Establishing strategies for sortase-catalyzed multi-peptide assemblies to profile T-cell selectivity

细菌转肽酶分选酶A通过催化多肽、蛋白质和合成探针的化学选择性连接反应而成为蛋白质化学的多功能工具。在催化过程中，分选酶在苏氨酸残基上切割一个由氨基酸序列LPxTG或LPxTA组成的所谓分选基序，同时形成一个分选酶结合的硫酯。通过将硫酯连接到作为亲核试剂的第二肽的甘氨酸残基上，使酶去酰化。虽然金黄色葡萄球菌的排序酶A在生物化学和化学生物学中被广泛应用，但由于每个排序基序和亲核试剂都被排序酶识别，因此多肽的组装受到限制，从而导致片段洗牌和聚合作为片段数量增加时的明显副反应。我们计划通过建立可切换的连接位点来解决这些问题。排序基序和/或N-Gly亲核试剂将被安装在激活的导通状态，允许排序酶介导的连接，或非激活的关闭状态。简单的化学转换将允许在状态之间切换，并启用多片段组装。这些发展将与排序酶选择性和活性的定向进化携手并进，旨在(a)设计针对连接位点切换方法的排序酶突变体，以及(b)产生具有正交底物选择性的新排序酶。我们计划使用这些新工具来建立量身定制的免疫学探针，特别是用于研究t细胞活化。配备生物物理探针的抗原肽四聚体将通过上述方法组装。组装抗原四聚体的亲和效应将用于随后与MHCII分子和t细胞的正交连接。这些工具应该对研究t细胞活化和追踪感染或自身免疫性疾病模型中的罕见t细胞群有用。

The bacterial transpeptidase sortase A has become a versatile tool for protein chemistry by catalyzing chemoselective ligation reactions of peptides, proteins and synthetic probes. During catalysis sortase cleaves a so-called sorting motif consisting of the amino acid sequence LPxTG or LPxTA at the threonine residue under concomitant formation of a sortase-bound thioester. The enzyme is deacylated by ligating the thioester to the glycine residue of a second peptide that serves as nucleophile. While sortase A of S. aureus is widely used in biochemistry and chemical biology the assembly of multiple peptides is limited by the fact that each sorting motif and nucleophile is recognized by sortase, thereby leading to fragment shuffling and polymerization as a manifesting side reaction when fragment numbers increase. We plan to address these problems by establishing switchable ligation sites. Sorting motif and/or N-Gly nucleophile will be installed in either active on-states, allowing sortase-mediated ligation, or inactive off-state. Simple chemical conversions will allow switching between states and enable multi-fragment assemblies. These developments will go hand-in-hand with directed evolution of sortase selectivity and activity aimed to (a) engineer sortase mutants specific for ligation site switching approaches as well as (b) to generate new sortases with orthogonal substrate selectivity. We plan to use these new tools for establishing tailor-made probes for immunology, in particular for the investigation of T-cell activation. Antigen peptide tetramers equipped with biophysical probes will be assembled by the methods described above. Avidity effects of the assembled antigen tetramers will be exploited for subsequent orthogonal ligation with MHCII molecules and T-cells. These tools should be useful to study T-cell activation and for tracking rare T-cell populations in infection or autoimmune disease models.