Crystal structure of the C-terminal peptidoglycan-binding domain of human peptidoglycan recognition protein Ialpha

Peptidoglycan recognition proteins (PGRPs) are pattern recognition receptors of the innate immune system that bind, and in some cases hydrolyze, peptidoglycans (PGNs) on bacterial cell walls. These molecules, which are highly conserved from insects to mammals, participate in host defense against both Gram-positive and Gram-negative bacteria. We report the crystal structure of the C-terminal PGN-binding domain of human PGRP-Ialpha in two oligomeric states, monomer and dimer, to resolutions of 2.80 and 1.65 A, respectively. In contrast to PGRPs with PGN-lytic amidase activity, no zinc ion is present in the PGN-binding site of human PGRP-Ialpha. The structure reveals that PGRPs exhibit extensive topological variability in a large hydrophobic groove, located opposite the PGN-binding site, which may recognize host effector proteins or microbial ligands other than PGN. We also show that full-length PGRP-Ialpha comprises two tandem PGN-binding domains. These domains differ at most potential PGN-contacting positions, implying different fine specificities. Dimerization of PGRP-Ialpha, which occurs through three-dimensional domain swapping, is mediated by specific binding of sodium ions to a flexible hinge loop, stabilizing the conformation found in the dimer. We further demonstrate sodium-dependent dimerization of PGRP-Ialpha in solution, suggesting a possible mechanism for modulating PGRP activity through the formation of multivalent adducts.     

Crystal structure of human peptidoglycan recognition protein S (PGRP-S) at 1.70 A resolution

Peptidoglycan recognition proteins (PGRPs) are pattern recognition receptors of the innate immune system that bind peptidoglycans (PGNs) of bacterial cell walls. These molecules, which are highly conserved from insects to mammals, contribute to host defense against infections by both Gram-positive and Gram-negative bacteria. Here, we present the crystal structure of human PGRP-S at 1.70A resolution. The overall structure of PGRP-S, which participates in intracellular killing of Gram-positive bacteria, is similar to that of other PGRPs, including Drosophila PGRP-LB and PGRP-SA and human PGRP-Ialpha. However, comparison with these PGRPs reveals important differences in both the PGN-binding site and a groove formed by the PGRP-specific segment on the opposite face of the molecule. This groove, which may constitute a binding site for effector or signaling proteins, is less hydrophobic and deeper in PGRP-S than in PGRP-IalphaC, whose PGRP-specific segments vary considerably in amino acid sequence. By docking a PGN ligand into the PGN-binding cleft of PGRP-S based on the known structure of a PGRP-Ialpha-PGN complex, we identified potential PGN-binding residues in PGRP-S. Differences in PGN-contacting residues and interactions suggest that, although PGRPs may engage PGNs in a similar mode, structural differences exist that likely regulate the affinity and fine specificity of PGN recognition.     

Elucidating the molecular interaction of Zebrafish (Danio rerio) peptidoglycan recognition protein 2 with diaminopimelic acid and lysine type peptidoglycans using in silico approaches

Abstract

Peptidoglycan recognition proteins (PGRPs) belong to the family of pattern recognition receptor, represent the major constituent of innate immunity. Although PGRPs are structurally conserved through evolution, their involvement in innate immunity is different in vertebrates and invertebrates. They are highly specific towards recognition of ligands and can hydrolyze bacterial peptidoglycans (PGNs). Zebrafish PGRPs (zPGRPs) have both peptidoglycans lytic amidase activity and broad-spectrum bactericidal activity, but far less is known about how these receptors recognize these microbial ligands. Such studies are hindered due to lack of structural and functional configuration of zPGRPs. Therefore, in this study, we predicted the three-dimensional structure of zPGRP2 through theoretical modeling, investigated the conformational and dynamic properties through molecular dynamics simulations. Molecular docking study revealed the microbial ligands, that is, muramyl pentapeptide–DAP , muramyl pentapeptide–LYS, muramyl tripeptide–DAP, muramyl tripeptide–Lys, muramyl tetrapeptide–DAP, muramyl tetrapeptide–LYS and tracheal cytotoxin interacts with the conserved amino acids of the ligand recognition site comprised of β1, α2, α4, β4 and loops connecting β1 ? α2, α2 ? β2, β3 ? β4 and α4 ? α5. Conserved His31, His32, Ala34, Ile35, Pro36, Lys38, Asp60, Trp61, Trp63, Ala89, His90, Asp106, His143 and Arg144 are predicted to essential for binding and provides stability to these zPGRP–PGN complexes. Our study provides basic molecular information for further research on the immune mechanisms of PGRP’s in Zebrafish. The plasticity of the zPGRP’s binding site revealed by these microbial ligands suggests an intrinsic capacity of the innate immune system to rapidly evolve specificities to meet new microbial challenges in the future.

