Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0378, USA.
NMRFAM University of Wisconsin, 433 Babcock Drive, Madison, WI 53706, USA.
Sci Rep. 2017 Jan 6;7:39575. doi: 10.1038/srep39575.
Although serine proteases are found ubiquitously in both eukaryotes and prokaryotes, and they comprise the largest of all of the peptidase families, their dynamic motions remain obscure. The backbone dynamics of the coagulation serine protease, apo-thrombin (S195M-thrombin), were compared to the substrate-bound form (PPACK-thrombin). R, R, N-{H}NOEs, and relaxation dispersion NMR experiments were measured to capture motions across the ps to ms timescale. The ps-ns motions were not significantly altered upon substrate binding. The relaxation dispersion data revealed that apo-thrombin is highly dynamic, with μs-ms motions throughout the molecule. The region around the N-terminus of the heavy chain, the Na-binding loop, and the 170 s loop, all of which are implicated in allosteric coupling between effector binding sites and the active site, were dynamic primarily in the apo-form. Most of the loops surrounding the active site become more ordered upon PPACK-binding, but residues in the N-terminal part of the heavy chain, the γ-loop, and anion-binding exosite 1, the main allosteric binding site, retain μs-ms motions. These residues form a dynamic allosteric pathway connecting the active site to the main allosteric site that remains in the substrate-bound form.
尽管丝氨酸蛋白酶广泛存在于真核生物和原核生物中，并且它们构成了所有肽酶家族中最大的家族，但它们的动态运动仍然不清楚。凝血丝氨酸蛋白酶，脱辅基凝血酶（S195M-凝血酶）的骨架动力学与底物结合形式（PPACK-凝血酶）进行了比较。R、R、N-{H}NOE 和弛豫分散 NMR 实验用于捕获 ps 到 ms 时间尺度的运动。底物结合后，ps-ns 运动没有明显改变。弛豫分散数据表明脱辅基凝血酶高度动态，分子内存在 μs-ms 运动。重链 N 端附近的区域、Na 结合环和 170s 环，所有这些都与效应物结合位点和活性位点之间的变构偶联有关，在apo 形式下主要是动态的。大多数围绕活性位点的环在与 PPACK 结合后变得更加有序，但重链 N 端的残基、γ-环和阴离子结合外位 1（主要变构结合位点）保留 μs-ms 运动。这些残基形成了一种动态变构途径，将活性位点与在底物结合形式中保留的主要变构位点连接起来。
