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.

Numerous variants of leucine rich repeats in proteins from nucleo-cytoplasmic large DNA viruses.

Leucine rich repeats (LRRs) occurring in tandem are 20-29 amino acids long. Eleven LRR types have been recognized. Sequence features of LRRs from viruses were investigated using over 600 LRR proteins from 89 species. Directly before, metagenome data of nucleo-cytoplasmic large dsDNA viruses (NCLDVs) have been published; the 2,074 NCLDVs encode 199,021 proteins. From the NCLDVs 547 LRR proteins were identified and 502 were used for analysis. Various variants of known LRR types were identified in viral LRRs. A comprehensive analysis of TpLRR and FNIP that belong to an LRR type was first performed. The repeating unit lengths (RULs) in five types are 19 residues which is the shortest among all LRRs. The RULs of eight LRR types including FNIP are one to five residues shorter than those of the known, corresponding LRR types. The conserved hydrophobic residues such as Leu, Val or Ile in the consensus sequences are frequently substituted by cysteine at one or two positions. Four unique LRR motifs that are different from those identified previously are observed. The present study enhances the previous result. An evolutionary scenario of short or unique LRR was discussed.

Withdrawal Notice: Novel Sequence Features of Leucine-rich Repeats in Proteins from Nucleocytoplasmic Large DNA Viruses

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Sequence features, structure, ligand interaction, and diseases in small leucine rich repeat proteoglycans.

Small leucine rich repeat proteoglycans (SLRPs) are a group of active components of the extracellular matrix in all tissues. SLRPs bind to collagens and regulate collagen fibril growth and fibril organization. SLRPs also interact with various cytokines and extracellular compounds, which lead to various biological functions such cell adhesion and signaling, proliferation, and differentiation. Mutations in SLRP genes are associated with human diseases. Now crystal structures of five SLRPs are available. We describe some features of amino acid sequence and structures of SLRPs. We also review ligand interactions and then discuss the interaction surfaces. Furthermore, we map mutations associated with human diseases and discuss possible effects on structures by the mutations.

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.

Shrinking of repeating unit length in leucine-rich repeats from double-stranded DNA viruses.

Leucine-rich repeats (LRRs) are present in over 563,000 proteins from viruses to eukaryotes. LRRs repeat in tandem and have been classified into fifteen classes in which the repeat unit lengths range from 20 to 29 residues. Most LRR proteins are involved in protein-protein or ligand interactions. The amount of genome sequence data from viruses is increasing rapidly, and although viral LRR proteins have been identified, a comprehensive sequence analysis has not yet been done, and their structures, functions, and evolution are still unknown. In the present study, we characterized viral LRRs by sequence analysis and identified over 600 LRR proteins from 89 virus species. Most of these proteins were from double-stranded DNA (dsDNA) viruses, including nucleocytoplasmic large dsDNA viruses (NCLDVs). We found that the repeating unit lengths of 11 types are one to five residues shorter than those of the seven known corresponding LRR classes. The repeating units of six types are 19 residues long and are thus the shortest among all LRRs. In addition, two of the LRR types are unique and have not been observed in bacteria, archae or eukaryotes. Conserved strongly hydrophobic residues such as Leu, Val or Ile in the consensus sequences are replaced by Cys with high frequency. Phylogenetic analysis indicated that horizontal gene transfer of some viral LRR genes had occurred between the virus and its host. We suggest that the shortening might contribute to the survival strategy of viruses. The present findings provide a new perspective on the origin and evolution of LRRs.

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.

Molecular Dynamics Study of the Changes in Conformation of Calmodulin with Calcium Binding and/or Target Recognition.

Calmodulin is a calcium binding protein with two lobes, N-lobe and C-lobe, which evolved from duplication and fusion of a single precursor lobe of a pair of EF-hand. These two lobes of calmodulin show subtle differences in calcium binding and target recognition; these are important for the functions of calmodulin. Since the structures, especially main chain conformations, of two EF-lobes in holo-form are quite similar; this is a good example to evaluate the effect of side chains for structural dynamics. We analyzed the structure of calmodulin using molecular dynamics and found differences in conformational ensembles between N- and C-lobes. We also showed the mutant structures created by homology modeling could reproduce the difference of dynamic motion between N- and C-lobes.

Polyproline II Helix as a Recognition Motif of Plant Peptide Hormones and Flagellin Peptide flg22.

