Dual Strategies for Peptidoglycan Discrimination by Peptidoglycan Recognition Proteins (Pgrps)

Dual strategies for peptidoglycan discrimination by peptidoglycan recognition proteins (PGRPs)

Chittoor P. Swaminathan*, Patrick H. Brown*, Abhijit Roychowdhury†, Qian Wang*, Rongjin Guan*, Neal Silverman‡, William E. Goldman§, Geert-Jan Boons†¶, and Roy A. Mariuzza*¶

*Center for Advanced Research in Biotechnology, W. M. Keck Laboratory for Structural Biology, University of Maryland Biotechnology Institute, Rockville, MD 20850; †Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602; ‡Division of Infectious Diseases, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605; and §Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110

Edited by Kathryn Anderson, Sloan–Kettering Institute, New York, NY, and approved November 21, 2005 (received for review September 1, 2005)

The innate immune system constitutes the first line of defense against microorganisms in both vertebrates and invertebrates. Although much progress has been made toward identifying key receptors and understanding their role in host defense, far less is known about how these receptors recognize microbial ligands. Such studies have been severely hampered by the need to purify ligands from microbial sources and a reliance on biological assays, rather than direct binding, to monitor recognition. We used syn- thetic peptidoglycan (PGN) derivatives, combined with microcalo- rimetry, to define the binding specificities of human and insect peptidogycan recognition proteins (PGRPs). We demonstrate that these innate immune receptors use dual strategies to distinguish between PGNs from different bacteria: one based on the composition of the PGN peptide stem and another that senses the peptide bridge crosslinking the stems. To pinpoint the site of PGRPs that mediates discrimination, we engineered structure-based variants having altered PGN-binding properties. The plasticity of the PGRP- binding site revealed by these mutants suggests an intrinsic capacity of the innate immune system to rapidly evolve specificities to meet new microbial challenges.

afﬁnity ͉ bacteria ͉ innate immunity ͉ calorimetry ͉ synthesis

he innate immune system recognizes invading microbes by Tmeans of conserved pattern recognition receptors that bind unique products of microbial metabolism not produced by the host (pathogen-associated molecular patterns) (1, 2). Examples Fig. 1. Structure of PGN and PGN derivatives. (A) General structure of natural 1 1 2 of microbial ligands recognized by pattern recognition receptors PGN. R , H (Lys) Gram-positive PGN; R , COOH (Dap) Gram-negative PGN; R , H or crosslink. (B) The monomeric muramyl pentapeptides, MPP (R1, H, com- such as Toll-like receptors, peptidoglycan recognition proteins pound 1) and MPP-Dap (R1, COOH, compound 2), are represented in a single (PGRPs), and NOD proteins include lipopolysaccharide of figure. (C) The crosslinked Lys-type PGN analog, CL-PGN (compound 3). (D) The Gram-negative bacteria, lipoteichoic acid of Gram-positive bac- natural Dap-type PGN fragment, TCT, which contains an anhydro bond be- teria, nonmethylated CpG sequences, flagellin, and peptidogly- tween C6 and C1 of MurNAc. can (PGN) of Gram-negative and -positive bacteria. Cellular activation by pattern recognition receptors results in acute inflammatory responses involving cytokine and chemokine pro- exists in the peptide moiety (8, 9). According to the residue at duction, direct local attack against the invading pathogen, and position 3 of the stems, PGNs are classified into two major groups: induction of the adaptive component of the immune system. In L-lysine type (Lys-type) and meso-diaminopimelic acid type (Dap- humans, overactivation of inflammatory responses can lead to type). Dap-type PGN peptides are usually directly crosslinked, septic shock, which accounts for 100,000 deaths annually in the whereas Lys-type PGN peptides are interconnected by a peptide United States alone. By sitting at the intersection of the pathways bridge that varies in length and amino acid composition in different of microbial recognition, inflammation, and cell death, the bacteria (Fig. 1A). Moreover, bacteria differ widely in the extent of innate immune system offers emerging opportunities for the development of therapeutics to modulate immune responses (3). PGRPs, a newly discovered class of pattern recognition recep- Conflict of interest statement: No conflicts declared. tors, are highly conserved from insects to mammals (4–7). By This paper was submitted directly (Track II) to the PNAS office. detecting PGN from both Gram-negative and -positive bacteria, Abbreviations: CL-PGN, crosslinked Lys-type peptidoglycan; Dap-type, meso-diamin- PGRPs are important contributors to host defense against micro- opimelic acid type; ITC, isothermal titration calorimetry; Lys-type, L-lysine type; MPP, bial infections (2, 4). PGNs are polymers of alternating N- MurNAc-L-Ala-D-isoGln-L-Lys-D-Ala-D-Ala; MTP, MurNAc-L-Ala-D-isoGln-L-Lys; MurNAc, N- acetyl muramic acid; PGN, peptidoglycan; PGRP, peptidoglycan recognition protein; PGRP- acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) I␣C, C-terminal PGN-binding domain of PGRP-I␣; TCT, GlcNAc-MurNAc(1,6-anhydro)-L-Ala- in ␤(134) linkage, crosslinked by short peptide stems composed of D-isoGlu-(2S,6R)-Dap-D-Ala. alternating L- and D-amino acids (8, 9) (Fig. 1A). Whereas the ¶To whom correspondence may be addressed. E-mail: [email protected] or mariuzza@ carbohydrate backbone is conserved among all bacteria, except for carb.nist.gov. de-N-acetylated or O-acetylated variants, considerable diversity © 2006 by The National Academy of Sciences of the USA

