The NLRP12 pyrin domain: structure, dynamics, and functional insights

The initial line of defense against infection is sustained by the innate immune system. Together, membrane-bound Toll-like receptors and cytosolic nucleotide-binding domain and leucine-rich repeat-containing receptors (NLR) play key roles in the innate immune response by detecting bacterial and viral invaders as well as endogenous stress signals. NLRs are multi-domain proteins with varying N-terminal effector domains that are responsible for regulating downstream signaling events. Here, we report the structure and dynamics of the N-terminal pyrin domain of NLRP12 (NLRP12 PYD) determined using NMR spectroscopy. NLRP12 is a non-inflammasome NLR that has been implicated in the regulation of Toll-like receptor-dependent nuclear factor-κB activation. NLRP12 PYD adopts a typical six-helical bundle death domain fold. By direct comparison with other PYD structures, we identified hydrophobic residues that are essential for the stable fold of the NLRP PYD family. In addition, we report the first in vitro confirmed non-homotypic PYD interaction between NLRP12 PYD and the pro-apoptotic protein Fas-associated factor 1 (FAF-1), which links the innate immune system to apoptotic signaling. Interestingly, all residues that participate in this protein:protein interaction are confined to the α2-α3 surface, a region of NLRP12 PYD that differs most between currently reported NLRP PYD structures. Finally, we experimentally highlight a significant role for tryptophan 45 in the interaction between NLRP12 PYD and the FAF-1 UBA domain.     

Mechanisms of NLRP1-Mediated Autoinflammatory Disease in Humans and Mice

NLRP1 was the first NOD-like receptor described to form an inflammasome, recruiting ASC to activate caspase-1, which processes interleukin-1β and interleukin-18 to their active form. A wealth of new genetic information has now redefined our understanding of this innate immune sensor. Specifically, rare loss-of-function variants in the N-terminal pyrin domain indicate that this part of NLRP1 is autoinhibitory and normally acts to prevent a familial autoinflammatory skin disease associated with cancer. In the absence of a ligand to trigger human NLRP1, these mutations have now confirmed the requirement of NLRP1 autolytic cleavage within the FIIND domain, which had previously been implicated in NLRP1 activation. Autolytic cleavage generates a C-terminal fragment of NLRP1 containing the CARD domain which then forms an ASC-dependent inflammasome. The CARD domain as an inflammasome linker is consistent with the observation that under some conditions, particularly for mouse NLRP1, caspase-1 can be engaged directly, and although it is no longer processed, it is still capable of producing mature IL-1β. Additional rare variants in a linker region between the LRR and FIIND domains of NLRP1 also cause autoinflammatory disease in both humans and mice. This new genetic information is likely to provide for more mechanistic insight in the years to come, contributing to our understanding of how NLRP1 functions as an innate immune sensor of infection and predisposes to autoimmune or autoinflammatory diseases.     

Designed leucine‐rich repeat proteins bind two muramyl dipeptide ligands

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 K _{d 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 K _d1 values ranging from 0.04 to 4.5 μM, and lower affinity K _d2 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.     

Structure of the NLRP1 caspase recruitment domain suggests potential mechanisms for its association with procaspase‐1

The NLRP1 inflammasome responds to microbial challenges such as Bacillus anthracis infection and is implicated in autoimmune disease such as vitiligo. Human NLRP1 contains both an N‐terminal pyrin domain (PYD) and a C‐terminal caspase recruitment domain (CARD), with the latter being essential for its association with the downstream effector procaspase‐1. Here we report a 2.0 Å crystal structure of the human NLRP1 CARD as a fusion with the maltose‐binding protein. The structure reveals the six‐helix bundle fold of the NLRP1 CARD, typical of the death domain superfamily. The charge surface of the NLRP1 CARD structure and a procaspase‐1 CARD model suggests potential mechanisms for their association through electrostatic attraction. Proteins 2013; 81:1266–1270. © 2013 Wiley Periodicals, Inc.     

