Structural diversity of the hagfish variable lymphocyte receptors

Variable lymphocyte receptors (VLRs) are recently discovered leucine-rich repeat (LRR) family proteins that mediate adaptive immune responses in jawless fish. Phylogenetically it is the oldest adaptive immune receptor and the first one with a non-immunoglobulin fold. We present the crystal structures of one VLR-A and two VLR-B clones from the inshore hagfish. The hagfish VLRs have the characteristic horseshoe-shaped structure of LRR family proteins. The backbone structures of their LRR modules are highly homologous, and the sequence variation is concentrated on the concave surface of the protein. The conservation of key residues suggests that our structures are likely to represent the LRR structures of the entire repertoire of jawless fish VLRs. The analysis of sequence variability, prediction of protein interaction surfaces, amino acid composition analysis, and structural comparison with other LRR proteins suggest that the hypervariable concave surface is the most probable antigen binding site of the VLR.     

Crystal structure of the TLR4-MD-2 complex with bound endotoxin antagonist Eritoran

TLR4 and MD-2 form a heterodimer that recognizes LPS (lipopolysaccharide) from Gram-negative bacteria. Eritoran is an analog of LPS that antagonizes its activity by binding to the TLR4-MD-2 complex. We determined the structure of the full-length ectodomain of the mouse TLR4 and MD-2 complex. We also produced a series of hybrids of human TLR4 and hagfish VLR and determined their structures with and without bound MD-2 and Eritoran. TLR4 is an atypical member of the LRR family and is composed of N-terminal, central, and C-terminal domains. The beta sheet of the central domain shows unusually small radii and large twist angles. MD-2 binds to the concave surface of the N-terminal and central domains. The interaction with Eritoran is mediated by a hydrophobic internal pocket in MD-2. Based on structural analysis and mutagenesis experiments on MD-2 and TLR4, we propose a model of TLR4-MD-2 dimerization induced by LPS.     

Toll-Like Receptor 4 Decoy,TOY, Attenuates Gram-Negative Bacterial Sepsis

Lipopolysaccharide (LPS), the Gram-negative bacterial outer membrane glycolipid, induces sepsis through its interaction with myeloid differentiation protein-2 (MD-2) and Toll-like receptor 4 (TLR4). To block interaction between LPS/MD-2 complex and TLR4, we designed and generated soluble fusion proteins capable of binding MD-2, dubbed TLR4 decoy receptor (TOY) using ‘the Hybrid leucine-rich repeats (LRR) technique’. TOY contains the MD-2 binding ectodomain of TLR4, the LRR motif of hagfish variable lymphocyte receptor (VLR), and the Fc domain of IgG1 to make it soluble, productive, and functional. TOY exhibited strong binding to MD-2, but not to the extracellular matrix (ECM), resulting in a favorable pharmacokinetic profile in vivo. TOY significantly extended the lifespan, when administered in either preventive or therapeutic manners, in both the LPS- and cecal ligation/puncture-induced sepsis models in mice. TOY markedly attenuated LPS-triggered NF-κB activation, secretion of proinflammatory cytokines, and thrombus formation in multiple organs. Taken together, the targeting strategy for sequestration of LPS/MD-2 complex using the decoy receptor TOY is effective in treating LPS- and bacteria-induced sepsis; furthermore, the strategy used in TOY development can be applied to the generation of other novel decoy receptor proteins.     

A leucine-rich repeat assembly approach for homology modeling of the human TLR5-10 and mouse TLR11-13 ectodomains

So far, 13 groups of mammalian Toll-like receptors (TLRs) have been identified. Most TLRs have been shown to recognize pathogen-associated molecular patterns from a wide range of invading agents and initiate both innate and adaptive immune responses. The TLR ectodomains are composed of varying numbers and types of leucine-rich repeats (LRRs). As the crystal structures are currently missing for most TLR ligand-binding ectodomains, homology modeling enables first predictions of their three-dimensional structures on the basis of the determined crystal structures of TLR ectodomains. However, the quality of the predicted models that are generated from full-length templates can be limited due to low sequence identity between the target and templates. To obtain better templates for modeling, we have developed an LRR template assembly approach. Individual LRR templates that are locally optimal for the target sequence are assembled into multiple templates. This method was validated through the comparison of a predicted model with the crystal structure of mouse TLR3. With this method, we also constructed ectodomain models of human TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10 and mouse TLR11, TLR12, and TLR13 that can be used as first passes for a computational simulation of ligand docking or to design mutation experiments. This template assembly approach can be extended to other repetitive proteins.     

