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Peptidoglycan recognition proteins of the innate immune system Rongjin Guan 1, 2 and Roy A. Mariuzza 1 1 Center for Advanced Research in Biotechnology, W.M. Keck Laboratory for Structural Biology, University of Maryland Biotechnology Institute, Rockville, MD 20850, USA 2 Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA Peptidoglycan (PGN) is the major component of bacterial cell walls and one of the main microbial products recog- nized by the innate immune system. PGN recognition is mediated by several families of pattern recognition mol- ecules, including Toll-like receptors, nucleotide-binding oligomerization domain-containing proteins, and pepti- doglycan 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 as a target for innate immune recognition The innate immune system is the first line of defense against invading microorganisms in vertebrates and the only line of defense in invertebrates and plants [1,2]. It recognizes microbes by means of pattern recognition mol- ecules, such as Toll-like receptors, collectins and peptido- glycan recognition proteins (PGRPs), which are conserved in evolution to bind unique products of microbial metab- olism not produced by the host (pathogen-associated mol- ecular patterns). Examples of microbial ligands recognized by pattern recognition molecules include lipopolysacchar- ide (LPS) of Gram-negative bacteria, lipoteichoic acid of Gram-positive bacteria, flagellin, mannans, nonmethy- lated CpG sequences and peptidoglycan (PGN), which is present in both Gram-positive and Gram-negative bacteria [1,2]. Cellular activation by pattern recognition molecules following ligand binding results in acute inflammatory responses involving direct local attack against the invad- ing pathogen, cytokine and chemokine production, and induction of the adaptive component of the immune system (in the case of vertebrates). As a major constituent of the cell wall of virtually all bacteria, PGN represents an excellent target for innate immune recognition. PGNs are polymers of alternating N- acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) in a b(1!4) linkage, crosslinked by short pep- tide stems composed of alternating L- and D- amino acids [3,4] (Figure 1). Whereas the carbohydrate backbone is conserved among all bacteria (except for de-N-acetylated or O-acetylated variants), the peptide moiety displays con- siderable diversity. According to the residue at position three of the peptide stems, PGNs are divided into two major categories: L-lysine type (Lys-type) and meso-diami- nopimelic acid type (Dap-type). Dap-type PGN stems are usually directly crosslinked, whereas Lys-type PGN stems are interconnected by a peptide bridge that varies in length and amino acid composition in different bacteria (Figure 1). Moreover, bacteria differ widely in the extent of cross- linking, thus introducing additional diversity in PGN structure [3,4]. Several families of pattern recognition molecules have been shown to detect PGNs, including CD14 [5], which also binds to LPS, Toll-like receptor 2 (although this is con- troversial) [6,7], nucleotide-binding oligomerization domain-containing proteins (NODs) [8] and PGRPs, which are the subject of this review. To date, however, only the interaction of PGN with PGRPs has been characterized at the molecular level by X-ray crystallography. Indeed, except for collectins [9], which bind to mannans, no struc- tural information is available on how any other pattern recognition molecules engage microbial ligands. Here, we present the general characteristics and biological functions of PGRPs (reviewed in greater detail elsewhere; see Ref. [10]). We describe recent structural studies of PGRPs that have revealed the basis for PGN recognition and discuss the results within the context of the role of PGRPs in immune defense. Distribution and characteristics of PGRPs PGRPs are found in most animals, including insects, echinoderms, molluscs and vertebrates, but not in lower metazoa (nematodes) or plants [10]. These highly con- served innate immunity molecules were first discovered in silkworm (Bombyx mori) as 20-kDa proteins with the ability to trigger the prophenoloxidase (PPO) cascade upon binding to PGN [11]. Subsequently, PGRPs were identified in other insects such as Drosophila and moths (Trichoplu- sia ni), and in mice and humans [12]. The PGRP family currently includes nearly 100 members [10], all of which have a homologous PGN-binding domain of 165 amino acids in length. This domain shares 30% sequence sim- ilarity with bacteriophage T7 lysozyme, a Zn 2+ -dependent amidase that hydrolyzes PGN [13]. Like T7 lysozyme, some PGRPs (referred to as catalytic PGRPs) hydrolyze PGN by Review TRENDS in Microbiology Vol.15 No.3 Corresponding author: Mariuzza, R.A. ([email protected]). Available online 1 February 2007. www.sciencedirect.com 0966-842X/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2007.01.006

Peptidoglycan recognition proteins of the innate immune system

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Page 1: Peptidoglycan recognition proteins of the innate immune system