Three-dimensional Structure of the NLRP7 Pyrin Domain: INSIGHT INTO PYRIN-PYRIN-MEDIATED EFFECTOR DOMAIN SIGNALING IN INNATE IMMUNITY*

The innate immune system provides an initial line of defense against infection. Nucleotide-binding domain- and leucine-rich repeat-containing protein (NLR or (NOD-like)) receptors play a critical role in the innate immune response by surveying the cytoplasm for traces of intracellular invaders and endogenous stress signals. NLRs themselves are multi-domain proteins. Their N-terminal effector domains (typically a pyrin or caspase activation and recruitment domain) are responsible for driving downstream signaling and initiating the formation of inflammasomes, multi-component complexes necessary for cytokine activation. However, the currently available structures of NLR effector domains have not yet revealed the mechanism of their differential modes of interaction. Here, we report the structure and dynamics of the N-terminal pyrin domain of NLRP7 (NLRP7 PYD) obtained by NMR spectroscopy. The NLRP7 PYD adopts a six-α-helix bundle death domain fold. A comparison of conformational and dynamics features of the NLRP7 PYD with other PYDs showed distinct differences for helix α3 and loop α2-α3, which, in NLRP7, is stabilized by a strong hydrophobic cluster. Moreover, the NLRP7 and NLRP1 PYDs have different electrostatic surfaces. This is significant, because death domain signaling is driven by electrostatic contacts and stabilized by hydrophobic interactions. Thus, these results provide new insights into NLRP signaling and provide a first molecular understanding of inflammasome formation.     

The activity of the RGA5 sensor NLR from rice requires binding of its integrated HMA domain to effectors but not HMA domain self-interaction

The rice nucleotide-binding (NB) and leucine-rich repeat (LRR) domain immune receptors (NLRs) RGA4 and RGA5 form a helper NLR/sensor NLR (hNLR/sNLR) pair that specifically recognizes the effectors AVR-Pia and AVR1-CO39 from the blast fungus Magnaporthe oryzae. While RGA4 contains only canonical NLR domains, RGA5 has an additional unconventional heavy metal-associated (HMA) domain integrated after its LRR domain. This RGA5_HMA domain binds the effectors and is crucial for their recognition. Investigation of the three-dimensional structure of the AVR1-CO39/RGA5_HMA complex by X-ray crystallography identified a candidate surface for effector binding in the HMA domain and showed that the HMA domain self-interacts in the absence of effector through the same surface. Here, we investigated the relevance of this HMA homodimerization for RGA5 function and the role of the RGA5_HMA effector-binding and self-interaction surface in effector recognition. By analysing structure-informed point mutations in the RGA5_HMA-binding surface in protein interaction studies and in Nicotiana benthamiana cell death assays, we found that HMA self-interaction does not contribute to RGA5 function. However, the effector-binding surface of RGA5_HMA identified by X-ray crystallography is crucial for both in vitro and in vivo effector binding as well as effector recognition. These results support the current hypothesis that noncanonical integrated domains of NLRs act primarily as effector traps and deepen our understanding of the sNLRs' function within NLR pairs.     

Zinc depletion regulates the processing and secretion of IL-1β

Sterile inflammation contributes to many common and serious human diseases. The pro-inflammatory cytokine interleukin-1β (IL-1β) drives sterile inflammatory responses and is thus a very attractive therapeutic target. Activation of IL-1β in sterile diseases commonly requires an intracellular multi-protein complex called the NLRP3 (NACHT, LRR, and PYD domains-containing protein 3) inflammasome. A number of disease-associated danger molecules are known to activate the NLRP3 inflammasome. We show here that depletion of zinc from macrophages, a paradigm for zinc deficiency, also activates the NLRP3 inflammasome and induces IL-1β secretion. Our data suggest that zinc depletion damages the integrity of lysosomes and that this event is important for NLRP3 activation. These data provide new mechanistic insight to how zinc deficiency contributes to inflammation and further unravel the mechanisms of NLRP3 inflammasome activation.     

