Xie Pujun, Li Yan, Lamon Gaëlle, Kuang Huihui, Wang Da-Neng, Traaseth Nathaniel J
Department of Chemistry, New York University, New York, NY, USA.
Department of Cell Biology, New York University School of Medicine, New York, NY, USA.
Nat Commun. 2025 Jan 2;16(1):7. doi: 10.1038/s41467-024-54986-5.
Advancements in cryo-EM have stimulated a revolution in structural biology. Yet, for membrane proteins near the cryo-EM size threshold of approximately 40 kDa, including transporters and G-protein coupled receptors, the absence of distinguishable structural features makes image alignment and structure determination a significant challenge. Furthermore, resolving more than one protein conformation within a sample, a major advantage of cryo-EM, represents an even greater degree of difficulty. Here, we describe a strategy for introducing a rigid fiducial marker (BRIL domain) at the C-terminus of membrane transporters from the Major Facilitator Superfamily (MFS) with AlphaFold2. This approach involves fusion of the last transmembrane domain helix of the target protein with the first helix of BRIL through a short poly-alanine linker to promote helicity. Combining this strategy with a BRIL-specific Fab, we elucidated four cryo-EM structures of the 42 kDa Staphylococcus aureus transporter NorA, three of which were derived from a single sample corresponding to inward-open, inward-occluded, and occluded conformations. Hence, this fusion construct facilitated experiments to characterize the conformational landscape of NorA and validated our design to position the BRIL/antibody pair in an orientation that avoids steric clash with the transporter. The latter was enabled through AlphaFold2 predictions, which minimized guesswork and reduced the need for screening several constructs. We further validated the suitability of the method to three additional MFS transporters (GlpT, Bmr, and Blt), results that supported a rigid linker between the transporter and BRIL. The successful application to four MFS proteins, the largest family of secondary transporters in nature, and analysis of predicted structures for the family indicates this strategy will be a valuable tool for studying other MFS members using cryo-EM.
冷冻电镜技术的进步推动了结构生物学的一场革命。然而，对于接近约40 kDa冷冻电镜尺寸阈值的膜蛋白，包括转运蛋白和G蛋白偶联受体，缺乏可区分的结构特征使得图像对齐和结构确定成为一项重大挑战。此外，解析样品中的多种蛋白质构象是冷冻电镜的一个主要优势，但这带来的难度更大。在这里，我们描述了一种策略，即利用AlphaFold2在主要易化子超家族（MFS）的膜转运蛋白C端引入刚性基准标记（BRIL结构域）。这种方法涉及通过短聚丙氨酸接头将目标蛋白的最后一个跨膜结构域螺旋与BRIL的第一个螺旋融合，以促进螺旋形成。将该策略与BRIL特异性Fab相结合，我们阐明了42 kDa金黄色葡萄球菌转运蛋白NorA的四种冷冻电镜结构，其中三种来自对应向内开放、向内堵塞和堵塞构象的单个样品。因此，这种融合构建体有助于表征NorA构象景观的实验，并验证了我们将BRIL/抗体对定位在避免与转运蛋白发生空间冲突的方向上的设计。后者通过AlphaFold2预测得以实现，该预测最大限度地减少了猜测工作，并减少了筛选多种构建体的需求。我们进一步验证了该方法对另外三种MFS转运蛋白（GlpT、Bmr和Blt）的适用性，结果支持了转运蛋白与BRIL之间存在刚性接头。该方法成功应用于自然界中最大的次级转运蛋白家族——四种MFS蛋白，并对该家族的预测结构进行了分析，表明该策略将成为使用冷冻电镜研究其他MFS成员的有价值工具。
