Localization of the binding site on plasma kallikrein for high-molecular-weight kininogen to both apple 1 and apple 4 domains of the heavy chain.
Localization of the binding site on plasma kallikrein for high-molecular-weight kininogen to both apple 1 and apple 4 domains of the heavy chain.
复制标题
高分子量激肽原在血浆激肽释放酶上的结合位点定位于重链的苹果 1 和苹果 4 结构域。
DOI：
10.1006/abbi.1994.1424
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发表时间：
1994
影响因子：
3.9
通讯作者：
Colman,RW
中科院分区：
文献类型：
--
作者：
Page,JD;You,JL;Harris,RB;Colman,RW
人血浆前激肽释放酶或激肽释放酶重链的C末端含有高分子量激肽原的结合位点，激肽原是接触活化的非酶促原辅因子。为了进一步定位该结合位点，合成了一系列重叠肽。通过激肽释放酶的酰胺分解活性测定在增加浓度的每种肽存在下结合到激肽原包被的微量滴定板威尔斯孔的激肽释放酶的量。一个包含激肽释放酶的Lys 266-Gly 295的肽段，在构象上受到二硫键的限制，显示最低的Kd值（~ 67 μM）。线性肽Leu 262-Gly 295显示出较低的亲和力（129 μM）。该序列的N-末端或C-末端截短/延伸肽降低了结合活性。由于密切相关的蛋白质因子XI已被证明可结合激肽原，因此检查了与FXI结合结构域同源的基于激肽释放酶的肽（Phe 56-Gly 86），发现其对激肽原具有较低但显著的结合亲和力（Kd 530 μM）。使用等温滴定量热法来评估基于激肽释放酶的肽与包含激肽原中的激肽释放酶结合结构域的肽（Ser 565-Lys 595）之间的结合。Leu 262-Gly 295具有强结合活性（Kd 52 μM），而Phe 56-Gly 86显示较差的结合活性（Kd 400 μM）。这些相互作用是吸热的且具有熵优势，表明结合后发生了构象重排。我们得出结论，激肽原内的结合位点是由不连续的线性片段，形成一个连续的表面折叠的蛋白质。
The C-terminal end of the heavy chain of human plasma prekallikrein or kallikrein contains a binding site for high-molecular-weight kininogen, the nonenzymatic procofactor of contact activation. To further map this binding site, a series of overlapping peptides were synthesized. The amount of kallikrein that bound to kininogen-coated microtiter plate wells in the presence of increasing concentrations of each peptide was determined by kallikrein amidolytic activity. A peptide encompassing Lys266-Gly295of kallikrein, conformationally constrained by a disulfide bond, displayed the lowestKdvalue (∼67 μM). The linear peptide, Leu262-Gly295, displayed lower affinity (129 μM). N-terminal or C-terminal truncation/extension peptides of this sequence diminished binding activity. Since the closely related protein, factor XI, has been shown to bind kininogen, a kallikrein-based peptide (Phe56-Gly86) homologous to the binding domain of FXI, was examined and found to possess less, but significant, binding affinity for kininogen (Kd530 μM). Isothermal titration calorimetry was used to assess binding between the kallikrein-based peptides and a peptide encompassing the kallikrein binding domain in kininogen (Ser565- Lys595). Leu262-Gly295possesses potent binding activity (Kd52 μM), while Phe56-Gly86displays poorer binding activity (Kd400 μM). These interactions are endothermic and entropically favored, suggesting that a conformational rearrangement takes place upon binding. We conclude that the binding site for kininogen within prekallikrein is composed of discontinuous linear segments that form a contiguous surface in the folded protein.