Crystal Structure of the F27G AIM2 PYD Mutant and Similarities of Its Self-Association to DED/DED Interactions

Absent in melanoma 2 (AIM2) is a cytoplasmic double-stranded DNA sensor involved in innate immunity. It uses its C-terminal HIN domain for recognizing double-stranded DNA and its N-terminal pyrin domain (PYD) for eliciting downstream effects through recruitment and activation of apoptosis-associated Speck-like protein containing CARD (ASC). ASC in turn recruits caspase-1 and/or caspase-11 to form the AIM2 inflammasome. The activated caspases process proinflammatory cytokines IL-1β and IL-18 and induce the inflammatory form of cell death pyroptosis. Here we show that AIM PYD (AIM2^PYD) self-oligomerizes. We notice significant sequence homology of AIM2^PYD with the hydrophobic patches of death effector domain (DED)-containing proteins and confirm that mutations on these residues disrupt AIM2^PYD self-association. The crystal structure at 1.82 Å resolution of such a mutant, F27G of AIM2^PYD, shows the canonical six-helix (H1–H6) bundle fold in the death domain superfamily. In contrast to the wild-type AIM2^PYD structure crystallized in fusion with the large maltose-binding protein tag, the H2–H3 region of the AIM2^PYD F27G is well defined with low B-factors. Structural analysis shows that the conserved hydrophobic patches engage in a type I interaction that has been observed in DED/DED and other death domain superfamily interactions. While previous mutagenesis studies of PYDs point to the involvement of charged interactions, our results reveal the importance of hydrophobic interactions in the same interfaces. These centrally localized hydrophobic residues within fairly charged patches may form the hot spots in AIM2^PYD self-association and may represent a common mode of PYD/PYD interactions in general.     

Crystal structure and role of glycans and dimerization in folding of neuronal leucine-rich repeat protein AMIGO-1

AMIGO-1 is the parent member of a novel family of three cell surface leucine-rich repeat (LRR) proteins. Its expression is induced by the binding of HMGB1 (high-mobility group box 1 protein) to RAGE (receptor for advanced glycation end products) on neurons. Binding of HMGB1 to RAGE is known to have a direct effect on cellular growth regulation and mobility, and AMIGO-1 directly supports growth of neuronal processes and fasciculation of neurites. In addition, the second member of the AMIGO-family, AMIGO-2, has been implicated in adhesion of tumor cells in adenocarcinoma and survival of neurons.We have determined the crystal structure of AMIGO-1 at 2.0 Å resolution, which reveals a typical cell surface LRR domain arrangement with N- and C-terminal capping domains with disulfide bridges, followed by a C2-type Ig domain. AMIGO-1 is a dimer, with the LRR regions forming the dimer interface, and sequence conservation analysis and static light-scattering measurements suggest that all three AMIGO family proteins form similar dimers. Based on the AMIGO-1 structure, we have also modeled AMIGO-2 and present small-angle X-ray scattering data on AMIGO-2 and AMIGO-3. Our mutagenesis studies show that AMIGO-1 dimerization is necessary for proper cell surface expression and thus probably for proper or stable folding in the endoplastic reticulum and for the function of the protein. Based on the data presented earlier, we also suggest that dimerization through the LRR-LRR interface is likely to be involved in cell-cell adhesion by AMIGO-1, while extensive glycosylation may have a role.     

LRRC4通过LRR结构域抑制脑胶质母细胞瘤U251细胞的生长和侵袭

LRRC4基因是候选的脑胶质瘤抑制基因,其细胞外区域含有一个保守的LRR和一个IgC2 结构域.本研究结合生物信息学分析,通过一步PCR法构建了不同结构域缺失的突变体（pc ΔLRR, pcΔIgC2或pcΔTm）.并将全长LRRC4（pcLRRC4）和各突变体转染至U251细胞,构建了稳定表达的U251细胞系.通过MTT,软琼脂集落形成及Transwell体外侵袭模型检测发现,全长LRRC4能够抑制U251细胞的生长和侵袭;LRR结构域缺失的突变体不再抑制U251细胞的生长和侵袭;而IgC2或Tm区缺失的突变体仍然可以抑制U251细胞的生长和侵袭.该结果表明,LRRC4抑制U251细胞的生长和侵袭依赖于它的LRR结构域,而不是IgC2或Tm结构域.     