684–689 ͉ PNAS ͉ January 17, 2006 ͉ vol. 103 ͉ no. 3 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0507656103 Downloaded by guest on October 1, 2021 and PGN (13), it now appears that these receptors recognize PGN alone (14, 15). Similarly, mammalian Toll-like receptor 2 is no longer thought to detect lipopolysaccharide (16, 17), but rather lipoteichoic acids, zymosan, and PGN (18), although PGN recognition by Toll-like receptor 2 has been disputed (19). Further complicating the analysis of receptor–ligand interactions is the exclusive reliance on biological assays to monitor recognition (1, 2), because these assays measure immunostimulatory capacity, not direct binding. To circumvent these difficulties, we synthesized muramyl pentapeptides that contain either Lys (1)orDap(2) as the third amino acid (Fig. 1B) and a crosslinked Lys-type PGN (3) (Fig. 1C). We used these compounds, in conjunction with isothermal titration calorimery (ITC), to elucidate the intrinsic PGN-binding specificities of human and insect PGRPs, the strategies these pattern recognition receptors employ to discriminate PGNs from different microbes, and the structural basis for this discrimination. Fig. 2. Calorimetric titrations of human PGRPs with monovalent and biva- Results lent PGN ligands at 275–277 K. (A) Raw data obtained from 50 automatic injections of 2-␮l aliquots of 12.3 mM MPP solution into 0.264 mM human Analysis of PGN Analog Binding to Human and Insect PGRPs. Use of PGRP-I␣C solution. (B) Nonlinear least-squares fit (solid line) of the incremen- classical fluorenylmethoxycarbonyl chemistry and solid phase syn- tal heat per mole of added ligand (open squares) for the titration in A. The thetic techniques enabled the assembly of compounds 1–3 (Fig. 1 B equilibrium binding constant obtained from this titration is Kb ϭ 45.0 Ϯ 1.0 ϫ and C). Having synthesized these compounds, attention was fo- 103 MϪ1 with n ϭ 1.01 Ϯ 0.01. (C) Raw data obtained from 45 automatic cused on determining thermodynamic parameters of their binding injections, first one of 1-␮l aliquot, and the remaining of 3-␮l aliquots of to human and Drosophila PGRPs. C-terminal PGN-binding domain 1.42 mM CL-PGN solution into 0.042 mM human PGRP-I␣C solution. (D) of PGRP-I␣ (PGRP-I␣C) recognizes MurNAc-L-Ala-D-isoGln-L- Nonlinear least-squares fit (solid line) of the incremental heat per mole of 3 Ϫ1 Lys-D-Ala-D-Ala (MPP) (Fig. 1B, 1), a muramyl pentapeptide added ligand (open squares) for the titration in C; Kb ϭ 74.2 Ϯ 4.6 ϫ 10 M with n ϭ 2.02 Ϯ 0.03. representing the conserved core of Lys-type PGN from Gram- 3 Ϫ1 positive bacteria (Fig. 1A), with a Kb of 45 ϫ 10 M . Despite the low affinity, there are measurable signal changes upon successive crosslinking (5–75%), thereby introducing additional variability in injections of ligand into the protein (Fig. 2 A and B) that allow for PGN structure (8, 9). accurate determination of Kb (20, 21). Furthermore, the fit of the In Drosophila, PGRPs activate two distinct signaling pathways ITC data to a single-site model returns an n value of 1.0 PGRP-I␣C that induce production of antimicrobial peptides: the Toll receptor per MPP, which indicates that we have accurately determined the pathway, which is primarily triggered by Lys-type PGNs from starting concentrations of both protein and ligand solutions and that ␣

