Srisantitham Suppachai, Walker Alyssa L, Markel Ulrich, Tezcan F Akif
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093, United States.
J Am Chem Soc. 2025 Apr 9;147(14):12338-12346. doi: 10.1021/jacs.5c03319. Epub 2025 Mar 26.
Isopeptide bonds (IPBs)─formed between the amine group of a Lys residue and the carboxamide/carboxy group of Asn/Gln or Asp/Glu─play essential roles in many biological processes, ranging from cellular signaling and regulation to blood clotting and bacterial pathogenesis. The formation of IPBs is not a spontaneous process and requires enzymatic machinery that provides a specialized active site environment to enable this challenging catalytic reaction. Here we report the de novo design and characterization of two proteins (dnIPB-1 and dnIPB-2) capable of autocatalytic IPB formation. While these designed proteins preserve the key active-site residues of their structural template (the bacterial pilin protein RrgA), they possess less than 31% sequence identity to RrgA. Extensive structural and Ala-scanning analyses indicate that IPB formation requires a solvent-protected core motif composed of several critical residues, yet there is also a large tolerance to different protein topologies and overall protein sizes in terms of accommodating an IPB-forming motif. Notably, the structural insights gained from the study of dnIPB-1 and dnIPB-2 also guided the redesign of an initially failed construct (dnIPB-3) and enabled it to form an IPB, highlighting the value of de novo design in examining sequence-structure-function relationships not explored in natural evolution. Our study highlights the versatility of IPBs as designable elements which can be used to construct functional proteins or protein-based materials with enhanced chemical, thermal, and mechanical stabilities.
异肽键（IPB）——在赖氨酸（Lys）残基的胺基与天冬酰胺/谷氨酰胺（Asn/Gln）或天冬氨酸/谷氨酸（Asp/Glu）的羧酰胺/羧基之间形成——在许多生物过程中发挥着重要作用，从细胞信号传导和调节到血液凝固和细菌致病机制。IPB的形成不是一个自发过程，需要酶机制提供一个特殊的活性位点环境来促成这种具有挑战性的催化反应。在此，我们报告了两种能够自催化形成IPB的蛋白质（dnIPB-1和dnIPB-2）的从头设计和表征。虽然这些设计的蛋白质保留了其结构模板（细菌菌毛蛋白RrgA）的关键活性位点残基，但它们与RrgA的序列同一性不到31%。广泛的结构和丙氨酸扫描分析表明，IPB的形成需要一个由几个关键残基组成的溶剂保护核心基序，然而，在容纳一个形成IPB的基序方面，对于不同的蛋白质拓扑结构和整体蛋白质大小也有很大的耐受性。值得注意的是，从对dnIPB-1和dnIPB-2的研究中获得的结构见解也指导了最初失败的构建体（dnIPB-3）的重新设计，并使其能够形成IPB，突出了从头设计在研究自然进化中未探索的序列-结构-功能关系方面的价值。我们的研究突出了IPB作为可设计元件的多功能性，其可用于构建具有增强的化学、热和机械稳定性的功能性蛋白质或蛋白质基材料。
