The sensitivity and specificity of a sensor are decidedly dependent upon high-affinity, specific molecular recognition between the recognition element, i.e. affinity probe and desired analyte. Analogous to man-made sensors, NLR proteins of the innate immune system have a tri-partite architecture where the recognition, transducer, and readout elements are easily identifiable. Previous studies of NLRs show that these proteins utilize leucine-rich repeat (LRR) domains to bind a large repertoire of ligands including bacterial cell-wall peptidoglycans, glycolipids, and bacterial RNA. Thus we hypothesized that the LRR motif (i.e., recognition element) of NLRs is especially well poised for development of sensors based on the direct binding of microbial peptidoglycans and nucleic acids. I will present design of the de novo protein scaffold, CLRR, based on the consensus sequence of the LRR domain from the NLR proteins. CLRR protein is a stable, monomeric, and cysteine free scaffold that without any affinity maturation displays micromolar binding to N-acetylated muramyl dipeptide (MDP), a gram-positive and gram-negative bacterial cell wall fragment. Interestingly, CLLR is a specific binder for the physiologically relevant L-D isomer of MDP, and does not bind D-D MDP or N-glycolylated MDP. Discovery of such strict discrimination between similar ligands prompts us to propose that CLLR scaffold is promising candidate for further design of microbial sensors.

Microbial sensing – learning from the innate immunity receptors

253rd National Meeting (2017)

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