Designed protein self-assembly by autocatalytic isopeptide bond formation

由于其结构的多样性，蛋白质在生命系统中发挥着多种功能，从催化作用到作为动态材料和分子机器组装的自然构建块。受这些自然例子的启发，研究人员一直在寻找方法，按照不同的非共价和共价组装策略，设计从离散寡聚结构到高阶结构（例如，1D纤维、2D阵列和3D晶格）的蛋白质组装。异肽键（IPB）是一个特别有趣的例子，说明大自然如何利用共价键来构建更高阶的蛋白质结构，并使它们具有非凡的稳定性。然而，这些天然的ipb形成系统仅限于少数蛋白质类。以前，这些天然ipb形成系统的工程变体已被用作基因融合标签来驱动自催化蛋白质组装。但这些系统来自致病菌，总是留下分子疤痕，这可能在潜在的治疗应用中引发不必要的免疫反应。因此，能够合理地将自催化IPB设计成选择的蛋白质-蛋白质界面将是非常可取的。该项目旨在i)合理化与天然IPB形成系统结构无关的蛋白质的从头设计IPB， ii)利用IPB的从头设计组装离散和高阶蛋白质组装。为此，同二聚体四螺旋束蛋白Rop将被用作原理证明。四螺旋结构代表了一种常见的基序，通常是独立观察到的，是较大蛋白质折叠的亚基，在结构上与任何天然ipb形成系统无关。计算设计和定向进化的结合方法将用于建立具有不同IPB数量的同源和异二聚体蛋白质组装，从而提供可调的组装稳定水平。使用这些离散组件作为构建块，IPB形成将用于构建具有高水平共价互联性的高阶结构以及1D组件（即线性蛋白质聚合物），这两种组件都有望具有卓越的结构稳定性。最后，在项目初始阶段建立的IPB设计原则将被转移到其他蛋白质上，以测试该方法的普遍性。这可能会带来有趣的新应用。例如，通过将IPB的结构稳定特性与正交和可切换的蛋白质组装策略（例如，金属模板组装或二硫键）相结合，可以设想稳定但具有潜在治疗相关性的刺激响应纳米结构。总的来说，该项目将展示IPB的从头设计，作为建立结构稳定共价相互作用的新手段，这将推动下一代蛋白质组装。

Owing to their structural diversity, proteins perform a variety of functions in living systems from catalysis to serving as nature’s building block for the assembly of dynamic materials and molecular machines. Inspired by these natural examples, researchers have sought ways to design protein assemblies from discrete oligomeric structures to higher-order structures (e.g., 1D fibers, 2D arrays, and 3D lattices) following different non-covalent and covalent assembly strategies. Isopeptide bonds (IPB) are a particularly intriguing example of how nature utilizes covalent bonds to build higher-order protein structures and equip them with exceptional stability. Yet, these natural IPB-forming systems are limited to few protein classes. Previously, engineered variants of these natural IPB-forming systems have been used as genetic fusion tags to drive autocatalytic protein assembly. But these systems are derived from pathogenic bacteria and always leave a molecular scar, which can prompt unwanted immune responses in potential therapeutic applications. Being able to rationally design autocatalytic IPB into a protein-protein interface of choice would thus be highly desirable. This project aims to i) rationalize de novo IPB design in proteins that are structurally unrelated to natural IPB-forming systems and ii) utilize the de novo design of IPB for the assembly of discrete and higher-order protein assemblies. To this end, the homodimeric four-helix bundle protein Rop will be used as proof-of-principle. The four-helix architecture represents a common motif that is often observed independently and as a subunit of larger protein folds and is structurally unrelated to any natural IPB-forming system. A combined approach of computational design and directed evolution will be used to establish homo- and heterodimeric protein assemblies with different numbers of IPB, thereby providing tunable levels of assembly stabilization. Using these discrete assemblies as building blocks, IPB formation will be used to build higher-order structures with high levels of covalent interconnectivity as well as 1D assemblies (i.e., linear protein polymers), both of which are expected to possess exceptional structural stability. Finally, the IPB design principles established in the initial phase of the project will be transferred to other proteins to test the generality of the approach. This could lead to interesting new applications. For instance, by combining the structure-stabilizing features of IPB with orthogonal and switchable protein assembly strategies (e.g., metal-templated assembly or disulfide bonds), stable but stimuli responsive nanostructures with potential therapeutic relevance are envisioned. Overall, the proposed project will demonstrate the de novo design of IPB as a novel means to establish structure-stabilizing covalent interactions that will drive the next generation of protein assemblies.