Gram-positive bacteria, and the Imd pathway, which is mainly PGRP-I C and MPP are each fully active. The solution state IMMUNOLOGY activated by Dap-type PGNs from Gram-negative bacteria (2). stoichiometry obtained by ITC is consistent with the single PGN- Mammalian PGRPs, located in neutrophil and eosinophil granules, binding site per PGRP-I␣C monomer observed by x-ray crystal- participate in the intracellular killing of both Gram-positive and lography (22). Truncation of the peptide stem of MPP at position -negative bacteria (4, 10, 11). 2 reduces affinity Ϸ60-fold based on the binding of GlcNAc- A major difficulty in identifying molecular determinants recog- MurNAc-L-Ala-D-Ala (GMDP) (Table 1). Injection of volumes, up nized by pattern recognition receptors such as PGRPs, or in simply to 10-␮l aliquots, of 10-fold molar excess of Dap into 0.09 mM establishing which general category of PAMP is recognized, arises PGRP-I␣C resulted in heats similar to those of dilution (Fig. 5A, from the usual practice of purifying these products from bacterial which is published as supporting information on the PNAS web site) cell walls, such that contamination with other cell wall components with no detectable change in the incremental heat per mole of has often led to contradictory conclusions. For example, although added Dap (Fig. 5B), indicating no binding. Under these experi- human NOD1 was initially believed to detect lipopolysaccharide mental conditions and those used throughout this study, interac- Ϫ1 (12), and Drosophila PGRP-LC to sense both lipopoysaccharide tions with Kb Ͼ 100 M should be detectable (21). These results

Table 1. Binding constants (؋103 M؊1) of PGN derivatives to human and Drosophila PGRPs at 275 K Protein MPP MPP-Dap TCT CL-PGN*

h-PGRP-I␣C 45.0 (Ϯ1.0)† NB 20.2 (Ϯ0.8)† 74.2 (Ϯ4.6) h-PGRP-S 6.3 (Ϯ0.4) 47.4 (Ϯ6.0) 23.4 (Ϯ1.9) NB d-PGRP-LCx NB† 63.5 (Ϯ4.7) 19.8 (Ϯ0.8)‡ NB d-PGRP-LCa 17.8 (Ϯ1.5)† NB 8.7 (Ϯ1.6) NB h-PGRP-S (G68N, W69F) 85.1 (Ϯ0.4) 3.6 (Ϯ0.9)‡ 45.4 (Ϯ2.1) 85.2 (Ϯ8.1) d-PGRP-LCx (G393N, W394F) 23.8 (Ϯ1.3)‡ NB‡ 18.8 (Ϯ1.8)‡ NB‡ d-PGRP-LCa (Q412N, K413F) 36.3 (Ϯ1.7)‡ 72.2 (Ϯ14.0) 33.8 (Ϯ1.9) 83.0 (Ϯ9.6)

*For CL-PGN, n ranged from 1.95 to 2.04 with uncertainties from 2.6% to 4.5%; for all other PGN derivatives, n 3 Ϫ1 ranged from 0.99 to 1.08 with uncertainties from 0.4% to 12.9%. Kb values ϫ 10 M . Values in parentheses Ϫ1 represent uncertainties of ﬁt. h, human; d, Drosophila; NB, no binding detectable (Kb Ͻ 100 M ); CL-PGN, crosslinked PGN (3, Fig. 1C); Dap, meso-diaminopimelic acid; GMDP, GlcNAc-MurNAc-L-Ala-D-isoGln; MPP, MurNAc-L-Ala-D-isoGln-(2S,6R)-L-Lys-D-Ala-D- Ala (1, Fig. 1B); MPP-Dap, MurNAc-L-Ala-D-isoGln-(2S,6R)-Dap-D- Ala-D-Ala (2, Fig. 1B); TCT, GlcNAc-MurNAc(1,6-anhydro)-L-Ala-D-isoGlu-(2S,6R)-Dap-D-Ala (Fig. 1D). † Kb at 277 K. ‡ Kb at 276 K.

