The PDZ (Postsynaptic density protein-95[PSD-95]/Discs-large) domain, prevalent as a recognition module, has attracted significant attention given its ability to specifically recognize ligands with consensus motifs (also termed PDZ binding motifs [PBMs]). PBMs typically bear a C-terminal carboxylate as a recognition handle and have been extensively characterized, whilst internal ligands are less well known. Here we characterize a short linear motif (SLiM) - EESTSFQGP - as an internal PBM based on its strong binding affinity towards the SHANK1 PDZ domain (SHANK1656-762 hereafter referred to as SHANK1). Using the acetylated analogue Ac-EESTSFQGP-CONH2 as a competitor for the interaction of SHANK1 with FAM-Ahx-EESTSFQGP-CONH2 or a typical fluorophore-labelled C-terminal PBM - GKAP - FITC-Ahx-EAQTRL-COOH - the internal SLiM was demonstrated to show comparable low-micromolar IC50 by competition fluorescent anisotropy. To gain further insight into the internal ligand interaction at the molecular level, we obtained the X-ray co-crystal structure of the Ac-EESTSFQGP-CONH2/SHANK1 complex and compared this to the Ac-EAQTRL-COOH/SHANK1 complex. The crystallographic studies reveal that the SHANK1 backbones for the two interactions overlap significantly. The main structural differences were shown to result from the flexible loops which reorganize to accommodate the two PBMs with distinct lengths and terminal groups. In addition, the two C-terminal residues Gly and Pro in Ac-EESTSFQGP-CONH2 were shown not to participate in interaction with the target protein, implying further truncation and structural modification using peptidomimetic approaches on this sequence may be feasible. Taken together, the SLiM Ac-EESTSFQGP-CONH2 holds potential as an internal ligand for targeting SHANK1.

中文翻译：

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SHANK1 PDZ 结构域和内部 SLiM 之间相互作用的生物物理和结构分析。


PDZ（突触后密度蛋白-95[PSD-95]/Discs-large）结构域作为一种普遍存在的识别模块，因其能够特异性识别具有共有基序（也称为 PDZ 结合基序 [PBM]）的配体而引起了极大的关注。 。 PBM 通常带有 C 端羧酸盐作为识别柄，并已被广泛表征，而内部配体则鲜为人知。在这里，我们将一个短线性基序 (SLiM) - EESTSFQGP - 作为内部 PBM，基于其对 SHANK1 PDZ 结构域（SHANK1656-762 以下称为 SHANK1）的强结合亲和力。使用乙酰化类似物 Ac-EESTSFQGP-CONH2 作为 SHANK1 与 FAM-Ahx-EESTSFQGP-CONH2 或典型荧光团标记的 C 端 PBM 相互作用的竞争者 - GKAP - FITC-Ahx-EAQTRL-COOH - 内部 SLiM通过竞争荧光各向异性证明显示出可比的低微摩尔 IC50。为了进一步了解分子水平上的内部配体相互作用，我们获得了 Ac-EESTSFQGP-CONH2/SHANK1 复合物的 X 射线共晶结构，并将其与 Ac-EAQTRL-COOH/SHANK1 复合物进行了比较。晶体学研究表明，两种相互作用的 SHANK1 主链显着重叠。主要的结构差异是由柔性环引起的，柔性环重新组织以适应具有不同长度和末端基团的两个 PBM。此外，Ac-EESTSFQGP-CONH2 中的两个 C 端残基 Gly 和 Pro 不参与与靶蛋白的相互作用，这意味着使用肽模拟方法对该序列进行进一步截断和结构修饰可能是可行的。综上所述，SLiM Ac-EESTSFQGP-CONH2 具有作为靶向 SHANK1 的内部配体的潜力。