Assessment of the ability to model proteins with leucine-rich repeats in light of the latest structural information

The three-dimensional structures of leucine-rich repeat (LRR)-containing proteins from five different families were previously predicted based on the crystal structure of the ribonuclease inhibitor, using an approach that combined homology-based modeling, structure-based sequence alignment of LRRs, and several rational assumptions. The structural models have been produced based on very limited sequence similarity, which, in general, cannot yield trustworthy predictions. Recently, the protein structures from three of these five families have been determined. In this report we estimate the quality of the modeling approach by comparing the models with the experimentally determined structures. The comparison suggests that the general architecture, curvature, "interior/exterior" orientations of side chains, and backbone conformation of the LRR structures can be predicted correctly. On the other hand, the analysis revealed that, in some cases, it is difficult to predict correctly the twist of the overall super-helical structure. Taking into consideration the conclusions from these comparisons, we identified a new family of bacterial LRR proteins and present its structural model. The reliability of the LRR protein modeling suggests that it would be informative to apply similar modeling approaches to other classes of solenoid proteins.     

PIRLs: a novel class of plant intracellular leucine-rich repeat proteins

Leucine-rich repeat (LRR) proteins feature tandem leucine-rich motifs that form a protein-protein interaction domain. Plants contain diverse classes of LRR proteins, many of which take part in signal transduction. We have identified a novel family of nine Arabidopsis LRR proteins that, based on predicted intracellular location and LRR motif consensus sequence, are related to Ras-binding LRR proteins found in signaling complexes in animals and yeast. This new class has been named plant intracellular Ras group-related LRR proteins (PIRLs). We have characterized PIRL cDNAs, rigorously defined gene and protein annotations, investigated gene family evolution and surveyed mRNA expression. While LRR regions suggested a relationship to Ras group LRR proteins, outside of their LRR domains PIRLs differed from Ras group proteins, exhibiting N- and C-terminal regions containing low complexity stretches and clusters of charged amino acids. PIRL genes grouped into three subfamilies based on sequence relationships and gene structures. Related gene pairs and dispersed chromosomal locations suggested family expansion by ancestral genomic or segmental duplications. Expression surveys revealed that all PIRL mRNAs are actively transcribed, with three expressed differentially in leaves, roots or flowers. These results define PIRLs as a distinct, plant-specific class of intracellular LRR proteins that probably mediate protein interactions, possibly in the context of signal transduction. T-DNA knock-out mutants have been isolated as a starting point for systematic functional analysis of this intriguing family.     

Sensing bacterial flagellin by membrane and soluble orthologs of Toll-like receptor 5 in rainbow trout (Onchorhynchus mikiss)

Rainbow trout (Onchorhynchus mikiss) possess two genes encoding putative leucine-rich repeat (LRR)-containing proteins similar to human TLR5. Molecular cloning of these two LRR proteins suggested the presence of a TLR5-like membrane form (rtTLR5M) and a soluble form (rtTLR5S). Here we elucidated the primary structures and the unique combinational functions of these fish versions of TLR5. The LRR regions of rtTLR5S and rtTLR5M exhibited 81% homology and relatively high (35.6 and 33.7%) homology to the extracellular domains of human TLR5 (huTLR5). Thus, two distinct genes encode the TLR5 orthologs in fish, one of which has a consensus intracellular domain (TIR). In order to test their functions, we constructed fusion proteins with the LRR region of rtTLR5S (S-chimera) or that of rtTLR5M and the TIR of huTLR5 (M-chimera). The S- and M-chimeras expressed in HeLa or CHO cells signaled the presence of Vibrio anguillarum flagellin, resulting in NF-kappaB activation. rtTLR5M was ubiquitously expressed, whereas rtTLR5S was predominantly expressed in the liver. In the hepatoma cell lines of the rainbow trout RTH-149, stimulation of rtTLR5M with V. anguillarum or its flagellin allowed the up-regulation of rtTLR5S. Flagellin-mediated NF-kappaB activation was more significant in the presence of or simultaneous expression of rtTLR5S. Therefore, a two-step flagellin response occurred for host defense against bacterial infection in fish: (a) flagellin first induced basal activation of NF-kappaB via membrane TLR5, facilitating the production of soluble TLR5 and minimal acute phase proteins, and (b) the inducible soluble TLR5 amplifies membrane TLR5-mediated cellular responses in a positive feedback fashion.     

