Genetically encoding proximal-reactive unnatural amino acids (PrUaas), such as fluorosulfate-l-tyrosine (FSY), into natural proteins of interest (POI) confer the POI with the ability to covalently bind to its interacting proteins (IPs). The PrUaa-incorporated POIs hold promise for blocking undesirable POI–IP interactions. Selecting appropriate PrUaa anchor sites is crucial, but it remains challenging with the current methodology, which heavily relies on crystallography to identify the proximal residues between the POIs and the IPs for the PrUaa anchorage. To address the challenge, here, we propose a footprinting-directed genetically encoded covalent binder (footprinting-GECB) approach. This approach employs carbene footprinting, a structural mass spectrometry (MS) technique that quantifies the extent of labeling of the POI following the addition of its IP, and thus identifies the responsive residues. By genetically encoding PrUaa into these responsive sites, POI variants with covalent bonding ability to its IP can be produced without the need for crystallography. Using the POI–IP model, KRAS/RAF1, we showed that engineering FSY at the footprint-assigned KRAS residue resulted in a KRAS variant that can bind irreversibly to RAF1. Additionally, we inserted FSY at the responsive residue in RAF1 upon footprinting the oncogenic KRAS^G12D/RAF1, which lacks crystal structure, and generated a covalent binder to KRAS^G12D. Together, we demonstrated that by adopting carbene footprinting to direct PrUaa anchorage, we can greatly expand the opportunities for designing covalent protein binders for PPIs without relying on crystallography. This holds promise for creating effective PPI inhibitors and supports both fundamental research and biotherapeutics development.

中文翻译：

---

卡宾足迹指导基因编码邻近反应蛋白结合剂的设计


将近端反应性非天然氨基酸 (PrUaas)（例如氟硫酸盐-L-酪氨酸 (FSY)）基因编码到天然目的蛋白 (POI) 中，赋予 POI 与其相互作用蛋白 (IP) 共价结合的能力。 PrUaa 合并的 POI 有望阻止不良的 POI-IP 交互。选择合适的 PrUaa 锚定位点至关重要，但目前的方法仍然具有挑战性，该方法严重依赖晶体学来识别 PrUaa 锚定的 POI 和 IP 之间的近端残基。为了应对这一挑战，我们在这里提出了一种足迹导向的基因编码共价结合剂（足迹-GECB）方法。该方法采用卡宾足迹法，这是一种结构质谱 (MS) 技术，可量化添加 IP 后 POI 的标记程度，从而识别响应残基。通过将 PrUaa 基因编码到这些响应位点中，可以产生与其 IP 具有共价键合能力的 POI 变体，而无需进行晶体学分析。使用 POI-IP 模型 KRAS/RAF1，我们表明在足迹分配的 KRAS 残基上改造 FSY 会产生可以与 RAF1 不可逆结合的 KRAS 变体。此外，我们在追踪缺乏晶体结构的致癌 KRAS ^G12D /RAF1 后，将 FSY 插入 RAF1 中的响应残基处，并生成 KRAS ^G12D 的共价结合物。我们共同证明，通过采用卡宾足迹来指导 PrUaa 锚定，我们可以大大扩展为 PPI 设计共价蛋白结合剂的机会，而无需依赖晶体学。这有望创造出有效的 PPI 抑制剂，并支持基础研究和生物治疗药物的开发。