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Consensus design of a NOD receptor leucine rich repeat domain with binding affinity for a muramyl dipeptide, a bacterial cell wall fragment.

ABSTRACT: Repeat proteins have recently emerged as especially well-suited alternative binding scaffolds due to their modular architecture and biophysical properties. Here we present the design of a scaffold based on the consensus sequence of the leucine rich repeat (LRR) domain of the NOD family of cytoplasmic innate immune system receptors. Consensus sequence design has emerged as a protein design tool to create de novo proteins that capture sequence-structure relationships and interactions present in nature. The multiple sequence alignment of 311 individual LRRs, which are the putative ligand-recognition domain in NOD proteins, resulted in a consensus sequence protein containing two internal and N- and C-capping repeats named CLRR2. CLRR2 protein is a stable, monomeric, and cysteine free scaffold that without any affinity maturation displays micromolar binding to muramyl dipeptide, a bacterial cell wall fragment. To our knowledge, this is the first report of direct interaction of a NOD LRR with a physiologically relevant ligand.

SUBMITTER: Parker R

PROVIDER: S-EPMC4093954 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Designed leucine-rich repeat proteins bind two muramyl dipeptide ligands.

Project description:Designed protein receptors hold diagnostic and therapeutic promise. We now report the design of five consensus leucine-rich repeat proteins (CLRR4-8) based on the LRR domain of nucleotide-binding oligomerization domain (NOD)-like receptors involved in the innate immune system. The CLRRs bind muramyl dipeptide (MDP), a bacterial cell wall component, with micromolar affinity. The overall Kd app values ranged from 1.0 to 57 μM as measured by fluorescence quenching experiments. Biphasic fluorescence quenching curves were observed in all CLRRs, with higher affinity Kd1 values ranging from 0.04 to 4.5 μM, and lower affinity Kd2 values ranging from 3.1 to 227 μM. These biphasic binding curves, along with the docking studies of MDP binding to CLRR4, suggest that at least two MDPs bind to each protein. Previously, only single MDP binding was reported. This high-capacity binding of MDP promises small, soluble, stable CLRR scaffolds as candidates for the future design of pathogen biosensors.

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Molecular Recognition of Muramyl Dipeptide Occurs in the Leucine-rich Repeat Domain of Nod2.

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Alternative splicing controls Leucine-rich repeat domain modularity in NOD-like receptors

Project description:Leucine-rich repeat (LRR) domains are evolutionarily conserved in proteins that function in development and immunity. Somatic recombination of LRR sequences evolved to create diversity in jawless vertebrate adaptive immunity, yet the role repetitive LRR-encoding exons in humans remains unknown. We performed this RNA Seq study to understand how alternative splicing of NOD-like receptors (NLRs) at the locus encoding a LRR domain can regulate NLRs function, with a focus on NLRP3.

2019-06-09 | GSE117206 | GEO

Identification and characterization of a leucine-rich repeat kinase 2 (LRRK2) consensus phosphorylation motif.

Project description:Mutations in LRRK2 (leucine-rich repeat kinase 2) have been identified as major genetic determinants of Parkinson's disease (PD). The most prevalent mutation, G2019S, increases LRRK2's kinase activity, therefore understanding the sites and substrates that LRRK2 phosphorylates is critical to understanding its role in disease aetiology. Since the physiological substrates of this kinase are unknown, we set out to reveal potential targets of LRRK2 G2019S by identifying its favored phosphorylation motif. A non-biased screen of an oriented peptide library elucidated F/Y-x-T-x-R/K as the core dependent substrate sequence. Bioinformatic analysis of the consensus phosphorylation motif identified several novel candidate substrates that potentially function in neuronal pathophysiology. Peptides corresponding to the most PD relevant proteins were efficiently phosphorylated by LRRK2 in vitro. Interestingly, the phosphomotif was also identified within LRRK2 itself. Autophosphorylation was detected by mass spectrometry and biochemical means at the only F-x-T-x-R site (Thr 1410) within LRRK2. The relevance of this site was assessed by measuring effects of mutations on autophosphorylation, kinase activity, GTP binding, GTP hydrolysis, and LRRK2 multimerization. These studies indicate that modification of Thr1410 subtly regulates GTP hydrolysis by LRRK2, but with minimal effects on other parameters measured. Together the identification of LRRK2's phosphorylation consensus motif, and the functional consequences of its phosphorylation, provide insights into downstream LRRK2-signaling pathways.

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Comparative Geometrical Analysis of Leucine-Rich Repeat Structures in the Nod-Like and Toll-Like Receptors in Vertebrate Innate Immunity.

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Leucine Rich Repeat Kinase 2 and Innate Immunity.

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C9orf72 arginine-rich dipeptide repeat proteins disrupt karyopherin-mediated nuclear import.

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