Crystal structure of a plant leucine rich repeat protein with two island domains

Leucine rich repeat(LRR)domain,characterized by a repetitive sequence pattern rich in leucine residues,is a universal protein-protein interaction motif present in all life forms.LRR repeats interrupted by sequences of 30 70 residues(termed island domain,ID)have been found in some plant LRR receptor-like kinases(RLKs)and animal Toll-like receptors(TLR7-9).Recent studies provide insight into how a single ID is structurally integrated into an LRR protein.However,structural information on an LRR protein with two IDs is lacking.The receptor-like protein kinase 2(RPK2)is an LRR-RLK and has important roles in controlling plant growth and development by perception and transduction of hormone signal.Here we present the crystal structure of the extracellular LRR domain of RPK2(RPK2-LRR)containing two IDs from Arabidopsis.The structure reveals that both of the IDs are helical and located at the central region of the single RPK2-LRR solenoid.One of them binds to the inner surface of the solenoid,whereas the other one mainly interacts with the lateral side.Unexpectedly,a long loop immediately following the N-terminal capping domain of RPK2-LRR is presented toward and sandwiched between the two IDs,further stabilizing their embedding to the LRR solenoid.A potential ligand binding site formed by the two IDs and the solenoid is located at the C-terminal side of RPK2-LRR.The structural information of RPK2-LRR broadens our understanding toward the large family of LRR proteins and provides insight into RPK2-mediated signaling.     

A perspective on the structural studies of inner membrane electrochemical potential-driven transporters

Electrochemical potential-driven transporters represent a vast array of proteins with varied substrate specificities. While diverse in size and substrate specificity, they are all driven by electrochemical potentials. Over the past five years there have been increasing numbers of X-ray structures reported for this family of transporters. Structural information is available for five subfamilies of electrochemical potential-driven transporters. No structural information exists for the remaining 91 subfamilies. In this review, the various subfamilies of electrochemical potential-driven transporters are discussed. The seven reported structures for the electrochemical potential-driven transporters and the methods for their crystallization are also presented. With a few exceptions, overall crystallization trends have been very similar for the transporters despite their differences in substrate specificity and topology. Also discussed is why the structural studies on these transporters were successful while others are not as fruitful. With the plethora of transporters with unknown structures, this review provides incentive for crystallization of transporters in the remaining subfamilies for which no structural information exists.     

A perspective on the structural studies of inner membrane electrochemical potential-driven transporters

Electrochemical potential-driven transporters represent a vast array of proteins with varied substrate specificities. While diverse in size and substrate specificity, they are all driven by electrochemical potentials. Over the past five years there have been increasing numbers of X-ray structures reported for this family of transporters. Structural information is available for five subfamilies of electrochemical potential-driven transporters. No structural information exists for the remaining 91 subfamilies. In this review, the various subfamilies of electrochemical potential-driven transporters are discussed. The seven reported structures for the electrochemical potential-driven transporters and the methods for their crystallization are also presented. With a few exceptions, overall crystallization trends have been very similar for the transporters despite their differences in substrate specificity and topology. Also discussed is why the structural studies on these transporters were successful while others are not as fruitful. With the plethora of transporters with unknown structures, this review provides incentive for crystallization of transporters in the remaining subfamilies for which no structural information exists.     

