Mineev Konstantin S, Kryukova Elena V, Kasheverov Igor E, Egorova Natalia S, Zhmak Maxim N, Ivanov Igor A, Senko Dmitry A, Feofanov Alexey V, Ignatova Anastasia A, Arseniev Alexander S, Utkin Yuri N, Tsetlin Victor I
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia.
Department of Physico-Chemical Biology and Biotechnology, Moscow Institute of Physics and Technology, 141700 Dolgoprudnyi, Russia.
Biomolecules. 2020 Dec 22;11(1):1. doi: 10.3390/biom11010001.
Lynx1, membrane-bound protein co-localized with the nicotinic acetylcholine receptors (nAChRs) and regulates their function, is a three-finger protein (TFP) made of three β-structural loops, similarly to snake venom α-neurotoxin TFPs. Since the central loop II of α-neurotoxins is involved in binding to nAChRs, we have recently synthesized the fragments of Lynx1 central loop, including those with the disulfide between Cys residues introduced at N- and C-termini, some of them inhibiting muscle-type nAChR similarly to the whole-size water-soluble Lynx1 (ws-Lynx1). Literature shows that the main fragment interacting with TFPs is the C-loop of both nAChRs and acetylcholine binding proteins (AChBPs) while some ligand-binding capacity is preserved by analogs of this loop, for example, by high-affinity peptide HAP. Here we analyzed the structural organization of these peptide models of ligands and receptors and its role in binding. Thus, fragments of Lynx1 loop II, loop C from the AChBP and HAP were synthesized in linear and Cys-cyclized forms and structurally (CD and NMR) and functionally (radioligand assay on nAChR) characterized. Connecting the C- and N-termini by disulfide in the ws-Lynx1 fragment stabilized its conformation which became similar to the loop II within the H-NMR structure of ws-Lynx1, the activity being higher than for starting linear fragment but lower than for peptide with free cysteines. Introduced disulfides did not considerably change the structure of HAP and of loop C fragments, the former preserving high affinity for α-bungarotoxin, while, surprisingly, no binding was detected with loop C and its analogs.
Lynx1是一种与烟碱型乙酰胆碱受体（nAChRs）共定位的膜结合蛋白，可调节其功能，它是一种由三个β结构环组成的三指蛋白（TFP），类似于蛇毒α-神经毒素TFP。由于α-神经毒素的中央环II参与与nAChRs的结合，我们最近合成了Lynx1中央环的片段，包括在N端和C端引入了半胱氨酸残基之间二硫键的片段，其中一些片段与全长水溶性Lynx1（ws-Lynx1）类似，可抑制肌肉型nAChR。文献表明，与TFP相互作用的主要片段是nAChRs和乙酰胆碱结合蛋白（AChBPs）的C环，而该环的类似物保留了一些配体结合能力，例如高亲和力肽HAP。在此，我们分析了这些配体和受体肽模型的结构组织及其在结合中的作用。因此，合成了Lynx1环II、AChBP的C环和HAP的线性和半胱氨酸环化形式的片段，并对其进行了结构（圆二色光谱和核磁共振）和功能（nAChR放射性配体测定）表征。在ws-Lynx1片段中通过二硫键连接C端和N端稳定了其构象，该构象变得类似于ws-Lynx1的H-NMR结构中的环II，活性高于起始线性片段，但低于具有游离半胱氨酸的肽。引入的二硫键对HAP和环C片段的结构影响不大，前者对α-银环蛇毒素仍保持高亲和力，而令人惊讶的是，未检测到环C及其类似物的结合。
