Distinct functional constraints driving conservation of the cofilin N-terminal regulatory tail. Distinct functional constraints driving conservation of the cofilin N-terminal regulatory tail

Cofilin family proteins have essential roles in remodeling the cytoskeleton through filamentous actin depolymerization and severing. The short unstructured N-terminal region of cofilin is critical for actin binding and harbors the major site of inhibitory phosphorylation. Atypically for a disordered sequence, the N-terminal region is highly conserved, but specifics aspects driving this conservation are unclear. Here, we screened a library of 16,000 human cofilin N-terminal sequence variants for their capacity to support growth in S. cerevisiae in the presence or absence of the upstream regulator LIM kinase. Results from the screen and biochemical analysis of individual variants revealed distinct sequence requirements for actin binding and regulation by LIM kinase. LIM kinase recognition only partly explained sequence constraints on phosphoregulation, which were instead driven to a large extent by the capacity for phosphorylation to inactivate cofilin. We found remarkably loose sequence requirements for actin binding and phosphoinhibition, but collectively they restricted the N-terminus to sequences found in natural cofilins. Our results illustrate how a phosphorylation site can balance potentially competing sequence requirements for function and regulation. Overall design: The S. cerevisiae TeTO7-COF1 strain31 (Horizon Discovery TH_5610) was transformed sequentially with pRS415-GAL1-FLAG-LIMK1 and the cofilin library plasmid pool and frozen in aliquots to conduct replicate screens. For each screen, transformed yeast were pooled and grown at 30 °C with shaking in liquid culture with selective media (SC-His- Leu) containing 2% glucose at a starting OD600 of 0.1. After 4 - 5 population doublings, a portion was diluted to an OD600 of 0.1 with SC-His-Leu media containing 2% raffinose and 10 mg/L DOX. After the culture reached an OD600 of 1 - 1.5, a portion was reserved for plasmid recovery (T0 sample), and the remaining culture was split and diluted into SC-His-Leu liquid media containing 10 mg/L DOX and either 2% glucose or 2% raffinose/1% galactose to OD600 = 0.1. Cultures were subjected to three growth and dilution cycles in which they were grown to an OD600 of 1 - 2 and diluted back to an OD600 of 0.1, saving a portion for plasmid recovery at each step (T1 – T3 samples). After the screen was complete, plasmids were recovered from cell pellets using a QIAGEN Spin Miniprep kit. The N-terminal variable region of the plasmid pool was PCR amplified to attach sequencing adaptors and add barcodes and sequenced on an Illumina NovaSeq instrument. The relative representation of each cofilin variant at each timepoint was calculated by dividing the number of reads that variant by the total read count. The enrichment or depletion of a cofilin-1 library sequence in the absence of LIMK1 induction was calculated as the ratio of its relative representation at T0 and T2 in glucose. Enrichment or depletion following LIMK1 induction was calculated from timepoints T1 and T3 in glucose or galactose. Sequences were ranked by the average log fold changes in representation across three replicate screens. Data were processed using Microsoft Excel.

丝切蛋白（Cofilin）家族蛋白通过解聚、切断丝状肌动蛋白，在细胞骨架重塑中发挥核心作用。丝切蛋白短小的固有无序N端区域是其结合肌动蛋白的关键位点，同时也是抑制性磷酸化的主要靶点。作为固有无序序列的特例，该N端区域具有高度保守性，但目前尚不明确驱动其保守性的具体机制。
本研究构建了包含16000个人源丝切蛋白N端序列变体的文库，筛选这些变体在酿酒酵母（S. cerevisiae）中，在存在或缺失上游调控因子LIM激酶（LIM kinase）时支持酵母生长的能力。筛选结果与单变体生化分析显示，肌动蛋白结合与LIM激酶调控分别具有截然不同的序列需求。LIM激酶的识别仅能部分解释磷酸化调控相关的序列约束，这类约束在很大程度上由磷酸化失活丝切蛋白的能力所决定。我们发现，肌动蛋白结合与磷酸化抑制的序列约束均极为宽松，但二者共同作用将N端序列限制在了天然丝切蛋白所拥有的序列范围内。本研究结果阐明了磷酸化位点如何平衡功能与调控之间潜在的竞争性序列需求。
整体实验设计：将酿酒酵母TeTO7-COF1菌株31（Horizon Discovery公司TH_5610）依次转化pRS415-GAL1-FLAG-LIMK1质粒与丝切蛋白文库质粒池，分装冻存以开展重复筛选实验。每次筛选时，将转化后的酵母混合接种于含2%葡萄糖的SC-His-Leu选择性液体培养基中，初始OD600为0.1，于30℃振荡培养。待完成4~5次群体倍增后，将部分菌液用含2%棉子糖与10mg/L多西环素（DOX）的SC-His-Leu培养基稀释至OD600=0.1。待培养液OD600达到1~1.5时，留存部分菌液用于质粒回收（T0样本）；剩余培养液均分后，接种至含10mg/L DOX的SC-His-Leu液体培养基中，分别添加2%葡萄糖或2%棉子糖/1%半乳糖，调整初始OD600至0.1。随后将培养液进行3轮生长-稀释循环：培养至OD600为1~2后，稀释至OD600=0.1，每轮均留存部分菌液用于质粒回收（T1~T3样本）。筛选结束后，使用QIAGEN离心柱微量质粒提取试剂盒从细胞沉淀中回收质粒。通过PCR扩增文库质粒的N端可变区域以连上测序接头并添加条码，随后在Illumina NovaSeq测序仪上完成测序。通过将某一变体的读段数除以总读段数，计算该变体在各时间点的相对丰度。在未诱导LIMK1表达的条件下，丝切蛋白-1文库序列的富集或缺失程度通过葡萄糖培养基中T0与T2时间点的相对丰度比值计算得出；诱导LIMK1表达后的富集或缺失程度则通过葡萄糖或半乳糖培养基中T1与T3时间点的相对丰度比值计算得出。根据三次重复筛选的平均对数倍变化值对序列进行排序。数据分析使用Microsoft Excel完成。