Peptidoglycan recognition proteins ofthe innate immune systemRongjin Guan1,2 and Roy A. Mariuzza1

1 Center for Advanced Research in Biotechnology, W.M. Keck Laboratory for Structural Biology, University of Maryland

Biotechnology Institute, Rockville, MD 20850, USA2 Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health,

Bethesda, MD 20892, USA

Review TRENDS in Microbiology Vol.15 No.3

Peptidoglycan (PGN) is the major component of bacterialcell walls and one of the main microbial products recog-nized by the innate immune system. PGN recognition ismediated by several families of pattern recognition mol-ecules, including Toll-like receptors, nucleotide-bindingoligomerization domain-containing proteins, and pepti-doglycan recognition proteins (PGRPs). However, onlythe interaction of PGN with PGRPs, which are highlyconserved from insects to mammals, has so far beencharacterized at the molecular level. Here, we describerecent structural studies of PGRPs that reveal the basis forPGN recognition and provide insights into the signaltransduction and antibacterial activities of these innateimmune proteins.

Peptidoglycan as a target for innate immunerecognitionThe innate immune system is the first line of defenseagainst invading microorganisms in vertebrates and theonly line of defense in invertebrates and plants [1,2]. Itrecognizes microbes by means of pattern recognition mol-ecules, such as Toll-like receptors, collectins and peptido-glycan recognition proteins (PGRPs), which are conservedin evolution to bind unique products of microbial metab-olism not produced by the host (pathogen-associated mol-ecular patterns). Examples of microbial ligands recognizedby pattern recognition molecules include lipopolysacchar-ide (LPS) of Gram-negative bacteria, lipoteichoic acid ofGram-positive bacteria, flagellin, mannans, nonmethy-lated CpG sequences and peptidoglycan (PGN), which ispresent in both Gram-positive and Gram-negative bacteria[1,2]. Cellular activation by pattern recognition moleculesfollowing ligand binding results in acute inflammatoryresponses involving direct local attack against the invad-ing pathogen, cytokine and chemokine production, andinduction of the adaptive component of the immune system(in the case of vertebrates).

As a major constituent of the cell wall of virtually allbacteria, PGN represents an excellent target for innateimmune recognition. PGNs are polymers of alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid(MurNAc) in a b(1!4) linkage, crosslinked by short pep-tide stems composed of alternating L- and D- amino acids[3,4] (Figure 1). Whereas the carbohydrate backbone is

Corresponding author: Mariuzza, R.A. ([email protected]).Available online 1 February 2007.

www.sciencedirect.com 0966-842X/$ – see front matter � 2007 Elsevier Ltd. All rights reserv

conserved among all bacteria (except for de-N-acetylated orO-acetylated variants), the peptide moiety displays con-siderable diversity. According to the residue at positionthree of the peptide stems, PGNs are divided into twomajor categories: L-lysine type (Lys-type) and meso-diami-nopimelic acid type (Dap-type). Dap-type PGN stems areusually directly crosslinked, whereas Lys-type PGN stemsare interconnected by a peptide bridge that varies in lengthand amino acid composition in different bacteria (Figure 1).Moreover, bacteria differ widely in the extent of cross-linking, thus introducing additional diversity in PGNstructure [3,4].

Several families of pattern recognition molecules havebeen shown to detect PGNs, including CD14 [5], which alsobinds to LPS, Toll-like receptor 2 (although this is con-troversial) [6,7], nucleotide-binding oligomerizationdomain-containing proteins (NODs) [8] and PGRPs, whichare the subject of this review. To date, however, only theinteraction of PGN with PGRPs has been characterized atthe molecular level by X-ray crystallography. Indeed,except for collectins [9], which bind to mannans, no struc-tural information is available on how any other patternrecognition molecules engage microbial ligands. Here, wepresent the general characteristics and biological functionsof PGRPs (reviewed in greater detail elsewhere; see Ref.[10]). We describe recent structural studies of PGRPs thathave revealed the basis for PGN recognition and discussthe results within the context of the role of PGRPs inimmune defense.