Communicated by Ramaswamy H. Sarma     

Mutagenesis and computer modelling approach to study determinants for recognition of signal peptides by the mitochondrial processing peptidase

Determinants for the recognition of a mitochondrial presequence by the mitochondrial processing peptidase (MPP) have been investigated using mutagenesis and bioinformatics approaches. All plant mitochondrial presequences with a cleavage site that was confirmed by experimental studies can be grouped into three classes. Two major classes contain an arginine residue at position -2 or -3, and the third class does not have any conserved arginines. Sequence logos revealed loosely conserved cleavage motifs for the first two classes but no significant amino acid conservation for the third class. Investigation of processing determinants for a class III precursor, Nicotiana plumbaginifolia F1beta precursor of ATP synthase (pF1beta), was performed using a series of pF1beta presequence mutants and mutant presequence peptides derived from the C-terminal portion of the presequence. Replacement of -2 Gln by Arg inhibited processing, whereas replacement of either the most proximally located -5 Arg or -15 Arg by Leu had only a low inhibitory effect. The C-terminal portion of the pF1beta presequence forms a helix-turn-helix structure. Mutations disturbing or prolonging the helical element upstream of the cleavage site inhibited processing significantly. Structural models of potato MPP and the C-terminal pF1beta presequence peptide were built by homology modelling and empirical conformational energy search methods, respectively. Molecular docking of the pF1beta presequence peptide to the MPP model suggested binding of the peptide to the negatively charged binding cleft formed by the alpha-MPP and beta-MPP subunits in close proximity to the H111XXE114H115X(116-190)E191 proteolytic active site on beta-MPP. Our results show for the first time that the amino acid at the -2 position, even if not an arginine, as well as structural properties of the C-terminal portion of the presequence are important determinants for the processing of a class III precursor by MPP.     

Peptidoglycan recognition proteins: a novel family of four human innate immunity pattern recognition molecules.

The innate immune system recognizes microorganisms through a series of pattern recognition receptors that are highly conserved in evolution. Insects have a family of 12 peptidoglycan recognition proteins (PGRPs) that recognize peptidoglycan, a ubiquitous component of bacterial cell walls. We report cloning of three novel human PGRPs (PGRP-L, PGRP-Ialpha, and PGRP-Ibeta) that together with the previously cloned PGRP-S, define a new family of human pattern recognition molecules. PGRP-L, PGRP-Ialpha, and PGRP-Ibeta have 576, 341, and 373 amino acids coded by five, seven, and eight exons on chromosomes 19 and 1, and they all have two predicted transmembrane domains. All mammalian and insect PGRPs have at least three highly conserved C-terminal PGRP domains located either in the extracellular or in the cytoplasmic (or in both) portions of the molecules. PGRP-L is expressed in liver, PGRP-Ialpha and PGRP-Ibeta in esophagus (and to a lesser extent in tonsils and thymus), and PGRP-S in bone marrow (and to a lesser extent in neutrophils and fetal liver). All four human PGRPs bind peptidoglycan and Gram-positive bacteria. Thus, these PGRPs may play a role in recognition of bacteria in these organs.     

昆虫肽聚糖识别蛋白研究进展

在脊椎动物和非脊椎动物中,识别非己是天生免疫反应中的第一步。肽聚糖是细菌细胞壁的必需成分,属于进化上保守的微生物表面病原相关分子模式(pathogen-associated molecular pattern, PAMP),可以被模式识别蛋白(pattern recognition proteins, PRRs)如肽聚糖识别蛋白(peptidoglycan recognition proteins, PGRPs)识别。 在昆虫的天生免疫系统中,有些PGRPs能够利用细菌独有的肽聚糖识别入侵细菌,并将细菌入侵信号传递给下游的抗菌肽(antimicrobial peptide, AMP)合成途径,启动抗菌肽基因的转录及合成;PGRPs对肽聚糖的识别也会启动酚氧化酶原途径的激活,引起黑化反应。有些具有酰胺酶活性的PGRPs可以促进吞噬作用;有些可以抑制抗菌肽合成以减弱过度免疫反应带来的损伤。还有一些PGRPs作为效应因子直接作用于细菌将细菌杀死。本文主要从昆虫PGRPs作为识别受体(recognition receptor)、调节子(regulator)和效应因子(effector) 3个方面进行了综述,并分析了目前PGRPs研究中仍不清楚的问题和未来研究的方向。     

High-resolution structure of a polyomavirus VP1-oligosaccharide complex: implications for assembly and receptor binding.

