A Dual Leucine-rich Repeat in Proteins from the Eukaryotic SAR Group.

BACKGROUND: Leucine-rich repeats (LRRs) occurring in tandem are 20-29 amino acids long. Eleven LRR types have been recognized; they include plant-specific (PS) type with the consensus of LxxLxLxxNxL SGxIPxxIxxLxx of 24 residues and SDS22-like type with the consensus of LxxLxLxxNxL xxIxxIxxLxx of 22 residues. OBJECTIVE: A viral LRR protein in metagenome data indicated that most of the LRRs (5/6 = 0.83) are represented by the consensus of LxxLDLxxTxV SGKLSDLxxLTN of 23 residues. This LRR shows a dual characteristic of PS and SDS22-like LRRs (called PS/SDS22-like LRR). A comprehensive similarity search was performed under the hypothesis that many proteins contain LRR domains consisting of only or mainly PS/SDS22-like LRR. METHODS: Sequence similarity search by the FASTA and BLAST programs was performed using the sequence of this PS/SDS22-like LRR domain as a query sequence. The presence of PS/SDS22-like LRR was screened within the LRR domains in known structures. RESULTS: Over 280 LRR proteins were identified from protists, fungi, and bacteria; ~ 40% come from the SAR group (the phyla Alveolate and Stramenopiles). The secondary structure analysis of PS/SDS22-like LRRs occurring sporadically in the known structures indicates three or four type patterns of secondary structures. CONCLUSION: PS/SDS22-like LRR forms an LRR class with PS, SDS22-like and Leptospira-like LRRs. It appears that PS/SDS22-like LRR is a chameleon-like sequence. A duality of two LRR types brings diversity.

A crucial residue in the hydrophobic core of the solenoid structure of leucine rich repeats.

Leucine rich repeats (LRRs) with 20-30 residues form a super helix arrangement. Individual LRRs are separated into a highly conserved segment with a highly conserved (HCS) and a variable segment (VS). In LRRs short ß-strands in HCS stack in parallel, while VS adopts various secondary structures. Among eleven classes recognized, only RI-like, Cysteine-containing (CC), and GALA classes adopt an α-helix. However, the repeat unit lengths are usually different from each other. We performed some analyses based on the atomic coordinates in the known LRR structures. In the VS consensuses of the three classes, position 8 in the VS part is, in common, occupied by conserved aliphatic residue adopting an α-helix. This aliphatic residue is near to the two conserved hydrophobic residues at position 4 (in the center of ß-strands) in two adjacent HCS parts. The conserved aliphatic residue plays a crucial role to preserve two parallel ß-strands.

A strong correlation between consensus sequences and unique super secondary structures in leucine rich repeats.

Leucine rich repeats (LRRs) are present in over 430 000 proteins from viruses to eukaryotes. The LRRs are 20 to 30 residues long and occur in tandem. Individual LRRs are separated into a highly conserved segment with the consensus of LxxLxLxxNxL or LxxLxLxxNxxL (HCS) and a variable segment (VS). In LRRs parallel stacking of short ß-strands (at positions 3-5 in HCS) form a super helix arrangement called a solenoid structure. Many classes have been recognized. All three classes of Plant specific, Leptospira-like, and SDS22-like LRRs which are 24, 23, and 22 residues long, respectively, form a 3(10)-helix in the VS part. To get a deeper understanding of sequence, structure correlations in LRR structures, we utilized secondary structure assignment and HELFIT analysis (calculating helix axis, pitch, radius, residues per turn, and handedness) based on the atomic coordinates in crystal structures of 43 LRR proteins. We also defined three structural parameters using the three unit vectors of the helix axes of 3(10)-helix, ß-turn, and LRR-domain calculated by HELFIT. The combination of the secondary structure assignment and HELFIT reveals that their LRRs adopt unique super secondary structures consisting of a 3(10)-helix and one or two Type I ß-turns. We propose one structural parameter as a geometrical invariant of LRR solenoid structures. The common LxxLxxL sequence (where "L" is Leu, Ile, Val, Phe or Cys) in the three classes is an essential determinant for the super secondary structures providing a medium range interaction.

Super Secondary Structure Consisting of a Polyproline II Helix and a ß-Turn in Leucine Rich Repeats in Bacterial Type III Secretion System Effectors.

Leucine rich repeats (LRRs) are present in over 100,000 proteins from viruses to eukaryotes. The LRRs are 20-30 residues long and occur in tandem. LRRs form parallel stacks of short ß-strands and then assume a super helical arrangement called a solenoid structure. Individual LRRs are separated into highly conserved segment (HCS) with the consensus of LxxLxLxxNxL and variable segment (VS). Eight classes have been recognized. Bacterial LRRs are short and characterized by two prolines in the VS; the consensus is xxLPxLPxx with Nine residues (N-subtype) and xxLPxxLPxx with Ten residues (T-subtype). Bacterial LRRs are contained in type III secretion system effectors such as YopM, IpaH3/9.8, SspH1/2, and SlrP from bacteria. Some LRRs in decorin, fribromodulin, TLR8/9, and FLRT2/3 from vertebrate also contain the motifs. In order to understand structural features of bacterial LRRs, we performed both secondary structures assignments using four programs-DSSP-PPII, PROSS, SEGNO, and XTLSSTR-and HELFIT analyses (calculating helix axis, pitch, radius, residues per turn, and handedness), based on the atomic coordinates of their crystal structures. The N-subtype VS adopts a left handed polyproline II helix (PPII) with four, five or six residues and a type I ß-turn at the C-terminal side. Thus, the N-subtype is characterized by a super secondary structure consisting of a PPII and a ß-turn. In contrast, the T-subtype VS prefers two separate PPIIs with two or three and two residues. The HELFIT analysis indicates that the type I ß-turn is a right handed helix. The HELFIT analysis determines three unit vectors of the helix axes of PPII (P), ß-turn (B), and LRR domain (A). Three structural parameters using these three helix axes are suggested to characterize the super secondary structure and the LRR domain.