Molecular evolution of vertebrate Toll-like receptors: evolutionary rate difference between their leucine-rich repeats and their TIR domains

Toll-like receptors (TLRs) that initiate an innate immune response contain an extracellular leucine rich repeat (LRR) domain and an intracellular Toll IL-receptor (TIR) domain. There are fifteen different TLRs in vertebrates. The LRR domains, which adopt a solenoid structure, usually have higher rates of evolution than do the TIR globular domains. It is important to understand the molecular evolution and functional roles of TLRs from this standpoint. Both pairwise genetic distances and Ka/Ks's (the ratios between non synonymous and synonymous substitution rates) were compared between the LRR domain and the TIR domain of 366 vertebrate TLRs from 96 species (from fish to primates). In fourteen members (TLRs 1, 2, 3, 4, 5, 6, 7, 8, 9, 11/12, 13, 14, 21, and 22/23) the LRR domains evolved significantly more rapidly than did the corresponding TIR domains. The evolutionary rates of the LRR domains are significantly different among these members; LRR domains from TLR3 and TLR7 from primates to fishes have the lowest rate of evolution. In contrast, the fifteenth member, TLR10, shows no significant differences; its TIR domain is not highly conserved. The present results suggest that TLR10 may have a different function in signaling from those other members and that a higher conservation of TLR3 and TLR7 may reflect a more ancient mechanism and/or structure in the innate immune response system. Gene conversions are suggested to have occurred in platypus TLR6 and TLR10. This study provides new insight about structural and functional diversification of vertebrate TLRs.     

Identification of Francisella tularensis lipoproteins that stimulate the toll-like receptor (TLR) 2/TLR1 heterodimer

The innate immune response to Francisella tularensis is primarily mediated by TLR2, though the bacterial products that stimulate this receptor remain unknown. Here we report the identification of two Francisella lipoproteins, TUL4 and FTT1103, which activate TLR2. We demonstrate that TUL4 and FTT1103 stimulate chemokine production in human and mouse cells in a TLR2-dependent way. Using an assay that relies on chimeric TLR proteins, we show that TUL4 and FTT1103 stimulate exclusively the TLR2/TLR1 heterodimer. Our results also show that yet unidentified Francisella proteins, possibly unlipi-dated, have the ability to stimulate the TLR2/TLR6 heterodimer. Through domain-exchange analysis, we determined that an extended region that comprises LRR 9-17 in the extra-cellular portion of TLR1 mediates response to Francisella lipoproteins and triacylated lipopeptide. Substitution of the corresponding LRR of TLR6 with the LRR derived from TLR1 enables TLR6 to recognize TUL4, FTT1103, and triacylated lipopeptide. This study identifies for the first time specific Fran-cisella products capable of stimulating a proinflammatory response and the cellular receptors they trigger.     

Transcriptional profiling of Actinobacillus pleuropneumoniae under iron-restricted conditions

Background

Toll-like receptors (TLRs) play a central role in innate immunity. TLRs are membrane glycoproteins and contain leucine rich repeat (LRR) motif in the ectodomain. TLRs recognize and respond to molecules such as lipopolysaccharide, peptidoglycan, flagellin, and RNA from bacteria or viruses. The LRR domains in TLRs have been inferred to be responsible for molecular recognition. All LRRs include the highly conserved segment, LxxLxLxxNxL, in which "L" is Leu, Ile, Val, or Phe and "N" is Asn, Thr, Ser, or Cys and "x" is any amino acid. There are seven classes of LRRs including "typical" ("T") and "bacterial" ("S"). All known domain structures adopt an arc or horseshoe shape. Vertebrate TLRs form six major families. The repeat numbers of LRRs and their "phasing" in TLRs differ with isoforms and species; they are aligned differently in various databases. We identified and aligned LRRs in TLRs by a new method described here.

Results

The new method utilizes known LRR structures to recognize and align new LRR motifs in TLRs and incorporates multiple sequence alignments and secondary structure predictions. TLRs from thirty-four vertebrate were analyzed. The repeat numbers of the LRRs ranges from 16 to 28. The LRRs found in TLRs frequently consists of LxxLxLxxNxLxxLxxxxF/LxxLxx ("T") and sometimes short motifs including LxxLxLxxNxLxxLPx(x)LPxx ("S"). The TLR7 family (TLR7, TLR8, and TLR9) contain 27 LRRs. The LRRs at the N-terminal part have a super-motif of STT with about 80 residues. The super-repeat is represented by STTSTTSTT or _TTSTTSTT. The LRRs in TLRs form one or two horseshoe domains and are mostly flanked by two cysteine clusters including two or four cysteine residue.

