A reverse binding motif that contributes to specific protease inhibition by antibodies.
A reverse binding motif that contributes to specific protease inhibition by antibodies.
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DOI：
10.1016/j.jmb.2011.11.036
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发表时间：
2012-01-27
影响因子：
5.6
通讯作者：
Craik, Charles S.
中科院分区：
文献类型：
--
作者：
Schneider, Eric L.;Lee, Melody S.;Baharuddin, Aida;Goetz, David H.;Farady, Christopher J.;Ward, Mick;Wang, Cheng-I;Craik, Charles S.
II型跨膜丝氨酸蛋白酶(TTSP)家族由18种密切相关的丝氨酸蛋白酶组成，参与多种功能。为了鉴定针对一种特定的TTSP的选择性、抑制性抗体，我们创建了一个带有来自人类幼稚B细胞的天然FEB的噬菌体展示文库。Fab A11的抑制常数为720皮摩尔，与其他胰酶折叠丝氨酸蛋白酶相比，该酶具有较高的特异性。里氏木霉表达A11的产量约为200 mg/L。测定了A11与麦曲霉酶复合体的晶体结构，并与另一抗体抑制剂(S4)的晶体结构进行了比较。S4以前是从合成的单链抗体文库中发现的，它也是一种高度选择性和有效的Mattritase抑制剂。A11/麦曲霉酶和S4/麦曲霉酶复合体的晶体结构分别为2.1？和1.5？尽管这些从不同文库中发现的抗体因其特异性而与蛋白酶表面环相互作用不同，但这些结构揭示了类似的蛋白酶抑制新机制。通过在底物结合口袋以相反方向插入H3可变环，这些抗体掩埋了大面积的有效抑制并避免了蛋白水解性失活。这一发现突显了抗体支架在定位环中发挥的关键作用，以高度选择性地结合和抑制蛋白酶功能。此外，Fab A11是一种完全的人类抗体，它比其他密切相关的蛋白水解酶更能特异性地抑制Mattritase，这表明这种方法可能有助于临床应用。
The type II transmembrane serine protease (TTSP) family consists of eighteen closely related serine proteases that are implicated in multiple functions. To identify selective, inhibitory antibodies against one particular TTSP, matriptase (MT-SP1), a phage display library with a natural repertoire of Fabs from human naïve B cells was created. Fab A11 was identified with a 720 picomolar inhibition constant and high specificity for matriptase over other trypsin-fold serine proteases. A Trichoderma reesei system expressed A11 with ~200 mg/L yield. The crystal structure of A11 in complex with matriptase has been determined and compared to the crystal structure of another antibody inhibitor (S4) in complex with matriptase. Previously discovered from a synthetic scFv library, S4 is also a highly selective and potent matriptase inhibitor. The crystal structures of the A11/matriptase and S4/matriptase complexes were solved to 2.1 Å and 1.5 Å respectively. Although these antibodies, discovered from separate libraries, interact differently with the protease surface loops for their specificity, the structures reveal a similar novel mechanism of protease inhibition. Through the insertion of the H3 variable loop in a reverse orientation at the substrate-binding pocket, these antibodies bury a large surface area for potent inhibition and avoid proteolytic inactivation. This discovery highlights the critical role the antibody scaffold plays in positioning loops to bind and inhibit protease function in a highly selective manner. Additionally, Fab A11 is a fully human antibody that specifically inhibits matriptase over other closely related proteases, suggesting this approach could be useful for clinical applications.
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影响因子：
11.2
作者：
Darragh MR;Schneider EL;Lou J;Phojanakong PJ;Farady CJ;Marks JD;Hann BC;Craik CS
通讯作者：
Craik CS
影响因子：
4.8
作者：
de Haard, HJ;van Neer, N;Hoogenboom, HR
通讯作者：
Hoogenboom, HR
影响因子：
5.6
作者：
Farady, Christopher J.;Sun, Jeonghoon;Craik, Charles S.
通讯作者：
Craik, Charles S.
影响因子：
5.6
作者：
Dickinson, CD;Shobe, J;Ruf, W
通讯作者：
Ruf, W
影响因子：
20.3
作者：
Kilpatrick, Lynette M.;Harris, Roger L.;Ellis, Vincent
通讯作者：
Ellis, Vincent