School of Life Science and Technology, Harbin Institute of Technology, Harbin 150001, China.
Brain Research Center and Department of Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen 518055, China.
Int J Mol Sci. 2023 Jan 11;24(2):1414. doi: 10.3390/ijms24021414.
SH3 domains are common protein binding modules. The target sequence of SH3 domains is usually a proline-rich motif (PRM) containing a minimal "PxxP" sequence. The mechanism of how different SH3 domains specifically choose their targets from vast PxxP-containing sequences is still not very clear, as many reported SH3/PRM interactions are weak and promiscuous. Here, we identified the binding of the SH3 domain of ASAP1 to the PRM of MICAL1 with a sub-μM binding affinity, and determined the crystal structure of ASAP1-SH3 and MICAL1-PRM complex. Our structural and biochemical analyses revealed that the target-binding pocket of ASAP1-SH3 contains two negatively charged patches to recognize the "xPx + Px+" sequence in MICAL1-PRM and consequently strengthen the interaction, differing from the typical SH3/PRM interaction. This unique PRM-binding pocket is also found in the SH3 domains of GTPase Regulator associated with focal adhesion kinase (GRAF) and Src kinase associated phosphoprotein 1 (SKAP1), which we named SH3. In addition, we searched the Swiss-Prot database and found ~130 proteins with the SH3-binding PRM in silico. Finally, gene ontology analysis suggests that the strong interaction between the SH3-containing proteins and their targets may play roles in actin cytoskeleton regulation and vesicle trafficking.
SH3 结构域是常见的蛋白质结合模块。SH3 结构域的靶序列通常是富含脯氨酸的基序 (PRM)，其中包含一个最小的“PxxP”序列。不同的 SH3 结构域如何从大量含有 PxxP 的序列中特异性选择其靶标，其机制尚不清楚，因为许多报道的 SH3/PRM 相互作用较弱且杂乱无章。在这里，我们确定了 ASAP1 的 SH3 结构域与 MICAL1 的 PRM 的结合，其结合亲和力为亚微摩尔级，并确定了 ASAP1-SH3 和 MICAL1-PRM 复合物的晶体结构。我们的结构和生化分析表明，ASAP1-SH3 的靶标结合口袋包含两个带负电荷的斑块，用于识别 MICAL1-PRM 中的“xPx + Px +”序列，并因此增强相互作用，这与典型的 SH3/PRM 相互作用不同。这种独特的 PRM 结合口袋也存在于与粘着斑激酶 (GRAF) 和Src 激酶相关磷酸蛋白 1 (SKAP1) 相关的 GTP 酶调节剂 (GRAF) 和 Src 激酶相关磷酸蛋白 1 (SKAP1) 的 SH3 结构域中，我们将其命名为 SH3。此外，我们在 Swiss-Prot 数据库中进行了搜索，并在计算机上发现了约 130 种具有 SH3 结合 PRM 的蛋白质。最后，基因本体分析表明，含 SH3 的蛋白质与其靶标的强相互作用可能在肌动蛋白细胞骨架调节和囊泡运输中发挥作用。
