Serine protease inhibitor Kazal type 3 (SPINK3) from mouse seminal vesicles is a Kazal-type trypsin inhibitor. It has been shown to bind to the sperm acrosome and modify sperm activity by influencing the sub-cellular Ca2+ influx. Previously, SPINK3 was reported to suppress in vitro sperm capacitation. However, under natural coitus, SPINK3 is removed from the mouse acrosome in the female reproductive tract, leading to successful fertilisation. Identification of the SPINK3 binding partner becomes essential to develop a contraceptive that works by prolonging the binding of SPINK3 to the sperm acrosome. We identified the SPINK3 receptor by using recombinant SPINK3 (rSPINK3). Testicular serine protease 1 (TESP1) was identified as the receptor for SPINK3 by 2D gel electrophoresis coupled with western blot analysis. To authenticate TESP1 as the receptor for SPINK3, sperm cells were incubated with TESP1 peptide antibody followed by determining the intracellular [Ca2+]i concentration by flow cytometry using Fluo-3 AM as a calcium probe. Furthermore, the 3D structures of SPINK3 and TESP1 were predicted by homology modelling (Schrodinger suite) using the crystal structure of pancreatic secretory trypsin inhibitor (PDB ID—1TGS) and human prostasin (PDB ID—3DFJ) as templates. The modelled protein structures were validated and subjected to molecular dynamics simulation (MDS) using GROMACS v5.0.5. Protein–protein docking was performed using HDOCK and the complex was validated by MDS. The results predicted that SPINK3 and TESP1 had strong binding affinity, with a dock score of −430.70 and 14 hydrogen bonds as key active site residues. If the binding affinity between SPINK3 and TESP1 could be increased, the SPINK3-TESP1 association will be prolonged, which will be helpful in the development of a male contraceptive.

中文翻译：

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小鼠睾丸丝氨酸蛋白酶，TESP1，作为小鼠精子顶体上潜在的 SPINK3 受体蛋白

来自小鼠精囊的丝氨酸蛋白酶抑制剂 Kazal type 3 (SPINK3) 是 Kazal 型胰蛋白酶抑制剂。它已被证明可与精子顶体结合并通过影响亚细胞 Ca2+ 流入来改变精子活性。此前，据报道 SPINK3 可抑制体外精子获能。然而，在自然交配下，SPINK3 会从雌性生殖道的小鼠顶体中去除，从而成功受精。鉴定 SPINK3 结合伴侣对于开发一种通过延长 SPINK3 与精子顶体的结合起作用的避孕药变得至关重要。我们通过使用重组 SPINK3 (rSPINK3) 鉴定了 SPINK3 受体。通过二维凝胶电泳结合蛋白质印迹分析，睾丸丝氨酸蛋白酶 1 (TESP1) 被鉴定为 SPINK3 的受体。为了验证 TESP1 是 SPINK3 的受体，将精子细胞与 TESP1 肽抗体一起孵育，然后使用 Fluo-3 AM 作为钙探针通过流式细胞术测定细胞内 [Ca2+]i 浓度。此外，SPINK3 和 TESP1 的 3D 结构通过同源建模（薛定谔套件）使用胰腺分泌胰蛋白酶抑制剂 (PDB ID-1TGS) 和人前列腺素 (PDB ID-3DFJ) 的晶体结构作为模板进行预测。使用 GROMACS v5.0.5 验证建模的蛋白质结构并进行分子动力学模拟 (MDS)。使用 HDOCK 进行蛋白质-蛋白质对接，并通过 MDS 验证复合物。结果预测SPINK3和TESP1具有很强的结合亲和力，dock score为-430.70，14个氢键作为关键活性位点残基。