Cantrell Donovan A, Urbauer Ramona J Bieber, Kim Hyun W, Woods Robert J, Urbauer Jeffrey L, Wood Zachary A, West Christopher M
Biochemistry. 2025 May 20;64(10):2262-2279. doi: 10.1021/acs.biochem.4c00859. Epub 2025 Apr 29.
Skp1/Cullin-1/F-Box protein (SCF) complexes represent a major class of E3 ubiquitin ligases responsible for proteomic control throughout eukaryotes. Target specificity is mediated by a large set of F-box proteins (FBPs) whose F-box domains interact with Skp1 in a conserved, well-organized fashion. In the social amoeba , Skp1 is regulated by oxygen-dependent glycosylation which alters Skp1's FBP interactome and inhibits homodimerization that is mediated in part by an ordered interface which overlaps with that of FBPs. Based on sedimentation velocity experiments, Skp1 from the intracellular pathogen exhibits a homodimerization comparable to that of a previously measured FBP/Skp1 interaction. Glycosylation of Skp1's disordered C-terminal region (CTR) distal to the ordered homodimer interface significantly weakens Skp1 homodimerization, an effect reproduced by CTR deletion. Replacement with a randomized CTR sequence retains high affinity excluding an extension of the ordered dimer interface. Substitution by poly serine weakens the homodimer to a degree equal to its deletion, indicating a composition dependent effect. The contribution of the CTR to Skp1 homodimerization is canceled by high salt consistent with an electrostatic mechanism. All-atom molecular dynamics simulations suggest that the CTR promotes homodimerization via charge cluster interactions. Taken together, the data indicate that glycosylation weakens homodimerization by disrupting a C-terminal fuzzy interaction that functions in tandem with the ordered dimer interface, thereby freeing Skp1 for FBP binding. Thus, the CTR contributes to Skp1/Skp1 and Skp1/FBP interactions via independent mechanisms that are each influenced by O, indicating multiple constraints on the evolution of its sequence.
Skp1/Cullin-1/F-Box蛋白（SCF）复合物是一类主要的E3泛素连接酶，负责真核生物中的蛋白质组控制。靶标特异性由大量F-Box蛋白（FBP）介导，其F-Box结构域以保守、有序的方式与Skp1相互作用。在社会性变形虫中，Skp1受氧依赖性糖基化调节，这会改变Skp1的FBP相互作用组，并抑制部分由与FBP重叠的有序界面介导的同二聚化。基于沉降速度实验，细胞内病原体的Skp1表现出与先前测量的FBP/Skp1相互作用相当的同二聚化。Skp1有序同二聚体界面远端无序的C末端区域（CTR）的糖基化显著削弱Skp1同二聚化，CTR缺失也会产生同样的效果。用随机CTR序列替代保留了高亲和力，排除了有序二聚体界面的延伸。用多聚丝氨酸替代会使同二聚体减弱到与其缺失程度相同，表明存在组成依赖性效应。CTR对Skp1同二聚化的贡献在高盐条件下被消除，这与静电机制一致。全原子分子动力学模拟表明，CTR通过电荷簇相互作用促进同二聚化。综合来看，数据表明糖基化通过破坏与有序二聚体界面协同作用的C末端模糊相互作用来削弱同二聚化，从而使Skp1能够与FBP结合。因此，CTR通过各自受氧影响的独立机制对Skp1/Skp1和Skp1/FBP相互作用有贡献，表明其序列进化受到多种限制。