Swaminathan et al. PNAS ͉ January 17, 2006 ͉ vol. 103 ͉ no. 3 ͉ 685 Downloaded by guest on October 1, 2021 and those presented below reveal that PGN recognition by PGRPs, 5 Ϫ1 although of low affinity (Kb Ͻ 10 M ), is nevertheless highly selective (see Discussion). In addition to MPP, PGRP-I␣C recognizes 3, an analog of crosslinked Lys-type PGN (CL-PGN) composed of MPP connected to the muramyl tetrapeptide MurNAc-L-Ala-D-isoGln-L-Lys-D-Ala 3 Ϫ1 via a pentaglycine bridge (Fig. 1C), with Kb ϭ 74 ϫ 10 M and stoichiometry of 2.0 PGRP-I␣C per CL-PGN (Fig. 2 C and D). The simplest model that best fits the binding isotherm comprises two independent, identical sites of each CL-PGN engaging two PGRP- I␣C, indicating accommodation of the connecting peptide. At the same time, a strict requirement for L-Lys at position 3 of the peptide stem is implicit in the inability of PGRP-I␣C to bind the muramyl pentapeptide MurNAc-L-Ala-D-isoGln-(2S,6R)-Dap-D-Ala-D-Ala (MPP-Dap) (Fig. 1B, 2), which corresponds to the core of Dap-type PGNs from Bacillus and Gram-negative bacteria (Table 1). By contrast, human PGRP-S preferentially recognizes MPP-Dap 3 Ϫ1 3 Ϫ1 (Kb ϭ 47 ϫ 10 M ) over MPP (6.3 ϫ 10 M ), and fails to bind CL-PGN (Fig. 5 C and D). The ability of PGRP-S to recognize both MPP and MPP-Dap, albeit with somewhat different affinities, is consistent with the finding that mice deficient in PGRP-S exhibit increased susceptibility to i.p. infections with B. subtilis (Dap-type PGN) and Micrococcus luteus (Lys-type PGN) (11). Notably, M. luteus PGN is only Ϸ25% crosslinked (8, 9), which should not preclude recognition by PGRP-S. The observed binding of human PGRP-S to MPP and MPP-Dap also agrees with recent data showing that this PGRP inhibits the in vitro growth of both Staphylococcus aureus (Lys-type PGN) and Escherichia coli (Dap- type PGN) (23). The Drosophila PGRP-LC receptor, which is mainly triggered by Gram-negative bacteria, exists on the cell surface as three splice isoforms (PGRP-LCa, -LCx, and -LCy), each comprising a unique PGN-binding extracellular domain linked to identical transmem- brane and cytoplasmic domains (13). These isoforms are believed Fig. 3. Calorimetric titrations of Drosophila PGRP receptors with PGN ligands to form homo- or heterodimers, via their membrane proximal and tracheal cytotoxin at 275–278 K. (A) Raw data obtained from 40 automatic injections, first one of 1-␮l aliquot, and the remaining of 4-␮l aliquots of 1.98 cytoplasmic domain (24), with distinct PGN binding characteristics mM MPP-Dap solution into 0.086 mM Drosophila PGRP-LCx solution. (B) (15, 25). To investigate the specificities of PGRP-LCx and PGRP- Nonlinear least-squares fit (solid line) of the incremental heat per mole of LCa, we expressed their extracellular domains in soluble form. added ligand (open squares) for the titration in A. The equilibrium binding Ϫ ϫ 3 1 3 Ϫ1 PGRP-LCx binds MPP-Dap with a Kb of 64 10 M (Fig. 3 A constant obtained from this titration is Kb ϭ 63.5 Ϯ 4.7 ϫ 10 M with n ϭ and B) but fails to recognize MPP (Table 1). Conversely, PGRP- 1.01 Ϯ 0.02. (C) Raw data obtained from 50 automatic injections of 1-␮l 3 Ϫ1 LCa binds MPP (Kb ϭ 18 ϫ 10 M ;Fig.3C and D) but not aliquots of 12.46 mM MPP solution into 0.032 mM Drosophila