The crystal structure of a recombinant polyomavirus VP1 pentamer (residues 32-320) in complex with a branched disialylated hexasaccharide receptor fragment has been determined at 1.9 A resolution. The result extends our understanding of oligosaccharide receptor recognition. It also suggests a mechanism for enhancing the fidelity of virus assembly. We have previously described the structure of the complete polyomavirus particle complexed with this receptor fragment at 3.65 A. The model presented here offers a much more refined view of the interactions that determine carbohydrate recognition and allows us to assign additional specific contacts, in particular those involving the (alpha2,6)-linked, branching sialic acid. The structure of the unliganded VP1 pentamer, determined independently, shows that the oligosaccharide fits into a preformed groove and induces no measurable structural rearrangements. A comparison with assembled VP1 in the virus capsid reveals a rearrangement of residues 32-45 at the base of the pentamer. This segment may help prevent the formation of incorrectly assembled particles by reducing the likelihood that the C-terminal arm will fold back into its pentamer of origin.     

Structural insights for MPP8 chromodomain interaction with histone H3 lysine 9: potential effect of phosphorylation on methyl-lysine binding

M-phase phosphoprotein 8 (MPP8) harbors an N-terminal chromodomain and a C-terminal ankyrin repeat domain. MPP8, via its chromodomain, binds histone H3 peptide tri- or di-methylated at lysine 9 (H3K9me3/H3K9me2) in submicromolar affinity. We determined the crystal structure of MPP8 chromodomain in complex with H3K9me3 peptide. MPP8 interacts with at least six histone H3 residues from glutamine 5 to serine 10, enabling its ability to distinguish lysine-9-containing peptide (QTARKS) from that of lysine 27 (KAARKS), both sharing the ARKS sequence. A partial hydrophobic cage with three aromatic residues (Phe59, Trp80 and Tyr83) and one aspartate (Asp87) encloses the methylated lysine 9. MPP8 has been reported to be phosphorylated in vivo, including the cage residue Tyr83 and the succeeding Thr84 and Ser85. Modeling a phosphate group onto the side-chain hydroxyl oxygen of Tyr83 suggests that the negatively charged phosphate group could enhance the binding of positively charged methyl-lysine or create a regulatory signal by allowing or inhibiting binding of other protein(s).     

Structure-function analysis of Arg-Gly-Asp helix motifs in alpha v beta 6 integrin ligands

Data relating to the structural basis of ligand recognition by integrins are limited. Here we describe the physical requirements for high affinity binding of ligands to alpha v beta6. By combining a series of structural analyses with functional testing, we show that 20-mer peptide ligands, derived from high affinity ligands of alpha v beta6 (foot-and-mouth-disease virus, latency associated peptide), have a common structure comprising an Arg-Gly-Asp motif at the tip of a hairpin turn followed immediately by a C-terminal helix. This arrangement allows two conserved Leu/Ile residues at Asp(+1) and Asp(+4) to be presented on the outside face of the helix enabling a potential hydrophobic interaction with the alpha v beta6 integrin, in addition to the Arg-Gly-Asp interaction. The extent of the helix determines peptide affinity for alpha v beta6 and potency as an alpha v beta6 antagonist. A major role of this C-terminal helix is likely to be the correct positioning of the Asp(+1) and Asp(+4) residues. These data suggest an explanation for several biological functions of alpha v beta6 and provide a structural platform for design of alpha v beta6 antagonists.     

Structure of a specific peptide complex of the carboxy-terminal SH2 domain from the p85 alpha subunit of phosphatidylinositol 3-kinase.

We have determined the solution structure of the C-terminal SH2 domain of the p85 alpha subunit of human phosphatidylinositol (PI) 3-kinase (EC 2.7.1.137) in complex with a phosphorylated tyrosine pentapeptide sequence from the platelet-derived growth factor receptor using heteronuclear nuclear magnetic resonance spectroscopy. Overall, the structure is similar to other SH2 domain complexes, but displays different detail interactions within the phosphotyrosine binding site and in the recognition site for the +3 methionine residue of the peptide, the side chain of which inserts into a particularly deep and narrow pocket which is displaced relative to that of other SH2 domains. The contacts made within this +3 pocket provide the structural basis for the strong selection for methionine at this position which characterizes the SH2 domains of PI3-kinase. Comparison with spectral and structural features of the uncomplexed domain shows that the long BG loop becomes less mobile in the presence of the bound peptide. In contrast, extreme resonance broadening encountered for most residues in the beta D', beta E and beta F strands and associated connecting loops of the domain in the absence of peptide persists in the complex, implying conformational averaging in this part of the molecule on a microsecond-to-millisecond time scale.     