Roles of NOD1 (NLRC1) and NOD2 (NLRC2) in innate immunity and inflammatory diseases

NOD1 {nucleotide-binding oligomerization domain 1; NLRC [NOD-LRR (leucine-rich repeat) family with CARD (caspase recruitment domain) 1]} and NOD2 (NLRC2) are among the most prominent members of the NLR (NOD-LRR) family –proteins that contain nucleotide-binding NACHT domains and receptor-like LRR domains. With over 20 members identified in humans, NLRs represent important components of the mammalian innate immune system, serving as intracellular receptors for pathogens and for endogenous molecules elaborated by tissue injury. NOD1 and NOD2 proteins operate as microbial sensors through the recognition of specific PG (peptidoglycan) constituents of bacteria. Upon activation, these NLR family members initiate signal transduction mechanisms that include stimulation of NF-κB (nuclear factor-κB), stress kinases, IRFs (interferon regulatory factors) and autophagy. Hereditary polymorphisms in the genes encoding NOD1 and NOD2 have been associated with an increasing number of chronic inflammatory diseases. In fact, potential roles for NOD1 and NOD2 in inflammatory disorders have been revealed by investigations using a series of animal models. In the present review, we describe recent experimental findings associating NOD1 and NOD2 with various autoimmune and chronic inflammatory disorders, and we discuss prospects for development of novel therapeutics targeting these NLR family proteins.     

Structural insights into the MDP binding and CARD–CARD interaction in zebrafish (Danio rerio) NOD2: a molecular dynamics approach

Nucleotide binding and oligomerization domain (NOD2) is a key component of innate immunity that is highly specific for muramyl dipeptide (MDP)—a peptidoglycan component of bacterial cell wall. MDP recognition by NOD2–leucine rich repeat (LRR) domain activates NF‐κB signaling through a protein–protein interaction between caspase activating and recruitment domains (CARDs) of NOD2 and downstream receptor interacting and activating protein kinase 2 (RIP2). Due to the lack of crystal/NMR structures, MDP recognition and CARD–CARD interaction are poorly understood. Herein, we have predicted the probable MDP and CARD–CARD binding surfaces in zebrafish NOD2 (zNOD2) using various in silico methodologies. The results show that the conserved residues Phe819, Phe871, Trp875, Trp929, Trp899, and Arg845 located at the concave face of zNOD2–LRR confer MDP recognition by hydrophobic and hydrogen bond (H‐bond) interactions. Molecular dynamics simulations reveal a stable association between the electropositive surface on zNOD2–CARDa and the electronegative surface on zRIP2–CARD reinforced mostly by H‐bonds and electrostatic interactions. Importantly, a 3.5 Å salt bridge is observed between Arg60 of zNOD2–CARDa and Asp494 of zRIP2–CARD. Arg11 and Lys53 of zNOD2–CARDa and Ser498 and Glu508 of zRIP2–CARD are critical residues for CARD–CARD interaction and NOD2 signaling. The 2.7 Å H‐bond between Lys104 of the linker and Glu508 of zRIP2–CARD suggests a possible role of the linker for shaping CARD–CARD interaction. These findings are consistent with existing mutagenesis data. We provide first insight into MDP recognition and CARD–CARD interaction in the zebrafish that will be useful to understand the molecular basis of NOD signaling in a broader perspective. Copyright © 2014 John Wiley & Sons, Ltd.     

Crystal structure and standardized geometric analysis of InlJ, a listerial virulence factor and leucine-rich repeat protein with a novel cysteine ladder

We report on the crystal structure of the internalin domain of InlJ, a virulence-associated surface protein of Listeria monocytogenes, at 2.7-Å resolution. InlJ is a member of the internalin family of listerial cell surface proteins characterized by a common N-terminal domain. InlJ bears 15 leucine-rich repeats (LRRs), the same number as in InlA, the prototypical internalin family member. The LRRs of InlJ differ from those of other internalins by having 21, rather than 22, residues and by replacing 1 LRR-defining hydrophobic residue with a conserved cysteine. These cysteines stack to form an intramolecular ladder and regular hydrophobic interactions in consecutive repeats. Analyzing the curvature, twist, and lateral bending angles of InlJ and comparing these with several other LRR proteins, we provide a systematic geometric comparison of LRR protein structures (http://bragi2.helmholtz-hzi.de/Angulator/). These indicate that both cysteine and asparagine ladders stabilize the LRR fold, whereas substitutions in some repeat positions are more likely than others to induce changes in LRR geometry.     