PGRP-LCa MPP-Dap. Neither PGRP recognizes CL-PGN. These findings solution. (D) Nonlinear least-squares fit (solid line) of the incremental heat per ϭ Ϯ ϫ implicate PGRP-LCx as the isoform responsible for detecting mole of added ligand (open squares) for the titration in C; Kb 17.8 1.4 3 Ϫ1 ϭ Ϯ Gram-negative bacteria while excluding PGRP-LCa from such a 10 M with n 1.01 0.13. (E) Raw data obtained from 130 automatic injections of 2-␮l aliquots of 2.89 mM TCT solution into 0.037 mM Drosophila role. PGRP-LCx solution. (F) Nonlinear least-squares fit (solid line) of the incremen- PGRP-LCx exhibits a similar overall specificity profile as tal heat per mole of added ligand (open squares) for the titration in E; Kb ϭ PGRP-S except in its greater capacity to distinguish Dap-type from 19.8 Ϯ 0.8 ϫ 103 MϪ1 with n ϭ 1.04 Ϯ 0.06. (G) Raw data obtained from 30 Lys-type PGN. On the other hand, PGRP-LCa, despite its resem- automatic injections of 2-␮l aliquots of 9.7 mM MPP solution into 0.023 mM blance to PGRP-I␣C in binding MPP but not MPP-Dap, differs Drosophila PGRP-LCx (G393N, W394F) solution. (H) Nonlinear least-squares fit from PGRP-I␣C in not recognizing CL-PGN. Thus, the four (solid line) of the incremental heat per mole of added ligand (open squares) ϭ Ϯ ϫ 3 Ϫ1 ϭ Ϯ PGRPs analyzed here represent three qualitatively distinct speci- for the titration in G; Kb 23.8 1.3 10 M with n 1.06 0.09. ficity profiles after accounting for the similarity of PGRP-S and PGRP-LCx. PGN crosslinking is supported by very recent data showing that These results demonstrate that PGRPs use dual strategies, one Ϸ based on Lys- or Dap-type specificity and another that relies on M. luteus PGN, which is only 25% crosslinked (8, 9), is capable sensing the PGN crossbridge, to achieve exquisitely selective PGN of triggering the Drosophila Imd pathway through the PGRP-LC recognition and discrimination. PGRP-S, PGRP-LCx, and PGRP- receptor, but that Staphylococcus aureus PGN, which is highly Ϸ LCa distinguish PGN ligands on the basis of both criteria, whereas ( 75%) crosslinked, is inactive (30). Moreover, S. aureus PGN PGRP-I␣C, although remarkably specific for Lys-type PGN, is became as stimulatory as M. luteus PGN after enzymatic diges- insensitive to crosslinking. Importantly, the identity of the amino tion of its pentaglycine crossbridges. acid at position 3 of the stem, coupled with differences in the type and amount of crosslinking between stems, account for almost all Interaction of Human and Insect PGRPs with Tracheal Cytotoxin. We variability in PGNs from different bacteria (8, 9). also measured PGRP binding to GlcNAc-MurNAc(1,6-anhydro)- The result that PGRP binding to PGN analogs highly depends L-Ala-D-isoGlu-(2S,6R)-Dap-D-Ala (TCT) (Fig. 1D), a natural on the composition of the peptide stem helps explain the ability monomeric fragment of Dap-type PGN containing an anhydro of PGRPs to distinguish PGNs from different bacteria, as form of MurNAc (Fig. 3 E and F) (31). TCT is the factor responsible measured in biological assays (1, 2, 26–29). In addition, the for tissue damage in whooping cough and gonorrhea infections (31, biological relevance of our finding that certain PGRPs detect 32). Acting through the PGRP-LC receptor, TCT is also a potent