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

The NOD-like receptors (NLRs) and Toll-like receptors (TLRs) are pattern recognition receptors that are involved in the innate, pathogen pattern recognition system. The TLR and NLR receptors contain leucine-rich repeats (LRRs) that are responsible for ligand interactions. In LRRs short ß-strands stack parallel and then the LRRs form a super helical arrangement of repeating structural units (called a coil of solenoids). The structures of the LRR domains of NLRC4, NLRP1, and NLRX1 in NLRs and of TLR1-5, TLR6, TLR8, TLR9 in TLRs have been determined. Here we report nine geometrical parameters that characterize the LRR domains; these include four helical parameters from HELFIT analysis. These nine parameters characterize well the LRR structures in NLRs and TLRs; the LRRs of NLR adopts a right-handed helix. In contrast, the TLR LRRs adopt either a left-handed helix or are nearly flat; RP105 and CD14 also adopt a left-handed helix. This geometrical analysis subdivides TLRs into four groups consisting of TLR3/TLR8/TLR9, TLR1/TLR2/TRR6, TLR4, and TLR5; these correspond to the phylogenetic tree based on amino acid sequences. In the TLRs an ascending lateral surface that consists of loops connecting the ß-strand at the C-terminal side is involved in protein, protein/ligand interactions, but not the descending lateral surface on the opposite side.

omega-Helices in proteins.

A modification of the alpha-helix, termed the omega-helix, has four residues in one turn of a helix. We searched the omega-helix in proteins by the HELFIT program which determines the helical parameters-pitch, residues per turn, radius, and handedness-and p = rmsd/(N - 1)(1/2) estimating helical regularity, where "rmsd" is the root mean square deviation from the best fit helix and "N" is helix length. A total of 1,496 regular alpha-helices 6-9 residues long with p < or = 0.10 A were identified from 866 protein chains. The statistical analysis provides a strong evidence that the frequency distribution of helices versus n indicates the bimodality of typical alpha-helix and omega-helix. Sixty-two right handed omega-helices identified (7.2% of proteins) show non-planarity of the peptide groups. There is amino acid preference of Asp and Cys. These observations and analyses insist that the omega-helices occur really in proteins.

HELFIT: Helix fitting by a total least squares method.

The problem of fitting a helix to data arises in analysis of protein structure, in nuclear physics, and in engineering. A continuous helix is described by five parameters: helix axis, helix radius, and helix pitch. One of these helix parameters is frequently predefined in the helix fitting. Other algorithms find only the helix axis or determine separately the helix axis, the helix radius, or the helix pitch. Here we describe a total least squares method, HELFIT, for helix fitting. HELFIT enables one to calculate simultaneously all five of the helix parameters with high accuracy. The minimum number of data points required for the analysis is only four. HELFIT is very insensitive to noise even in short helices. HELFIT also calculates a parameter, p=rmsd/(N-1)(1/2), which estimates the regularity of helical structures independent of the number of data points, where rmsd is the root mean square distance from the best-fit helix to data points and N is the number of data points. It should become a basic tool of structural bioinformatics.

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.

Comparative sequence analysis of leucine-rich repeats (LRRs) within vertebrate toll-like receptors.

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.

3(10)-helices in proteins are parahelices.

The 3(10)-helix is characterized by having at least two consecutive hydrogen bonds between the main-chain carbonyl oxygen of residue i and the main-chain amide hydrogen of residue i + 3. The helical parameters--pitch, residues per turn, radius, and root mean square deviation (rmsd) from the best-fit helix--were determined by using the HELFIT program. All 3(10)-helices were classified as regular or irregular based on rmsd/(N - 1)1/2 where N is the helix length. For both there are systematic, position-specific shifts in the backbone dihedral angles. The average phi, psi shift systematically from approximately -58 degrees, approximately -32 degrees to approximately -90 degrees, approximately -4 degrees for helices 5, 6, and 7 residues long. The same general pattern is seen for helices, N = 8 and 9; however, in N = 9, the trend is repeated with residues 6, 7, and 8 approximately repeating the phi, psi of residues 2, 3, and 4. The residues per turn and radius of regular 3(10)-helices decrease with increasing length of helix, while the helix pitch and rise per residue increase. That is, regular 3(10)-helices become thinner and longer as N increases from 5 to 8. The fraction of regular 3(10)-helices decreases linearly with helix length. All longer helices, N > or = 9 are irregular. Energy minimizations show that regular helices become less stable with increasing helix length. These findings indicate that the definition of 3(10)-helices in terms of average, uniform dihedral angles is not appropriate and that it is inherently unstable for a polypeptide to form an extended, regular 3(10)-helix. The 3(10)-helices observed in proteins are better referred to parahelices.

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 Möbius strip.