NMR-based binding screen and structural analysis of the complex formed between alpha-cobratoxin and an 18-mer cognate peptide derived from the alpha 1 subunit of the nicotinic acetylcholine receptor from Torpedo californica

The alpha18-mer peptide, spanning residues 181-198 of the Torpedo nicotinic acetylcholine receptor alpha1 subunit, contains key binding determinants for agonists and competitive antagonists. To investigate whether the alpha18-mer can bind other alpha-neurotoxins besides alpha-bungarotoxin, we designed a two-dimensional (1)H-(15)N heteronuclear single quantum correlation experiment to screen four related neurotoxins for their binding ability to the peptide. Of the four toxins tested (erabutoxin a, erabutoxin b, LSIII, and alpha-cobratoxin), only alpha-cobratoxin binds the alpha18-mer to form a 1:1 complex. The NMR solution structure of the alpha-cobratoxin.alpha18-mer complex was determined with a backbone root mean square deviation of 1.46 A. In the structure, alpha-cobratoxin contacts the alpha18-mer at the tips of loop I and II and through C-terminal cationic residues. The contact zone derived from the intermolecular nuclear Overhauser effects is in agreement with recent biochemical data. Furthermore, the structural models support the involvement of cation-pi interactions in stabilizing the complex. In addition, the binding screen results suggest that C-terminal cationic residues of alpha-bungarotoxin and alpha-cobratoxin contribute significantly to binding of the alpha18-mer. Finally, we present a structural model for nicotinic acetylcholine receptor-alpha-cobratoxin interaction by superimposing the alpha-cobratoxin.alpha18-mer complex onto the crystal structure of the acetylcholine-binding protein (Protein Data Bank code ).     

Evolutionary origin of peptidoglycan recognition proteins in vertebrate innate immune system

Background  

Innate immunity is the ancient defense system of multicellular organisms against microbial infection. The basis of this first line of defense resides in the recognition of unique motifs conserved in microorganisms, and absent in the host. Peptidoglycans, structural components of bacterial cell walls, are recognized by Peptidoglycan Recognition Proteins (PGRPs). PGRPs are present in both vertebrates and invertebrates. Although some evidence for similarities and differences in function and structure between them has been found, their evolutionary history and phylogenetic relationship have remained unclear. Such studies have been severely hampered by the great extent of sequence divergence among vertebrate and invertebrate PGRPs. Here we investigate the birth and death processes of PGRPs to elucidate their origin and diversity.     

Structural and biochemical analysis of sliding clamp/ligand interactions suggest a competition between replicative and translesion DNA polymerases

Most DNA polymerases interact with their cognate processive replication factor through a small peptide, this interaction being absolutely required for their function in vivo. We have solved the crystal structure of a complex between the beta sliding clamp of Escherichia coli and the 16 residue C-terminal peptide of Pol IV (P16). The seven C-terminal residues bind to a pocket located at the surface of one beta monomer. This region was previously identified as the binding site of another beta clamp binding protein, the delta subunit of the gamma complex. We show that peptide P16 competitively prevents beta-clamp-mediated stimulation of both Pol IV and alpha subunit DNA polymerase activities, suggesting that the site of interaction of the alpha subunit with beta is identical with, or overlaps that of Pol IV. This common binding site for delta, Pol IV and alpha subunit is shown to be formed by residues that are highly conserved among many bacterial beta homologs, thus defining an evolutionarily conserved hydrophobic crevice for sliding clamp ligands and a new target for antibiotic drug design.     