Green tea polyphenol epigallocatechin-3-gallate suppresses melanoma growth by inhibiting inflammasome and IL-1β secretion

Epigallocatechin-3-gallate (EGCG), the major polyphenolic component of green tea, has been demonstrated to possess anti-inflammatory, antioxidant, anti-mutagenic and anti-carcinogenic properties. The anti-melanoma effect of EGCG has been previously suggested, but no clear mechanism of action has been established. In this study, we demonstrated that EGCG inhibits melanoma cell growth at physiological doses (0.1–1 μM). In the search for mechanisms of EGCG-mediated melanoma cell suppression, we found that NF-κB was inhibited, and that reduced NF-κB activity was associated with decreased IL-1β secretion from melanoma cells. Since inflammasomes are involved in IL-1β secretion, we investigated whether IL-1β suppression was mediated by inflammasomes, and found that EGCG treatment led to downregulation of the inflammasome component, NLRP1, and reduced caspase-1 activation. Furthermore, silencing the expression of NLRP1 abolished EGCG-induced inhibition of tumor cell proliferation both in vitro and in vivo, suggesting a key role of inflammasomes in EGCG efficacy. This paper provides a novel mechanism for EGCG-induced melanoma inhibition: inflammasome downregulation → decreased IL-1β secretion → decreased NF-κB activities → decreased cell growth. In addition, it suggests inflammasomes and IL-1β could be potential targets for future melanoma therapeutics.     

Promyelocytic Leukemia Protein Interacts with the Apoptosis-associated Speck-like Protein to Limit Inflammasome Activation

The apoptosis-associated speck-like protein containing a caspase-activating recruitment domain (ASC) is an essential component of several inflammasomes, multiprotein complexes that regulate caspase-1 activation and inflammation. We report here an interaction between promyelocytic leukemia protein (PML) and ASC. We observed enhanced formation of ASC dimers in PML-deficient macrophages. These macrophages also display enhanced levels of ASC in the cytosol. Furthermore, IL-1β production was markedly enhanced in these macrophages in response to both NLRP3 and AIM2 inflammasome activation and following bone marrow-derived macrophage infection with herpes simplex virus-1 (HSV-1) and Salmonella typhimurium. Collectively, our data indicate that PML limits ASC function, retaining ASC in the nucleus.     

A Pocket on the Surface of the N-Terminal BRCT Domain of Mcph1 Is Required to Prevent Abnormal Chromosome Condensation

Mcph1 is mutated in autosomal recessive primary microcephaly and premature chromosome condensation (PCC) syndrome. Increased chromosome condensation is a common feature of cells isolated from patients afflicted with either disease. Normal cells depleted of Mcph1 also exhibit PCC phenotype. Human Mcph1 contains three BRCA1-carboxyl terminal (BRCT) domains, the first of which (Mcph1N) is necessary for the prevention of PCC. The only known disease-associated missense mutation in Mcph1 resides in this domain (T27R). We have determined the X-ray crystal structure of human Mcph1N to 1.6 Å resolution. Compared with other BRCT domain structures, the most striking differences are an elongated, ordered β1-α1 loop and an adjacent hydrophobic pocket. This pocket is in the equivalent structural position to the phosphate binding site of BRCT domains that recognize phospho-proteins, although the phosphate-binding residues are absent in Mcph1N. Mutations in the pocket abrogate the ability of full-length Mcph1 to rescue the PCC phenotype of Mcph1^−/− mouse embryonic fibroblast cells, suggesting that it forms an essential part of a protein-protein interaction site necessary to prevent PCC.     

