Computational design of protein-based modular small-molecule biosensors

描述（由申请人提供）：感知小分子的能力在探测生物过程、生物工程和医学诊断方面具有重要而广泛的应用。这个R21项目旨在证明一个计算设计平台的可行性，以设计基于蛋白质（因此基因可编码）的小分子生物传感器。为了实现这一目标，我们将开发和评估将小分子结合位点设计成异二聚体蛋白质-蛋白质界面的方法，使蛋白质-蛋白质相互作用依赖于小分子。这两个蛋白伴侣中的每一个都将被连接到一个分裂报告基因的片段上。然后一个功能性传感器通过报告蛋白的补充来检测小分子的存在。在这种方式下，传感器输出原则上是模块化的：不同的报告片段可以连接到小分子传感器组件上，并进行检测（例如通过分裂- gfp）或驱动（分裂酶或基因表达）的测试。我们的新贡献将是开发一种设计方法，该方法使用四步过程将小分子结合位点从配体蛋白质结构移植到蛋白质-蛋白质界面中：(1)给定小分子靶标，我们定义结合“基序”（通常是蛋白质侧链的一部分）的几何约束，以协调配体蛋白质单体结构中的靶标。(2)我们搜索了bb0 600个异二聚体蛋白-蛋白界面（“支架”），寻找那些可以容纳靶标及其结合基序的界面。(3)我们利用我们最近开发的机器人启发的方法，在目标结合位点周围计算重构和设计支架界面，以稳定基序残基的结合构象。(4)我们预测传感器序列的文库，并仅在小分子存在的情况下使用体外结合试验和使用生物传感器输出在细菌中测试这些序列的二聚化。计算机辅助设计模块化小分子诱导二聚化将是第一次。我们的实验计划旨在通过几种方式改善这个设计驱动项目中固有的风险：(i)使用基序导向的方法将适度的目标结合亲和力预编程到界面中，这种方法最近成功地设计了新的酶。（ii）计算与实验测试相辅相成，不仅对最高预测，而且对序列库。（iii）生物传感器平台促进了这些文库的测试，其中预期输出本身将用于筛选、选择和改进传感器。虽然小分子诱导的蛋白质二聚化，如雷帕霉素系统，存在于自然界中，并已被重新设计，但这些系统仅限于少数可以被感知的分子。该项目的成功完成将展示一种平台技术，通过为许多目标提供模块化生物传感器的途径，可以极大地扩大小分子传感和驱动的应用。

DESCRIPTION (provided by applicant): The ability to sense small molecules has important and wide-ranging applications for probing biological processes, for biological engineering and for medical diagnostics. This R21 project seeks to demonstrate feasibility of a computational design platform to engineer protein-based (thus genetically encodable) small molecule biosensors. To achieve this goal, we will develop and assess methods to engineer small molecule binding sites into heterodimeric protein-protein interfaces such that the protein-protein interaction becomes dependent on the small molecule. Each of the two protein partners will be linked to a fragment of a split reporter. A functional sensor then detects the presence of the small molecule by complementation of the reporter. In this fashion, the sensor output is in principle modular: different reporter fragments can be attached to the small molecule sensor components and tested for either detection (via for example split-GFP) or actuation (split-enzymes or gene expression). Our novel contribution will be to develop a design approach that uses a four-step process to transplant small molecule binding sites from liganded protein structures into protein-protein interfaces: (1) Given a small molecule target, we define geometric constraints for a binding "motif" (generally parts of protein side chains) that coordinates the target in a liganded protein monomer structure. (2) We search >600 heterodimeric protein-protein interfaces ("scaffolds") for those that could accommodate the target and its binding motif. (3) We computationally remodel and design the scaffold interface around the target-binding site to stabilize the motif residues in their binding conformation, using robotics-inspired approaches we recently developed. (4) We predict libraries of sensor sequences and test these for dimerization only in the presence of the small molecule using in vitro binding assays and in bacteria using the biosensor output. Computer-aided design of modular small-molecule induced dimerization would be a first. Our experimental plan seeks to ameliorate the risk inherent in this design-driven project in several ways: (i) Moderate target binding affinity is intended to be pre-programmed into the interface using a motif- directed approach that has recently led to successful design of new enzymes. (ii) Computation is complemented with experimental testing of not only the top predictions, but libraries of sequences. (iii) Testing these libraries is facilitated by the biosensor platform, where the intended output itself will be used to screen, select and improve sensors. While small-molecule induced protein dimerization, such as in the rapamycin system, exists in nature and has been reengineered, these systems are limited to a few molecules that can be sensed. Successful completion of this project would demonstrate a platform technology that could greatly broaden application of small-molecule sensing and actuation by providing a route to modular biosensors for many targets. PUBLIC HEALTH RELEVANCE: This research develops new technologies to engineer sensors made out of biological components that allow real-time detection of molecules in living cells and organisms. Such new biosensors have many practical applications in biomedical research and biological engineering, and will also help to advance our understanding of fundamental cellular processes by monitoring molecules in health and disease states. The software and technology developed in this project will be widely available to researchers to improve biosensor design.