Distribution and characteristics of PGRPsPGRPs are found in most animals, including insects,echinoderms, molluscs and vertebrates, but not in lowermetazoa (nematodes) or plants [10]. These highly con-served innate immunity molecules were first discoveredin silkworm (Bombyx mori) as 20-kDa proteins with theability to trigger the prophenoloxidase (PPO) cascade uponbinding to PGN [11]. Subsequently, PGRPs were identifiedin other insects such as Drosophila and moths (Trichoplu-sia ni), and in mice and humans [12]. The PGRP familycurrently includes nearly 100 members [10], all of whichhave a homologous PGN-binding domain of �165 aminoacids in length. This domain shares �30% sequence sim-ilarity with bacteriophage T7 lysozyme, a Zn2+-dependentamidase that hydrolyzes PGN [13]. Like T7 lysozyme, somePGRPs (referred to as catalytic PGRPs) hydrolyze PGN by

ed. doi:10.1016/j.tim.2007.01.006

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Figure 1. Structure of PGNs. (a) Schematic representation of Lys-type PGNs. In Staphylococcus aureus, Lys-type PGN peptides are crosslinked through a peptide bridge

composed of one to five glycines. However, this bridge varies in length and amino acid composition in different bacteria [3,4]. The number of amino acids (n) can range

from zero to five, depending on the organism. (b) In Dap-type PGNs, L-lysine at position three of the peptide stems is replaced by meso-diaminopimelic acid and the peptide

stems are usually directly connected.

128 Review TRENDS in Microbiology Vol.15 No.3

cleaving the amide bond between MurNAc and L-Ala[14–17]. Non-catalytic PGRPs, which comprise the largemajority of this protein family, bind to PGN but do nothave amidase activity owing to the lack of a key cysteineresidue for Zn2+ binding [12,15,18]. With respect to PGNrecognition, both catalytic and non-catalytic PGRPs showdistinct specificities for PGNs from Gram-positive orGram-negative bacteria.

Insect PGRPs are grouped into two classes: shortPGRPs (PGRP-S), which are small (20 kDa) extracellularproteins, and long PGRPs (PGRP-L) of up to 90 kDa, whichcan be intracellular, extracellular or transmembrane. Dro-sophila melanogaster has 13 PGRP genes that are tran-scribed into at least 17 proteins [19] and a species ofmosquito (Anopheles gambiae) has seven PGRP genes thatare transcribed into at least nine proteins [20]. Many of theinsect PGRPs are expressed in immune competent organs,such as the fat body, gut and hemocytes [2]. In most cases,PGRP expression is up-regulated by exposure to PGN orbacteria, suggesting an important role in antimicrobialdefense (see later). Indeed, all functions of insect PGRPsidentified so far involve innate immune responses. Phylo-genetic analysis of insect PGRPs has suggested a possibleearly separation into catalytic PGRPs, which hydrolyzePGN, and non-catalytic PGRPs, which activate proteolyticcascades or signaling pathways [10].

Mammals have four PGRPs, termed PGRP-S, PGRP-L,and PGRP-Ia and PGRP-Ib (for short, long and intermedi-ate, respectively). (Recently, the Human Genome Organiz-ation Gene Nomenclature Committee changed their namesto PGLYRP-1, PGLYRP-2, PGLYRP-3, and PGLYRP-4,respectively; however, to avoid confusion, we retain the

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original nomenclature here.) Of these, only PGRP-L hasamidase activity [14,17]. PGRP-L also contains a large N-terminal domain of unknown function. By contrast to insectPGRPs, some of which are transmembrane receptors, allmammalian PGRPs are soluble intracellular or secretedmolecules. Thus, PGRP-S is found in phagocytic granules[21–24], PGRP-Ia and PGRP-Ib are present on skin andmucous membranes [25,26] and PGRP-L is constitutivelyproduced by the liver [27]. Some mammals such as pigsexpressmultiple splice variants of PGRP-L thatmight havedistinct roles [28].

Functions of PGRPs in innate immunityInsect PGRPs are involved in important signalingpathways in innate immunity. Two Drosophila PGRPs,PGRP-SA and PGRP-SD, recognize bacterial PGN and acti-vate the Toll receptor [29,30]. PGRP-SA is required fortriggering the Toll pathwaywhereas PGRP-SD is not essen-tial but enhances signaling. Activation of Toll initiates asignal transduction cascade that results in the expression ofdrosomycin and other antimicrobial peptides, which areprimarily active against Gram-positive bacteria and fungi[2]. Toll is mainly triggered by Lys-type PGN from Gram-positive bacteria and only weakly triggered by Dap-typePGN from Gram-negative bacteria [31].

