 with alanine abolished exosite activity, similarly to replacement of the entire A3 domain with the A3 domain from PK (FXIa/PKA3). Reintroducing FXI residues 183-185 into FXIa/PKA3 partially restored the exosite, and replacing residues 183-185 and 260-264 completely restored exosite function. FIX in which the Ω-loop (residues 4-11) was replaced with the FVII Ω-loop was activated poorly by FXIa, suggesting that the FIX Ω-loop binds to FXIa.
CONCLUSIONS
The results support a model in which the Ω-loop of FIX binds to an area on FXIa composed of residues from the N-terminus and C-terminus of the A3 domain. These residues are buried in zymogen FXI, and must be exposed upon conversion to FXIa to permit FIX binding.
背景
曾报道过一名因子 XI（FXI）缺乏症患者，其 FXI A3 结构域存在 Arg184Gly 取代。A3 结构域包含一个 FXIa 与激活的 FXI（FXIa）结合所需的外位。
目的
检测 Arg184Gly 取代对外位 FXI 激活的影响，并对 FXIa 上的 FIX 结合位点进行特征分析。
方法
使用重组 FXIa 和 FIX 变体鉴定 FXIa 激活 FIX 涉及的残基。FXI 结构分析用于鉴定潜在的 FIX 结合位点。
结果
FXIa-Gly184 对 FIX 的 Km 约为 FXIa 的三倍，提示 Arg184 是外位的一部分。Arg184 及其相邻残基 Ile183 和 Asp185 有助于形成电荷和疏水性区域，而在 FXI 同源物激肽释放酶（PK）中不存在这些区域。用丙氨酸取代残基 183-185 可消除外位活性，类似地，用 PK 的 A3 结构域取代整个 A3 结构域（FXIa/PKA3）也可消除外位活性。将 FXI 残基 183-185 重新引入 FXIa/PKA3 可部分恢复外位，而取代残基 183-185 和 260-264 可完全恢复外位功能。用 FVII 的 Ω 环（残基 4-11）取代 FIX 的 Ω 环的 FIX 被 FXIa 激活的能力较差，提示 FIX 的 Ω 环与 FXIa 结合。
结论
结果支持这样一种模型，即 FIX 的 Ω 环与 FXIa 上由 A3 结构域的 N 端和 C 端的残基组成的区域结合。这些残基在酶原 FXI 中被掩埋，只有在转化为 FXIa 时才能暴露出来，以允许 FIX 结合。
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