BACKGROUND: Plant peptide hormones play a crucial role in plant growth and development. A group of these peptide hormones are signaling peptides with 5 - 23 amino acids. Flagellin peptide (flg22) also elicits an immune response in plants. The functions are expressed through recognition of the peptide hormones and flg22. This recognition relies on membrane localized receptor kinases with extracellular leucine rich repeats (LRR-RKs). The structures of plant peptide hormones - AtPep1, IDA, IDL1, RGFs 1- 3, TDIF/CLE41 - and of flg22 complexed with LRR domains of corresponding LRR-RKs and co-receptors SERKs have been determined. However, their structures are well not analyzed and characterized in detail. The structures of PIP, CEP, CIF, and HypSys are still unknown. OBJECTIVE: Our motivation is to clarify structural features of these plant, small peptides and Flg22 in their bound states. METHODS: In this article, we performed secondary structure assignments and HELFIT analyses (calculating helix axis, pitch, radius, residues per turn, and handedness) based on the atomic coordinates from the crystal structures of AtPep1, IDA, IDL1, RGFs 1- 3, TDIF/CLE41 - and of flg22. We also performed sequence analysis of the families of PIP, CEP, CIF, and HypSys in order to predict their secondary structures. RESULTS: Following AtPep1 with 23 residues adopts two left handed polyproline helices (PPIIs) with six and four residues. IDA, IDL1, RGFs 1 - 2, and TDIF/CLE41 with 12 or 13 residues adopt a four residue PPII; RGF3 adopts two PPIIs with four residues. Flg22 with 22 residues also adopts a six residue PPII. The other peptide hormones - PIP, CEP, CIF, and HypSys - that are rich in proline or hydroxyproline presumably prefer PPII. CONCLUSION: The present analysis indicates that PPII helix in the plant small peptide hormones and in flg22 is crucial for recognition of the LRR domains in receptors.

Interaction sites of PEF proteins for recognition of their targets.

The EF-hand is a helix-loop-helix motif observed mainly in intracellular calcium binding proteins. The EF-hand usually occurs as a pair, EF-lobe, which is a unit of evolution and structure. Penta EF-hand (PEF) proteins form a unique group including calpain, sorcin, grancalcin, ALG-2, and peflin. The fifth EF-hand of PEF proteins makes a pair with that of another PEF protein. The members of PEF family have diverse functions and their evolution is complex. The interaction of PEF proteins with target occurs at several sites. Here, we analyzed the ancestral sequences of each group of PEF proteins and determined the interfaces for the specific and selective interaction to the target among several PEF proteins. The shape of the groove for interaction at common site is different among PEF proteins. We found that the changes at limited sites induced the divergence of interaction sites that determines the selectivity of targets. The residues involved the changes at limited sites are important for the drug design selective to each PEF protein.

Leucine Rich Repeat Proteins: Sequences, Mutations, Structures and Diseases.

Mutations in the genes encoding Leucine Rich Repeat (LRR) containing proteins are associated with over sixty human diseases; these include high myopia, mitochondrial encephalomyopathy, and Crohn's disease. These mutations occur frequently within the LRR domains and within the regions that shield the hydrophobic core of the LRR domain. The amino acid sequences of fifty-five LRR proteins have been published. They include Nod-Like Receptors (NLRs) such as NLRP1, NLRP3, NLRP14, and Nod-2, Small Leucine Rich Repeat Proteoglycans (SLRPs) such as keratocan, lumican, fibromodulin, PRELP, biglycan, and nyctalopin, and F-box/LRR-repeat proteins such as FBXL2, FBXL4, and FBXL12. For example, 363 missense mutations have been identified. Replacement of arginine, proline, or cysteine by another amino acid, or the reverse, is frequently observed. The diverse effects of the mutations are discussed based on the known structures of LRR proteins. These mutations influence protein folding, aggregation, oligomerization, stability, protein-ligand interactions, disulfide bond formation, and glycosylation. Most of the mutations cause loss of function and a few, gain of function.

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.

Conformational landscape mapping the difference between N-lobes and C-lobes of calmodulin.

Calmodulin is a calcium binding protein that consists of four EF-hand domains. The two EF-lobes of calmodulin, called the N-lobe and the C-lobe, arose from duplication and fusion of a precursor EF-hand. The amino acid sequences and the structures of the N-lobe and of the C-lobe are quite similar to each other. The N-lobe and the C-lobe, however, have subtle differences in structure and function. We analyzed the helix positions of calmodulin lobes by the alignment with the pseudo-two fold axis of the EF-lobe. We made a map of conformational landscape of helix positions. The four states of the EF-lobe appeared on two lines in the landscape; these two lines show the trajectory of opening and closing of the EF-lobe. For the N-lobe of calmodulin, the calcium bound form and the apo-forms are on the lower line. The two apo-forms of the C-lobe of calmodulin, with target and without target, are on the upper line. The calcium bound form of the C-lobe is on the lower line. The rearrangement of helix interaction between two the EF-hands is necessary for calcium binding in the C-lobe. The hydrophobic packing in the apo-form of the N-lobe is similar to the packing of the N- and C-lobes of the calcium bound form. However, the packing of C-lobe side chains in the apo-form is different from these other three structures. Our detailed analysis should serve as an example that can be applied to other proteins that undergo changes in conformation upon binding effectors.