The Drosophila Imd pathway, which regulatesexpression of diptericin and other antimicrobial peptidesactive against Gram-negative bacteria, is triggered by thebinding of Dap-type PGN to PGRP-LC in cooperation withPGRP-LE [32–34]. PGRP-LC is a transmembrane receptorwith three alternative splice isoforms (PGRP-LCa, -LCxand -LCy) that have somewhat different specificities,

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Review TRENDS in Microbiology Vol.15 No.3 129

whereas PGRP-LE exists as either a soluble intracellularor extracellular protein [34]. PGN binding to PGRP-LCinduces formation of PGRP-LCa–PGRP-LCx heterodimerson the cell surface, which in turn induces downstreamsignaling [35–37]. Unlike non-catalytic PGRPs, catalyticPGRPs are thought to function as scavengers that areimplicated in terminating the immune response to PGN[16]. In this regard, the Drosophila amidases PGRP-LB,PGRP-SC1 and PGRP-SC2 negatively regulate the Imdpathway to adjust immune activation to infection byGram-negative bacteria [38,39].

A third pathway in insects triggered by PGRPs is thePPO cascade, a proteolytic cascade in the hemolymph thatleads to localized wound healing and melanization [40,41].Several PGRPs, including PGRP-S from both B. mori [11]and mealworm (Tenebrio molitor) [42], and PGRP-LE fromDrosophila [43], activate the PPO pathway following PGNrecognition. In addition, PGRP-1 from a beetle (Holotrichiadiomphalia) triggers this pathway by sensing 1,3-b-D-glucan, which is a component of fungal cell walls [44].

Mammalian PGRPs are selectively expressed in avariety of tissues including bone marrow (PGRP-S), skinand intestinal tract (PGRP-Ia and -Ib), and liver (PGRP-L)[12,25,26,45]. Initially, mammalian PGRPs were thoughtto activate signaling pathways in ways analogous to insectPGRPs [46]; however,more recent data have demonstratedthat the mammalian proteins are bactericidal (PGRP-S, -Ia and -Ib) or function as scavengers (PGRP-L) [14,17,21–25]. The bactericidal activity of PGRP-S requires Ca2+,without which it is only bacteriostatic [21,23,24]. HumanPGRP-S, which is present in polymorphonuclear leukocytegranules, participates in the intracellular killing of bac-teria [21] and is an effector of neutrophil-mediated innateimmunity [24]. In addition, mice deficient in PGRP-Sexhibit increased susceptibility to intraperitoneal infec-tions with Gram-positive bacteria [23].

HumanPGRP-Ia and -Ib are secreted as disulfide-linkedhomo- or heterodimers and are directly bactericidal againstpathogenic and nonpathogenic Gram-positive bacteria butnot against normal flora bacteria [25]. They are also bacter-iostatic for Gram-negative bacteria and for normal floraGram-positive bacteria. In sharp contrast to antimicrobialpeptides such as defensins, which permeabilize bacterialmembranes, PGRP-Ia and -Ib kill bacteria by interactingwith the cell wall where they presumably inhibit PGNbiosynthesis [25]. If so, this mechanism would resemblethose of peptide antibiotics such as vancomycin and acta-gardine, which interfere with PGN synthesis by binding toPGN or its precursors. PGRP-L, a catalytic PGRP producedin the liver and secreted into the bloodstream [27], mightprevent excessive inflammation or attenuate immuneresponses to bacteria by hydrolyzing PGN. It is also possiblethat PGRP-L, by hydrolyzing PGNs, can generate ligandsfor other innate immunity proteins, such as NOD1.

Structure of PGRPsEach PGRP contains a single PGN-binding domain(C-terminal in the case of long PGRPs), except for mam-malian PGRP-Ia and -Ib and Drosophila PGRP-LF inwhich there are two tandem PGN-binding domains [18].It was predicted that PGRPs would have a similar fold to

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T7 lysozyme and this was first confirmed by the crystalstructure of Drosophila PGRP-LB [15]. Subsequently, thePGN-binding domain structures of six additional PGRPswere determined: PGRP-Ia C-terminal PGN-bindingdomain (designated PGRP-IaC) [18,47,48] and PGRP-Sfrom humans [49] and PGRP-SA [50,51], PGRP-LCa[52], PGRP-LE [53] and PGRP-LCx [54] from Drosophila.All of these structures exhibit a similar overall scaffoldthat contains a central b-sheet composed of five b-strands[four parallel and one (b5) antiparallel] and three a-helices(Figure 2a). The structures are cross-linked by 1–3 dis-ulfide bonds, which probably serve to stabilize PGRPs inthe harsh environment of the extracellular compartmentor phagocytic granules. One of these disulfides (corre-sponding to Cys214–Cys220 in PGRP-IaC) is conservedin all PGRPs except Drosophila PGRP-LE. The Cys194–Cys238 disulfide in PGRP-IaC is unique to mammalianPGRPs, in which it is retained in all noncatalytic PGRPsbut is absent from PGRPs with known PGN-hydrolyzingactivity. All PGRPs share a conserved L-shaped PGN-binding groove (Figure 2b) with one deep and one shallowend [47].