Conclusion

Each of the six major TLR families is characterized by their constituent LRR motifs, their repeat numbers, and their patterns of cysteine clusters. The central parts of the TLR1 and TLR7 families and of TLR4 have more irregular or longer LRR motifs. These central parts are inferred to play a key role in the structure and/or function of their TLRs. Furthermore, the super-repeat in the TLR7 family suggests strongly that "bacterial" and "typical" LRRs evolved from a common precursor.     

Homology modeling of human Toll‐like receptors TLR7, 8, and 9 ligand‐binding domains

Toll‐like receptors (TLRs) play a key role in the innate immune system. The TLR7, 8, and 9 compose a family of intracellularly localized TLRs that signal in response to pathogen‐derived nucleic acids. So far, there are no crystallographic structures for TLR7, 8, and 9. For this reason, their ligand‐binding mechanisms are poorly understood. To enable first predictions of the receptor–ligand interaction sites, we developed three‐dimensional structures for the leucine‐rich repeat ectodomains of human TLR7, 8, and 9 based on homology modeling. To achieve a high sequence similarity between targets and templates, structural segments from all known TLR ectodomain structures (human TLR1/2/3/4 and mouse TLR3/4) were used as candidate templates for the modeling. The resulting models support previously reported essential ligand‐binding residues. They also provide a basis to identify three potential receptor dimerization mechanisms. Additionally, potential ligand‐binding residues are identified using combined procedures. We suggest further investigations of these residues through mutation experiments. Our modeling approach can be extended to other members of the TLR family or other repetitive proteins.     

Quantitative analysis and prediction of curvature in leucine‐rich repeat proteins

Leucine‐rich repeat (LRR) proteins form a large and diverse family. They have a wide range of functions most of which involve the formation of protein–protein interactions. All known LRR structures form curved solenoids, although there is large variation in their curvature. It is this curvature that determines the shape and dimensions of the inner space available for ligand binding. Unfortunately, large‐scale parameters such as the overall curvature of a protein domain are extremely difficult to predict. Here, we present a quantitative analysis of determinants of curvature of this family. Individual repeats typically range in length between 20 and 30 residues and have a variety of secondary structures on their convex side. The observed curvature of the LRR domains correlates poorly with the lengths of their individual repeats. We have, therefore, developed a scoring function based on the secondary structure of the convex side of the protein that allows prediction of the overall curvature with a high degree of accuracy. We also demonstrate the effectiveness of this method in selecting a suitable template for comparative modeling. We have developed an automated, quantitative protocol that can be used to predict accurately the curvature of leucine‐rich repeat proteins of unknown structure from sequence alone. This protocol is available as an online resource at http://www.bioinf.manchester.ac.uk/curlrr/ . Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Comparative analysis of species-specific ligand recognition in Toll-like receptor 8 signaling: a hypothesis

Toll-like receptors (TLRs) play a central role in the innate immune response by recognizing conserved structural patterns in a variety of microbes. TLRs are classified into six families, of which TLR7 family members include TLR7, 8, and 9, which are localized to endolysosomal compartments recognizing viral infection in the form of foreign nucleic acids. In our current study, we focused on TLR8, which has been shown to recognize different types of ligands such as viral or bacterial ssRNA as well as small synthetic molecules. The primary sequences of rodent and non-rodent TLR8s are similar, but the antiviral compound (R848) that activates the TLR8 pathway is species-specific. Moreover, the factors underlying the receptor's species-specificity remain unknown. To this end, comparative homology modeling, molecular dynamics simulations refinement, automated docking and computational mutagenesis studies were employed to probe the intermolecular interactions between this anti-viral compound and TLR8. Furthermore, comparative analyses of modeled TLR8 (rodent and non-rodent) structures have shown that the variation mainly occurs at LRR14-15 (undefined region); hence, we hypothesized that this variation may be the primary reason for the exhibited species-specificity. Our hypothesis was further bolstered by our docking studies, which clearly showed that this undefined region was in close proximity to the ligand-binding site and thus may play a key role in ligand recognition. In addition, the interface between the ligand and TLR8s varied depending upon the amino acid charges, free energy of binding, and interaction surface. Therefore, our current work provides a hypothesis for previous in vivo studies in the context of TLR signaling.     