3,4-Methylenedioxy-β-nitrostyrene Inhibits NLRP3 Inflammasome Activation by Blocking Assembly of the Inflammasome

The NLRP3 inflammasome is a critical component of the innate immune system. NLRP3 activation is induced by diverse stimuli associated with bacterial infection or tissue damage, but its inappropriate activation is involved in the pathogenesis of inherited and acquired inflammatory diseases. However, the mechanism by which NLRP3 is activated remains poorly understood. In this study, we explored the role of kinases in NLRP3 inflammasome activation by screening a kinase inhibitor library and identified 3,4-methylenedioxy-β-nitrostyrene (MNS) as an inhibitor for NLRP3 inflammasome activation. Notably, MNS did not affect the activation of the NLRC4 or AIM2 (absent in melanoma 2) inflammasome. Mechanistically, MNS specifically prevented NLRP3-mediated ASC speck formation and oligomerization without blocking potassium efflux induced by NLRP3 agonists. Surprisingly, Syk kinase, the reported target of MNS, did not mediate the inhibitory activity of MNS on NLRP3 inflammasome activation. We also found that the nitrovinyl group of MNS is essential for the inhibitory activity of MNS. Immunoprecipitation, mass spectrometry, and mutation studies suggest that both the nucleotide binding oligomerization domain and the leucine-rich repeat domain of NLRP3 were the intracellular targets of MNS. Administration of MNS also inhibited NLRP3 ATPase activity in vitro, suggesting that MNS blocks the NLRP3 inflammasome by directly targeting NLRP3 or NLRP3-associated complexes. These studies identified a novel chemical probe for studying the molecular mechanism of NLRP3 inflammasome activation which may advance the development of novel strategies to treat diseases associated with abnormal activation of NLRP3 inflammasome.     

The leucine-rich repeat domains of BK channel auxiliary γ subunits regulate their expression,trafficking, and channel-modulation functions

As high-conductance calcium- and voltage-dependent potassium channels, BK channels consist of pore-forming, voltage-, and Ca²⁺-sensing α and auxiliary subunits. The leucine-rich repeat (LRR) domain–containing auxiliary γ subunits potently modulate the voltage dependence of BK channel activation. Despite their dominant size in whole protein masses, the function of the LRR domain in BK channel γ subunits is unknown. We here investigated the function of these LRR domains in BK channel modulation by the auxiliary γ1–3 (LRRC26, LRRC52, and LRRC55) subunits. Using cell surface protein immunoprecipitation, we validated the predicted extracellular localization of the LRR domains. We then refined the structural models of mature proteins on the membrane via molecular dynamic simulations. By replacement of the LRR domain with extracellular regions or domains of non-LRR proteins, we found that the LRR domain is nonessential for the maximal channel-gating modulatory effect but is necessary for the all-or-none phenomenon of BK channel modulation by the γ1 subunit. Mutational and enzymatic blockade of N-glycosylation in the γ1–3 subunits resulted in a reduction or loss of BK channel modulation by γ subunits. Finally, by analyzing their expression in whole cells and on the plasma membrane, we found that blockade of N-glycosylation drastically reduced total expression of the γ2 subunit and the cell surface expression of the γ1 and γ3 subunits. We conclude that the LRR domains play key roles in the regulation of the expression, cell surface trafficking, and channel-modulation functions of the BK channel γ subunits.     

Structural basis for recognition of an endogenous peptide by the plant receptor kinase PEPR1

The endogenous peptides AtPep1-8 in Arabidopsis mature from the conserved C-terminal portions of their precursor proteins PROPEP1-8, respectively. The two homologous leucine-rich repeat-receptor kinases (LRR-RKs) PEPR1 and PEPR2 act as receptors of AtPeps. AtPep binding leads to stable association of PEPR1,2 with the shared receptor LRR-RK BAK1, eliciting immune responses similar to those induced by pathogens. Here we report a crystal structure of the extracellular LRR domain of PEPR1 (PEPR1LRR) in complex with AtPep1. The structure reveals that AtPep1 adopts a fully extended conformation and binds to the inner surface of the superhelical PEPR1LRR. Biochemical assays showed that AtPep1 is capable of inducing PEPR1LRR-BAK1LRR heterodimerization. The conserved C-terminal portion of AtPep1 dominates AtPep1 binding to PEPR1LRR, with the last amino acid of AtPep1 Asn23 forming extensive interactions with PEPR1LRR. Deletion of the last residue of AtPep1 significantly compromised AtPep1 interaction with PEPR1LRR. Together, our data reveal a conserved structural mechanism of AtPep1 recognition by PEPR1, providing significant insight into prediction of recognition of other peptides by their cognate LRR-RKs.