Structure of an affinity-matured inhibitory recombinant fab against urokinase plasminogen activator reveals basis of potency and specificity.
Structure of an affinity-matured inhibitory recombinant fab against urokinase plasminogen activator reveals basis of potency and specificity.
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尿激酶型纤溶酶原激活剂亲和力成熟的抑制性重组fab的结构揭示了其效力和特异性的基础。
DOI：
10.1016/j.bbapap.2020.140562
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发表时间：
2021-03
期刊：
影响因子：
--
通讯作者：
Craik CS
中科院分区：
文献类型：
--
作者：
Sevillano N;Bohn MF;Zimanyi M;Chen Y;Petzold C;Gupta S;Ralston CY;Craik CS
U33是uPA的重组Fab抑制剂，其亲和力成熟用于与亲本Fab相比提高亲和力和抑制效果。进行U33的六个CDR环的精氨酸扫描以鉴定初始结合决定簇，因为uPA在其主要底物结合口袋中偏好精氨酸。选择两个CDR环以产生围绕ArgL 91（CDR L3）和ArgH 52（CDR H2）多样化的U33的工程化亲和力成熟文库。在严格条件下对随机化的U33文库进行生物淘选，得到8个具有改善的结合特性的Fab。最有效的抑制剂之一AB 2与U33相比，IC 50降低了13倍，这主要是由于其解离速率降低。为了确定界面残基的贡献，可能会发生结构重排界面形成后，我们使用X射线足迹和质谱（XFMS）。四个残基显示出溶剂可及性的显著降低，并且它们的聚类表明AB 2靶向活性位点并且还接合人uPA特有的相邻口袋中的残基。AB 2与uPA结合的2.9 nm分辨率晶体结构显示了一种结合模式，其中CDR L1环插入活性位点裂缝并作为抑制的决定因素。这种结合模式的选择性决定因素不同于先前鉴定的针对uPA相关丝氨酸蛋白酶MTSP-I、HGFA和FXIa的抑制性Fab。CDR H2和L3环有助于界面形成，并提供关键的盐桥以重塑围绕uPA活性位点的环，从而提供特异性和进一步的证据表明抗体可以是蛋白水解酶的有效和选择性抑制剂。
Affinity maturation of U33, a recombinant Fab inhibitor of uPA, was used to improve the affinity and the inhibitory effect compared to the parental Fab. Arginine scanning of the six CDR loops of U33 was done to identify initial binding determinants since uPA prefers arginine in its primary substrate binding pocket. Two CDR loops were selected to create an engineered affinity maturation library of U33 that was diversified around ArgL91 (CDR L3) and ArgH52 (CDR H2). Biopanning of the randomized U33 library under stringent conditions resulted in eight Fabs with improved binding properties. One of the most potent inhibitors, AB2, exhibited a 13-fold decrease in IC50 when compared to U33 largely due to a decrease in its off rate. To identify contributions of interfacial residues that might undergo structural rearrangement upon interface formation we used X-ray footprinting and mass spectrometry (XFMS). Four residues showed a pronounced decrease in solvent accessibility, and their clustering suggests that AB2 targets the active site and also engages residues in an adjacent pocket unique to human uPA. The 2.9 Å resolution crystal structure of AB2-bound to uPA shows a binding mode in which the CDR L1 loop inserts into the active site cleft and acts as a determinant of inhibition. The selectivity determinant of this binding mode is unlike previously identified inhibitory Fabs against uPA related serine proteases, MTSP-1, HGFA and FXIa. CDRs H2 and L3 loops aid in interface formation and provide critical salt-bridges to remodel loops surrounding the active site of uPA providing specificity and further evidence that antibodies can be potent and selective inhibitors of proteolytic enzymes.
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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：
5.6
作者：
HAWKINS, RE;RUSSELL, SJ;WINTER, G
通讯作者：
WINTER, G
影响因子：
5.6
作者：
Halgren, Thomas A.
通讯作者：
Halgren, Thomas A.
DOI：
10.1007/978-1-4939-7595-2_26
发表时间：
2018-01-01
期刊：
PROTEASES AND CANCER: METHODS AND PROTOCOLS
影响因子：
--
作者：
Dong Hyun Nam;Ge, Xin
通讯作者：
Ge, Xin
DOI：
10.1016/j.str.2017.12.010
发表时间：
2018-02-06
期刊：
Structure (London, England : 1993)
影响因子：
--
作者：
Ely LK;Lolicato M;David T;Lowe K;Kim YC;Samuel D;Bessette P;Garcia JL;Mikita T;Minor DL Jr;Coughlin SR
通讯作者：
Coughlin SR