The leucine-rich repeat as a protein recognition motif

Leucine-rich repeats (LRRs) are 20-29-residue sequence motifs present in a number of proteins with diverse functions. The primary function of these motifs appears to be to provide a versatile structural framework for the formation of protein-protein interactions. The past two years have seen an explosion of new structural information on proteins with LRRs. The new structures represent different LRR subfamilies and proteins with diverse functions, including GTPase-activating protein rna1p from the ribonuclease-inhibitor-like subfamily; spliceosomal protein U2A', Rab geranylgeranyltransferase, internalin B, dynein light chain 1 and nuclear export protein TAP from the SDS22-like subfamily; Skp2 from the cysteine-containing subfamily; and YopM from the bacterial subfamily. The new structural information has increased our understanding of the structural determinants of LRR proteins and our ability to model such proteins with unknown structures, and has shed new light on how these proteins participate in protein-protein interactions.     

Rationalizing alpha-helical membrane protein crystallization

X-ray crystallography is currently the most successful method for determining the three-dimensional structure of membrane proteins. Nevertheless, growing the crystals required for this technique presents one of the major bottlenecks in this area of structural biology. This is especially true for the alpha-helical type membrane proteins that are of particular interest due to their medical relevance. To address this problem we have undertaken a detailed analysis of the crystallization conditions from 121 alpha-helical membrane protein structures deposited in the Protein Data Bank. This information has been analyzed so that the success of different parameters can be easily compared for different membrane protein families. Concurrent with this analysis, we also present the new sparse matrix crystallization screen MemGold.     

Comprehensive modeling and functional analysis of Toll‐like receptor ligand‐recognition domains

Toll‐like receptors (TLRs) are innate immune pattern‐recognition receptors endowed with the capacity to detect microbial pathogens based on pathogen‐associated molecular patterns. The understanding of the molecular principles of ligand recognition by TLRs has been greatly accelerated by recent structural information, in particular the crystal structures of leucine‐rich repeat‐containing ectodomains of TLR2, 3, and 4 in complex with their cognate ligands. Unfortunately, for other family members such as TLR7, 8, and 9, no experimental structural information is currently available. Methods such as X‐ray crystallography or nuclear magnetic resonance are not applicable to all proteins. Homology modeling in combination with molecular dynamics may provide a straightforward yet powerful alternative to obtain structural information in the absence of experimental (structural) data, provided that the generated three‐dimensional models adequately approximate what is found in nature. Here, we report the development of modeling procedures tailored to the structural analysis of the extracellular domains of TLRs. We comprehensively compared secondary structure, torsion angles, accessibility for glycosylation, surface charge, and solvent accessibility between published crystal structures and independently built TLR2, 3, and 4 homology models. Finding that models and crystal structures were in good agreement, we extended our modeling approach to the remaining members of the TLR family from human and mouse, including TLR7, 8, and 9.     

Structural principles of leucine-rich repeat (LRR) proteins

LRR-containing proteins are present in over 2000 proteins from viruses to eukaryotes. Most LRRs are 20-30 amino acids long, and the repeat number ranges from 2 to 42. The known structures of 14 LRR proteins, each containing 4-17 repeats, have revealed that the LRR domains fold into a horseshoe (or arc) shape with a parallel beta-sheet on the concave face and with various secondary structures, including alpha-helix, 3(10)-helix, and pII helix on the convex face. We developed simple methods to charactere quantitatively the arc shape of LRR and then applied them to all known LRR proteins. A quantity of 2Rsin(phi/2), in which R and phi are the radii of the LRR arc and the rotation angle about the central axis per repeating unit, respectively, is highly conserved in all the LRR proteins regardless of a large variety of repeat number and the radius of the LRR arc. The radii of the LRR arc with beta-alpha structural units are smaller than those with beta-3(10) or beta-pII units. The concave face of the LRR beta-sheet forms a surface analogous to a part of a M?bius strip.