PADI4 是一组干预精氨酸转化为瓜氨酸的酶的人类亚型之一。它参与多种类型肿瘤以及其他免疫疾病的发生，例如牛皮癣、多发性硬化症或类风湿性关节炎。 PADI4 在其序列的几个区域中自动瓜氨酸化，即对应于残基 Arg205、Arg212、Arg218 和 Arg383。我们想要研究瓜氨酸部分是否影响附近区域的构象及其与完整 PADI4 的结合。我们设计了两个系列的合成肽，其包含此类区域的野生型或相对瓜氨酸化版本——即，第一系列肽包含前三个精氨酸，第二系列包含Arg383。我们通过使用荧光、远紫外 (UV) 圆二色性 (CD) 和 2D1H NMR 单独研究了它们的构象特性。此外，我们通过使用等温滴定量热法 (ITC)、荧光和生物层干涉法 (BLI) 以及分子对接模拟，表征了两个系列中野生型和瓜氨酸肽与完整 PADI4 的结合。我们观察到瓜氨酸化并没有改变分离肽的局部构象倾向。然而，对于这两个系列中的所有肽，瓜氨酸化减慢了与 PADI4 的结合反应的动力学 koff 速率，这可能是由于与精氨酸的存在相比静电效应的差异。 PADI4对未修饰肽的亲和力略大于两个系列中相应瓜氨酸肽的亲和力，但它们都在同一范围内，表明由于序列效应，结合热力学不存在相关变化。这些结果突出显示了 PADI4 自身瓜氨酸化的细节，更普遍的是，其他瓜氨酸酶在体内发生的可能的自催化机制，或者被动经历瓜氨酸化的蛋白质。版权所有 © 2024 作者。由 Elsevier B.V. 出版。保留所有权利。

PADI4 is one of the human isoforms of a group of enzymes intervening in the conversion of arginine to citrulline. It is involved in the development of several types of tumors, as well as other immunological illnesses, such as psoriasis, multiple sclerosis, or rheumatoid arthritis. PADI4 auto-citrullinates in several regions of its sequence, namely in correspondence of residues Arg205, Arg212, Arg218, and Arg383. We wanted to study whether the citrullinated moiety affects the conformation of nearby regions and its binding to intact PADI4. We designed two series of synthetic peptides comprising either the wild-type or the relative citrullinated versions of such regions - i.e., a first series of peptides comprising the first three arginines, and a second series comprising Arg383. We studied their conformational properties in isolation by using fluorescence, far-ultraviolet (UV) circular dichroism (CD), and 2D1H NMR. Furthermore, we characterized the binding of the wild-type and citrullinated peptides in the two series to the intact PADI4, by using isothermal titration calorimetry (ITC), fluorescence, and biolayer interferometry (BLI), as well as by molecular docking simulations. We observed that citrullination did not alter the local conformational propensities of the isolated peptides. Nevertheless, for all the peptides in the two series, citrullination slowed down the kinetic koff rates of the binding reaction to PADI4, probably due to differences in electrostatic effects compared to the presence of arginine. The affinities of PADI4 for unmodified peptides were slightly larger than those of the corresponding citrullinated ones in the two series, but they were all within the same range, indicating that there were no relevant variations in the thermodynamics of binding due to sequence effects. These results highlight details of the self-citrullination of PADI4 and, more generally, of possible auto-catalytic mechanisms taking place in vivo for other citrullinating enzymes or, alternatively, in proteins undergoing citrullination passively.Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.