Lipid Biofunction Section, Faculty of Advanced Life Science, Hokkaido University, 001-0021 Sapporo, Hokkaido, Japan.
Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 062-8517 Sapporo, Hokkaido, Japan.
Cells. 2020 Feb 24;9(2):517. doi: 10.3390/cells9020517.
Konjac ceramide (kCer) is a plant-type ceramide composed of various long-chain bases and a-hydroxyl fatty acids. The presence of d4t,8t-sphingadienine is essential for semaphorin 3A (Sema3A)-like activity. Herein, we examined the three neuropilin 1 (Nrp1) domains (a1a2, b1b2, or c), and found that a1a2 binds to d4t,8t-kCer and possesses Sema3A-like activity. kCer binds to Nrp1 with a weak affinity of mM dissociation constant (Kd). We wondered whether bovine serum albumin could influence the ligand-receptor interaction that a1a2 has with a single high affinity binding site for kCer (Kd in nM range). In the present study we demonstrated the influence of bovine serum albumin. Thermal denaturation indicates that the a1a2 domain may include intrinsically disordered region (IDR)-like flexibility. A potential interaction site on the a1 module was explored by molecular docking, which revealed a possible Nrp1 activation mechanism, in which kCer binds to Site A close to the Sema3A-binding region of the a1a2 domain. The a1 module then accesses a2 as the IDR-like flexibility becomes ordered via kCer-induced protein rigidity of a1a2. This induces intramolecular interaction between a1 and a2 through a slight change in protein secondary structure.
魔芋神经酰胺（kCer）是一种植物型神经酰胺，由各种长链碱基和 α-羟脂肪酸组成。d4t、8t-鞘氨醇的存在对于类 Sema3A 活性是必需的。在此，我们研究了三个神经纤毛蛋白 1（Nrp1）结构域（a1a2、b1b2 或 c），发现 a1a2 与 d4t、8t-kCer 结合并具有类 Sema3A 活性。kCer 与 Nrp1 的结合亲和力较弱，解离常数（Kd）为 mM。我们想知道牛血清白蛋白是否会影响 a1a2 与 kCer 单一高亲和力结合位点（Kd 在 nM 范围内）的配体-受体相互作用。在本研究中，我们证明了牛血清白蛋白的影响。热变性表明 a1a2 结构域可能包含固有无序区域（IDR）样的灵活性。通过分子对接探索了 a1 模块上的潜在相互作用位点，揭示了一种可能的 Nrp1 激活机制，其中 kCer 结合到靠近 a1a2 结构域的 Sema3A 结合区域的 A 位点。然后，a1 模块通过 kCer 诱导的 a1a2 蛋白刚性获得 IDR 样灵活性，从而访问 a2。这通过蛋白二级结构的微小变化诱导 a1 和 a2 之间的分子内相互作用。