Structural basis for interaction of O-acetylserine sulfhydrylase and serine acetyltransferase in the Arabidopsis cysteine synthase complex

In plants, association of O-acetylserine sulfhydrylase (OASS) and Ser acetyltransferase (SAT) into the Cys synthase complex plays a regulatory role in sulfur assimilation and Cys biosynthesis. We determined the crystal structure of Arabidopsis thaliana OASS (At-OASS) bound with a peptide corresponding to the C-terminal 10 residues of Arabidopsis SAT (C10 peptide) at 2.9-A resolution. Hydrogen bonding interactions with key active site residues (Thr-74, Ser-75, and Gln-147) lock the C10 peptide in the binding site. C10 peptide binding blocks access to OASS catalytic residues, explaining how complex formation downregulates OASS activity. Comparison with bacterial OASS suggests that structural plasticity in the active site allows binding of SAT C termini with dissimilar sequences at structurally similar OASS active sites. Calorimetric analysis of the effect of active site mutations (T74S, S75A, S75T, and Q147A) demonstrates that these residues are important for C10 peptide binding and that changes at these positions disrupt communication between active sites in the homodimeric enzyme. We also demonstrate that the C-terminal Ile of the C10 peptide is required for molecular recognition by At-OASS. These results provide new insights into the molecular mechanism underlying formation of the Cys synthase complex and provide a structural basis for the biochemical regulation of Cys biosynthesis in plants.     

Peptidoglycan recognition proteins of the innate immune system

Peptidoglycan (PGN) is the major component of bacterial cell walls and one of the main microbial products recognized by the innate immune system. PGN recognition is mediated by several families of pattern recognition molecules, including Toll-like receptors, nucleotide-binding oligomerization domain-containing proteins, and peptidoglycan recognition proteins (PGRPs). However, only the interaction of PGN with PGRPs, which are highly conserved from insects to mammals, has so far been characterized at the molecular level. Here, we describe recent structural studies of PGRPs that reveal the basis for PGN recognition and provide insights into the signal transduction and antibacterial activities of these innate immune proteins.     

Peptidoglycan recognition protein (PGRP) from eri-silkworm, Samia cynthia ricini; protein purification and induction of the gene expression

Peptidoglycan recognition protein (PGRP) was isolated from immunized hemolymph of the wild silkworm, Samia cynthia ricini, detecting the biding activity with (125)I-labeled peptidoglycan (PGN). The binding specificity of PGRP was tested by competitive inhibition of the binding to (125)I-labeled-PGN by a large excess amount of non-labeled-PGN or other glucans. The binding to labeled uncross-linked Lys-type PGN from Micrococcus luteus was strongly inhibited by non-labeled-PGN of the same structure and meso-diaminopimelic acid (DAP)-type cross-linked PGN from Bacillus cell wall, but only a little by cross-linked PGN from M. luteus cell wall. The PGRP cDNA encodes a 193 amino acid open reading frame. The deduced amino acid sequence had 62 to 91% identities to known lepidopteran PGRPs, but less than 40% to Drosophila PGRPs. The PGRP gene constitutively expressed at a low level in naive fat body, and strongly induced by an injection of DAP-type cross-linked and Lys-type uncross-linked PGNs, but only weakly by Lys-type cross-linked PGN from M. luteus. The silkworm possibly distinguish between PGNs based on the structure of cross-linking peptide, but has less if any preference for the diamino acid residue of the stem peptide.     

The methyl group of N(alpha)(Me)Arg-containing peptides disturbs the active-site geometry of thrombin, impairing efficient cleavage

Bivalent peptidic thrombin inhibitors consisting of an N-terminal d-cyclohexylalanine-Pro-N(alpha)(Me)Arg active-site fragment, a flexible polyglycine linker, and a C-terminal hirugen-like segment directed towards the fibrinogen recognition exosite inhibit thrombin with K(i) values in the picomolar range, remaining stable in buffered solution at pH 7.8 for at least 15 hours. In order to investigate the structural basis of this increased stability, the most potent of these inhibitors, I-11 (K(i)=37pM), containing an N(alpha)(Me)Arg-Thr bond, was crystallized in complex with human alpha-thrombin. X-ray data were collected to 1.8A resolution and the crystal structure of this complex was determined. The Fourier map displays clear electron density for the N-terminal fragment and for the exosite binding segment. It indicates, however, that in agreement with Edman sequencing, the peptide had been cleaved in the crystal, presumably due to the long incubation time of 14 days needed for crystallization and data collection. The N(alpha)(Me) group is directed toward the carbonyl oxygen atom of Ser214, pushing the Ser195 O(gamma) atom out of its normal site. This structure suggests that upon thrombin binding, the scissile peptide bond of the intact peptide and the Ser195 O(gamma) are separated from each other, impairing the nucleophilic attack of the Ser195 O(gamma) toward the N(alpha)(Me)Arg carbonyl group. In the time-scale of two weeks, however, cleavage geometries favoured by the crystal allow catalysis at a slow rate.