In addition, all PGN-binding domains contain anN-terminal segment of 30–50 residues, characterized byhigh sequence and conformational variability, which isabsent from T7 lysozyme and, hence, is known as thePGRP-specific segment (Figure 2c). This segment, withhelix a2, forms a broad hydrophobic groove on the faceopposite the PGN-binding site of each protein (Figure 2d).The possibility exists that this topologically variable sur-face might serve as a docking site for different effector orsignaling proteins [15,18,49] that are still largely unde-fined. In this regard,Drosophila PGRP-SA has been shownto associate with Gram-negative binding protein 1 (GNBP-1) in activating the Toll receptor pathway [55–57]. Thisinteraction might be mediated by the PGRP-specific seg-ment of PGRP-SA.

The catalytic PGRPs include Drosophila PGRP-LB [15]and PGRP-SC1b [16] and the vertebrate PGRP-L [14,17].In the Drosophila PGRP-LB structure [15], a zinc ion isfound in the PGN-binding groove, coordinated by threeresidues (His42, His152, Cys160) that are strictly con-served in all catalytic PGRPs (Figure 2e). A general mech-anism for PGN hydrolysis by PGRPs has been proposed, inwhich the bound Zn2+ functions as an electrophilic catalystto facilitate cleavage of the amide bond between MurNAcand L-Ala. [47]. Although most non-catalytic PGRPs retainresidues corresponding to His42 and His152 of PGRP-LB,they invariably lack the zinc-coordinating residue Cys160(Figure 2f), which has been shown by mutagenesis to benecessary for PGN hydrolysis. As a consequence, non-catalytic PGRPs, which include most PGRPs, do not con-tain Zn2+ and bind to PGNs but do not hydrolyze them.

Interaction of PGRPs with PGNUnderstanding the molecular basis for PGN recognition byPGRPs requires direct information on PGRP–PGN inter-actions in the binding site. Accordingly, several crystalstructures have recently been determined of non-catalyticPGRPs in complex with small PGN fragments, eithersynthetic or naturally occurring [47,48,53,54]. The use of

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Figure 2. Overall structure of PGRPs. (a) Structure of human PGRP-IaC. Secondary structure elements are numbered sequentially. The N and C termini are labeled. The view

is looking down on the PGN-binding cleft, whose walls are formed by helix a1 and four loops extending above the b-sheet platform. The N-terminal PGRP-specific segment

is red. Disulfide bonds are yellow. The Cys214–Cys220 disulfide (labeled) is conserved in all PGRPs; Cys194–Cys238 is found exclusively in mammalian PGRPs. (b) Surface

representation of the PGN-binding site of PGRP-IaC. The orientation is the same as in part (a). The molecular surface is colored according to the percentage identities of

residues lining the PGN-binding groove based on sequence alignments of 45 PGRPs. Red, >80%; purple, 60–80%; yellow, 40–60%; and green, <40%. (c) View of the opposite

face of PGRP-IaC from that in part (a), looking down onto the PGRP-specific segment. (d) Surface analysis of a potential protein-binding site of PGRP-IaC showing a

hydrophobic groove (outlined in yellow) formed by the PGRP-specific segment and helix a2. Hydrophobic regions are green; polar regions are red. The protein is in a similar

orientation to that in part (c). (e) The PGN-binding site of Drosophila PGRP-LB, a catalytic PGRP with PGN hydrolyzing activity. Side chains of residues contributing to PGN

recognition or hydrolysis are shown. Carbon atoms are yellow, nitrogen atoms are blue, oxygen atoms are red and sulfur atoms are green. The bound zinc ion (purple

sphere) is coordinated by His42, His152 and Cys160. (f) The PGN binding site of PGRP-IaC, a non-catalytic PGRP lacking a bound zinc ion because one of the residues

coordinating Zn2+ in PGRP-LB (Cys160) is replaced by Ser324 in PGRP-IaC. The view is the same as in part (e).