Structural and functional diversity of EF-hand proteins: Evolutionary perspectives.

We have classified 865 sequences of EF-hand proteins from five proteomes into 156 subfamilies. These subfamilies were put into six groups. Evolutionary relationships among subfamilies and groups were analyzed from the inferred ancestral sequence for each subfamily. CTER, CPV, and PEF groups arose from a common EF-lobe (pair of adjacent EF-hands). They have two or more EF-lobes; the relative positions of their EF-lobes differ from each other. Comparisons of the ancestral sequences and the inferred structures of the EF-lobes of these groups indicate that the mutual positions of EF-lobes were established soon after divergence of an EF-lobe for each group and before the duplication and fusion of EF-lobe gene(s). These ancestral sequences reveal that some subfamilies in low similarity and isolated groups did not evolve from the EF-lobe precursor, even if their conformations are similar to the canonical EF-hand. This is an example of convergent evolution.

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.

Structural differences among subfamilies of EF-hand proteins--a view from the pseudo two-fold symmetry axis.

We have analyzed the conformations of EF-lobes, adjacent pairs of EF-hand domains, in a coordinate system based on the approximate two-fold (z) axis that relates the two EF-hands. Two parameters - dE(ø), the azimuthal angle between the y-axis and the projection of the offset vector to helix E onto the yz-plane, and δdF(ø), the difference angle between the two helices (F1 and F2) of odd and even domains--characterize the openness of a single EF-hand domain and of an EF-lobe, respectively. We describe and compare values of dE(ø) and of δdF(ø) for EF-hand proteins of five subfamilies--CTER, CPV, S100, PARV, CALP--in calci- and apo- forms, with and without bound target proteins. Each subfamily has characteristic changes associated with binding calcium and/or target proteins.

HVM: a web-based tool for alignment of EF-hand lobes relative to their local pseudo two-fold axes.

We have developed a method to align a pair of EF-hand domains, an EF-lobe, using the symmetry axis that is intrinsic to the EF-lobe itself. The coordinate system for the alignment is dependent only on the symmetry of the EF-lobe. The absolute positions of each component in the structure can be described in this coordinate system. Our site provides the foundation for the analyses and comparisons of structural features of EF-lobes.

Analysis of the movements of helices in EF-hands.

We have developed a method to place an EF-lobe in a coordinate system that recognizes the similarity of its two EF-hand domains as well as their relationship by a pseudo-two fold axis, z. The x-axis connects the center of mass, calculated from α-carbons of helices E1 and F1, with the center of mass of E2 and F2. The resulting coordinate system is intrinsic to each EF-lobe and requires no comparison with other EF-lobes. It has provided an intuitive and informative way to compare EF-lobes and especially those changes associated with calcium and/or target binding. We analyzed the EF-lobes of calmodulin and of other subfamilies with four EF-hands. We have rationalized a complex pattern of changes of conformation associated with calcium coordination and effector binding as observed in different subfamilies of EF-hand proteins.

Structure and characterization of the 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Aeropyrum pernix.

The first enzyme in the shikimic acid biosynthetic pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS), varies significantly in size and complexity in the bacteria and plants that express it. The DAH7PS from the archaebacterium Aeropyrum pernix (DAH7PS(Ap)) is among the smallest and least complex of the DAH7PS enzymes, leading to the hypothesis that DAH7PS(Ap) would not be subject to feedback regulation by shikimic acid pathway products. We overexpressed DAH7PS(Ap) in Escherichia coli, purified it, and characterized its enzymatic activity. We then solved its X-ray crystal structure with a divalent manganese ion and phosphoenolpyruvate bound (PDB ID: 1VS1). DAH7PS(Ap) is a homodimeric metalloenzyme in solution. Its enzymatic activity increases dramatically above 60 °C, with optimum activity at 95 °C. Its pH optimum at 60 °C is 5.7. DAH7PS(Ap) follows Michaelis-Menten kinetics at 60 °C, with a K(M) for erythrose 4-phosphate of 280 µM, a K(M) for phosphoenolpyruvate of 891 µM, and a k(cat) of 1.0 s(-1). None of the downstream products of the shikimate biosynthetic pathway we tested inhibited the activity of DAH7PS(Ap). The structure of DAH7PS(Ap) is similar to the structures of DAH7PS from Thermatoga maritima (PDB ID: 3PG8) and Pyrococcus furiosus (PDB ID: 1ZCO), and is consistent with its designation as an unregulated DAH7PS.