Non-combinatorial library screening reveals subsite cooperativity and identifies new high-efficiency substrates for kallikrein-related peptidase 14
Non-combinatorial library screening reveals subsite cooperativity and identifies new high-efficiency substrates for kallikrein-related peptidase 14
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DOI：
10.1515/bc-2011-250
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发表时间：
2012-05-01
影响因子：
3.7
通讯作者：
Harris, Jonathan M.
中科院分区：
文献类型：
--
作者：
de Veer, Simon J.;Swedberg, Joakim E.;Harris, Jonathan M.
一系列底物将胰蛋白酶丝氨酸蛋白酶、激肽释放酶相关肽酶14（KLK 14）与生理功能（包括与炎症和癌症相关的信号分子的脱皮和活化）联系起来。蛋白酶切割序列的识别是由暴露的底物基序和酶活性位点裂缝的物理化学特征之间的互补性驱动的。然而，传统的底物筛选方法产生了冲突的亚位点配置文件KLK 14。本研究利用最近开发的筛选技术，稀疏矩阵库，以确定五个新的高效序列KLK 14。最佳序列YASR的切割效率（k（cat）/K-M=3.81 +/-0.4x10（6）M-1 s（-1））分别比来自位置扫描和噬菌体展示的有利底物高2倍和10倍。结合位点协同性在优选序列中是突出的，这使得在所有亚位点处的最佳相互作用成为可能，如KLK 14/底物复合物的预测建模所示。这些模拟构成了KLK 14的第一个分子动力学分析，并为KLK 14和密切相关的KLK，KLK 4和KLK 5之间明显的不同亚位点偏好提供了结构依据。总的来说，这些研究结果突出了结合位点协同性在蛋白酶底物识别中的重要性，这对发现最佳底物和工程高效蛋白酶抑制剂具有重要意义。
An array of substrates link the tryptic serine protease, kallikrein-related peptidase 14 (KLK14), to physiological functions including desquamation and activation of signaling molecules associated with inflammation and cancer. Recognition of protease cleavage sequences is driven by complementarity between exposed substrate motifs and the physicochemical signature of an enzyme's active site cleft. However, conventional substrate screening methods have generated conflicting subsite profiles for KLK14. This study utilizes a recently developed screening technique, the sparse matrix library, to identify five novel high-efficiency sequences for KLK14. The optimal sequence, YASR, was cleaved with higher efficiency (k(cat)/K-M=3.81 +/- 0.4x10(6) M-1 s(-1)) than favored substrates from positional scanning and phage display by 2- and 10-fold, respectively. Binding site cooperativity was prominent among preferred sequences, which enabled optimal interaction at all subsites as indicated by predictive modeling of KLK14/substrate complexes. These simulations constitute the first molecular dynamics analysis of KLK14 and offer a structural rationale for the divergent subsite preferences evident between KLK14 and closely related KLKs, KLK4 and KLK5. Collectively, these findings highlight the importance of binding site cooperativity in protease substrate recognition, which has implications for discovery of optimal substrates and engineering highly effective protease inhibitors.