130 Review TRENDS in Microbiology Vol.15 No.3

such fragments is necessitated by the polymeric structureof PGN, which renders this large, heterogeneous mole-cule unsuitable for crystallization with PGRPs in its nativeform. The first reported PGRP–PGN structure was that ofa complex between human PGRP-IaC and MurNAc-L-Ala-D-isoGln-L-Lys, a synthetic muramyl tripeptide (MTP)representing the conserved core of Lys-type PGNs fromGram-positive bacteria [47]. Subsequently, the crystalstructure of PGRP-IaC in complex with a muramyl penta-peptide (MPP) containing a complete peptide stem (Mur-NAc-L-Ala-D-isoGln-L-Lys-D-Ala-D-Ala) was determined,providing a more comprehensive picture of PGRP–PGN

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interactions [48]. In this complex (Figure 3a), the peptidestem of the ligand is buried at the deep end of the PGN-binding groove and theN-acetylmuramic acid is situated inthe middle of the groove with its pyranose ring orientedperpendicularly to the base of the pocket.

In the PGRP-IaC–MPP complex (Figure 3a), the ligandis�60% buried in the PGN-binding site with its glycan andpeptide portions buried to similar extents. Comparison ofthe PGRP-IaC–MPP and PGRP-IaC–MTP structuresrevealed several conformational differences that contrib-ute to MPP recognition, including large side-chainrotations that lock the peptide stem of MPP into the

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Figure 3. Structure of PGRP–PGN complexes. (a) Stereo view of the human PGRP-IaC–MPP complex. Helices are red, strands are yellow and coils are gray; disulfide bonds

are purple. Secondary structural elements are labeled following the numbering for unbound PGRP-IaC (see Figure 2a). The bound MPP is drawn in stick representation with

carbon atoms in green, nitrogen atoms in blue and oxygen atoms in red. (b) Schematic representation of the interaction between PGRP-IaC and MPP. MPP is red; hydrogen

bonds are drawn as blue dashed lines. Residues making van der Waals contacts with MPP are indicated by gray arcs with spokes radiating towards the ligand moieties they

contact. Water-mediated interactions between PGRP-IaC and MPP are omitted for clarity. (c) The Drosophila PGRP-LCa–PGRP-LCx–TCT ternary complex. TCT bound to

PGRP-LCx is drawn in stick representation; PGRP-LCa does not bind TCT. Helices are labeled as for PGRP-IaC. (d) The Drosophila PGRP-LE–TCT complex. The two PGRP-LE

molecules associate similarly to PGRP-LCx–TCT and PGRP-LCa, except that both molecules bind to TCT.

Review TRENDS in Microbiology Vol.15 No.3 131

binding groove [48]. MPP makes extensive interactionswith 20 PGRP residues, including numerous van derWaalscontacts and both direct and solvent-mediated hydrogenbonds (Figure 3b). Most of these interactions are with thepeptide, rather than glycan, portion of the PGN analog;however, both sets of contacts are essential for specificligand recognition. In addition, the majority of hydrogenbonds to the pentapeptide stem of MPP involve main-chain, rather than side-chain, atoms of the peptide.Although none of the PGN-contacting residues in thecomplex is completely invariant among the almost 100PRGP sequences reported to date, five of them (His208,His231, Tyr242, His264, Asn269) are >80% identical.These residues form a nearly contiguous patch on the floorof the binding groove (Figure 2b), strongly suggesting thatall PGRPs employ this same basic PGN-binding mode.

This conclusion is supported by the crystal structure ofa heterodimeric receptor formed by Drosophila PGRP-LCaand -LCx in complex with tracheal cytotoxin (TCT), anaturally occurring monomeric fragment of Dap-typePGN containing an anhydro form of MurNAc [54]. Thisstructure also provides important new insights into PGRPsas both recognition and signalingmolecules. TCT [GlcNAc-MurNAc(1,6-anhydro)-L-Ala-g-D-Glu-meso-Dap-D-Ala] is a

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potent activator of the Drosophila Imd pathway [35] and aprincipal contributor to tissue damage in whooping coughand gonorrhea [58,59], although the mammalian receptorfor TCT is unknown. Recognition of TCT in Drosophilarequires both PGRP-LCa and -LCx [35,37], which are typeII transmembrane proteins that have identical cytoplasmicand transmembrane domains but different PGRP domainsin their extracellular regions. TCT binds directly to theectodomain of PGRP-LCx, but not to PGRP-LCa, whosePGN-binding groove is partially occluded by amino acidinsertions [52]. Upon binding PRGP-LCx, TCT inducesheterodimerization of PGRP-LCx and -LCa, resulting injuxtaposition of their cytoplasmic domains and receptoractivation [35–37]. In the PGRP-LCa–PGRP-LCx–TCTternary complex (Figure 3c), PGRP-LCx (which has atypical PGN-binding cleft) engages TCT in a similar man-ner to the binding of MPP by PGRP-IaC (Figure 3a). Theexposed GlcNAc-MurNAc(1,6-anhydro) moiety of boundTCT and adjacent elements of PGRP-LCx together formthe recognition surface for PGRP-LCa, which contacts TCTand PGRP-LCx using a site that is spatially separate fromits atypical PGN-binding groove. Thus, TCT forms anintegral part of the interface between the two proteins,stabilizing the ternary complex.

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132 Review TRENDS in Microbiology Vol.15 No.3

Drosophila PGRP-LE is a soluble protein that lacks atransmembrane domain or signal sequence and is anintracellular receptor for TCT [34]. In the complex betweenTCT and PGRP-LE (Figure 3d), TCT is bound by twoPGRP-LE molecules in a manner closely resembling thatobserved in the PGRP-LCa–PGRP-LCx–TCT complex [53].Thus, one PGRP-LE interacts with TCT at the PGN-bind-ing groove as described for PGRP-LCx. At the same time, asecond PGRP-LE interacts with the disaccharide unit ofthe ligand through a site equivalent to the one employed byPGRP-LCa. Importantly, dimerization of PGRG-LEincreases its binding affinity (KD) for TCT by 30-fold to�30 nM [53]. In addition, TCT was shown to induce multi-merization of PGRP-LE in solution.

Human PRGP-Ia and -Ib each contain two PGN-bindingdomains and are secreted as disulfide-linked homo- or het-erodimers [25]. Two possibilities can be considered for thefunctional role of tandem domains. First, incorporation ofmultiple PGN-bindingdomainswithin a singlePGRPmightserve to augment avidity by permitting multivalent attach-ment to the bacterial cell wall. Second, tandem domainsmight confer multiple PGN-binding specificities to individ-ual PGRPs, thereby expanding the ability of a relativelysmall number of proteins to detect a broad spectrum ofmicrobial pathogens.

Specificity of PGRPs: discrimination between Dap- andLys-type PGNsSome PGRPs specifically or preferentially recognizeDap-type or Lys-type PGNs. For example, the DrosophilaImd pathway is activated by Dap-type PGN from Gram-negative bacteria through PGRP-LC or -LE, whereas theToll pathway is activated by Lys-type PGN from Gram-positive bacteria through PGRP-SA or -SD [2,29–35]. Sim-ilarly, human PGRP-IaC exhibits a strict requirementfor L-Lys at position three of the peptide stem, whereasPGRP-S preferentially binds Dap-type PGN [60]. Crystal-lographic studies of PGRP–PGN complexes combined with

Figure 4. Discrimination between Lys-type and Dap-type PGNs. Left: Structure of the hu

MPP is drawn in stick representation; carbon atoms are green, nitrogen atoms are blue

which form van der Waals contacts with the side chain of L-Lys. Val256 is also shown for

complex, showing the bidentate salt bridge between the side-chain carboxylate group

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site-directed mutagenesis have revealed the molecularbasis for this discrimination, which seems to be vital forthe biological function of PGRPs.

In the PGRP-IaC–MPP structure, Asn236 and Phe237interact with the side chain of L-Lys by forming several vander Waals contacts (Figure 4a) [47,48], suggesting thatsequence variability at these two positions might explainspecificity differences. Consistent with this hypothesis,sequence alignments showed that, like PGRP-IaC, PGRPsthat are known to recognize Lys-type but not Dap-typePGN (e.g. Drosophila PGRP-SA) contain Asn-Phe at pos-itions 236 and 237 (or homologous combinations such asAsp-Phe or Asn-Tyr) [60]. By comparison, the correspond-ing residues are Gly-Trp in PGRPs that preferentiallyrecognize Dap-type PGN (e.g. Drosophila PGRP-LCx, -LB and -LE, human PGRP-S). Mutation of residues Asn-Phe to Gly-Trp (or vice versa) in several PGRPs resulted inthe expected changes in PGN-binding specificity, asmeasured by microcalorimetry, thereby pinpointing thesite of PGRPs that mediates discrimination [60]. Thecrystal structures of PGRPs provide an explanation forthe effects of these mutations. Thus, in the PGRP-IaC–MPP complex (Figure 4a), the side chain of Asn236 pro-trudes from thewall of the binding groove, contacting the L-Lys side chain. Attachment of a carboxylate group to thisside chain, which distinguishes Dap from L-Lys, wouldcreate steric clashes with Asn236, decreasing affinity.The corresponding Gly of PGRPs that prefer Dap wouldnot interfere with binding because Gly lacks a side chain.This analysis provides a basis for predicting the PGN-binding specificity of PGRPs that have not been charac-terized experimentally [60].

Further insights into the basis for differential PGNrecognition come from the structures of DrosophilaPGRP-LE and -LCx bound to TCT [53,54], which hasDap in its peptide stem [58]. In both complexes, the si-de-chain carboxylate of Dap forms a bidentate salt bridgewith the guanidinium group of a conserved Arg (residue

man PGRP-IaC–MPP complex, showing interactions at the binding site. The bound

and oxygen atoms are red. PGRP-IaC is yellow. In purple are Asn236 and Phe237,

comparison to Arg254 in PGRP-LE. Right: Structure of the Drosophila PGRP-LE–TCT

of Dap and Arg254.

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Review TRENDS in Microbiology Vol.15 No.3 133

254 in PGRP-LE) (Figure 4b). Notably, nearly all PGRPscontaining the Gly-Trp motif described earlier also containa corresponding Arg, indicating that these amino acidsfunction in concert to stabilize the interaction with Dap-type PGN.

In addition to distinguishing between PGNs based onL-Lys or Dap in the peptide stem, PGRPs can also detectthe peptide bridge crosslinking the stems [60]. Thus,PGRPs have evolved dual strategies for PGN discrimi-nation. The length and composition of the connectingbridge vary in different bacteria, and bacteria differ widelyin the extent of crosslinking (5–75%) [3,4]. In experimentswith synthetic PGN fragments, some PGRPs (e.g. humanPGRP-IaC) bind equally well to crosslinked and non-cross-linked PGN, whereas others (e.g. human PGRP-S, Droso-phila PGRP-LC) recognize only non-crosslinked PGN [60].Importantly, the identity of the amino acid at positionthree of the stem, coupled with differences in the typeand amount of crosslinking between stems, accounts fornearly all the structural variability in PGNs from differentbacteria. The biological relevance of the finding that cer-tain PGRPs sense PGN crosslinking is supported by datashowing that lightly crosslinked (�25%) PGN is capable oftriggering theDrosophila Imd pathway through PGRP-LC,whereas heavily crosslinked (�75%) PGN is inactive [61].

Concluding remarks and future perspectivesRemarkable progress has been made during the pastseveral years towards understanding the role of PGRPs inhost defense, and its structural underpinnings. These stu-dies have revealed that all PGRPsprobablybind toPGNinasimilar manner but insect and mammalian PGRPs differmarkedly in how they accomplish antimicrobial defense:whereas insect PGRPs activate signal transduction path-ways, mammalian PGRPs are directly bactericidal. How-ever, substantial challenges remain. These include theidentification and structural characterization of moleculessuch as GNBP1 that link insect PGRPs to signaling path-ways, and the functions ofPGRPs in other invertebratesandinnon-mammalian vertebrates. SomePGRPs (e.g.mamma-lian PGRP-L and Drosophila PGRP-LE) contain large N-terminal domains with no homology to other knownproteins,whose structures and functions remain tobe estab-lished. Mammalian PGRPs are less well understood thantheir insect counterparts,particularlyregardingtheir role invivo. Moreover, although mammalian PGRPs have beenshown to be bactericidal, the underlying mechanism is stillunclear. In this regard, Drosophila PGRP-SB1 was alsofound to be bactericidal, with activity dependent on PGNhydrolysis by this catalytic PGRP [62]. By contrast, mam-malian PGRPs kill bacteria in the absence of enzymaticactivity [25] and must therefore employ a different mech-anism. To date, structural studies of PGN recognition byPGRPs have been restricted to complexes between PGRPsandmonomeric PGN fragments. However, because cell wallPGN is polymeric, the existing crystal structures might notfully capture the interaction of PGRPs with PGN. Advancesin the synthesis of discrete multimeric PGN fragments[60,63], including ones with crosslinked peptide stems,might open the way to crystallographic analysis of higherorder PGRP–PGN complexes that could exhibit cooperative

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binding interactions. Finally, the recent discovery of mouseandhumanC-type lectins that, likePGRPs,bindtoPGNandaredirectlybactericidal [64] suggests that thesestructurallyunrelated pattern recognition molecules (and possiblyothers as well) mediate innate antimicrobial defense inmammals through functionally related mechanisms.

AcknowledgementsR.A.M. is supported by grants from the National Institutes of Health.

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