686 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0507656103 Swaminathan et al. Downloaded by guest on October 1, 2021 IMMUNOLOGY

Fig. 4. PGN-binding site of PGRPs. (A) Crystal structure of human PGRP-I␣C in complex with MTP, showing interactions at the binding site (22). MTP is drawn in stick representation. Carbon atoms are cyan, nitrogen atoms are dark blue, and oxygen atoms are red. PGRP-I␣C is yellow; residues making hydrogen bonds (dashed lines) with MTP are green. In purple are Asn-236 and Phe-237, drawn in ball-and-stick representation, which form van der Waals contacts with the side chain of L-lysine. MurNAc, N-acetylmuramic acid; ALA, L-alanine; IDG, D-isoglutamine; LYS, L-lysine. (B) View of the PGN-binding site of human PGRP-S (35). The orientation is the same as in A. In green are residues of PGRP-S corresponding to MTP-contacting residues in the PGRP-I␣C–MTP complex. Gly-68 and Trp-69 (purple) are predicted to contact the side chain of Lys-type PGN or Dap-type PGN. Gly-68 is represented by its carbonyl oxygen. (C) Structure-based sequence alignment of speciﬁcity-determining residues of mammalian and insect PGRPs. Residues corresponding to Asn-236 and Phe-237 of human PGRP-I␣CinA are highlighted in yellow. Mammals: Bt, Bos taurus; Cd, Camelus dromedarius; Hs, Homo sapiens; Mm, Mus musculus; Rn, Rattus norvegicus; Ss, Sus scrofa. For human and mouse PGRP-I␣ and PGRP-I␤, C and N indicate the C-terminal and N-terminal PGRP domains, respectively. Insects: Ag, Anopheles gambiae; Bm, Bombyx mori; Ce, Calpodes ethlius; Dm, Drosophila melanogaster; Ms, Manduca sexta; Tn, Trichoplusia ni. Sequence alignments were performed by using the program CLUSTALW at ExPASy (www.expasy.ch). The ﬁgure was generated by using ESPRIPT (http:͞͞espript.ibcp.fr͞ESPript͞ESPript).

activator of the Drosophila Imd pathway (15). Surprisingly, TCT noncrosslinked PGN, we engineered structure-based variants of binds all four PGRPs with similar affinities (Table 1), ranging from human and Drosophila PGRPs. In the crystal structure of PGRP- Ϫ Ϫ 8.7 ϫ 103 M 1 (PGRP-LCa) to 23 ϫ 103 M 1 (PGRP-S). The I␣C bound to the muramyl tripeptide MurNAc-L-Ala-D-isoGln-L- reported inability of PGRP-LCa to bind TCT in pull-down assays Lys (MTP) (22), the ligand is bound in a deep groove, where the (25, 33) may be explained by the lower sensitivity of this nonquan- side chain of L-Lys packs against Asn-236 and Phe-237 at one titative detection method compared to ITC. extremity (Fig. 4A). Sequence variability at these two positions Because PGRP-I␣C and PGRP-LCa both bind TCT, yet show no among Ͼ40 PGRPs suggests that they may account for the ability detectable reactivity toward MPP-Dap, we hypothesize that the of these proteins to distinguish PGNs from different microbes. To 1 1,6-anhydro bond of TCT, which locks MurNAc into the C4 test this hypothesis, we asked whether the specificity profile of conformation, alters the interaction of the peptide stem with PGRP-S could be converted to that of PGRP-I␣C by mutating PGRPs in a manner preventing discrimination against Dap at Gly-68 and Trp-69 of PGRP-S to the corresponding residues of position 3. Promiscuous binding of TCT to PGRPs, or PGRP-like PGRP-I␣C (Asn and Phe, respectively). In sharp contrast to molecules, could contribute to the diverse biological effects of this wild-type PGRP-S, which binds MPP Ϸ7-fold less tightly than PGN fragment, which include triggering normal light organ mor- PGRP-I␣C, the PGRP-S double mutant binds MPP Ϸ2-fold better 3 Ϫ1 3 Ϫ1 phogenesis in the squid (34). than PGRP-I␣C(Kb ϭ 85 ϫ 10 M versus 45 ϫ 10 M ) (Table 1). The PGRP-S mutant also resembles PGRP-I␣C in binding Structural Basis for PGN Discrimination by PGRPs. To identify the site MPP-Dap with Ϸ25-fold lower affinity than MPP, whereas the (or sites) of PGRPs responsible for discriminating between L-Lys wild-type PGRP-S exhibits a preferential recognition of MPP-Dap. and Dap at peptide position 3, and between crosslinked and Unlike wild-type PGRP-S, for which no interaction with CL-PGN

Swaminathan et al. PNAS ͉ January 17, 2006 ͉ vol. 103 ͉ no. 3 ͉ 687 Downloaded by guest on October 1, 2021 could be detected (Fig. 5 C and D), the mutant binds this ligand with of mammalian and insect PGRPs in the region encompassing 3 Ϫ1 Kb ϭ 85 ϫ 10 M (Table 1), an affinity similar to that of PGRP-I␣C residues 236 and 237, which we have shown by PGRP-I␣C (74 ϫ 103 MϪ1;Fig.2C and D). At the same time, the site-directed mutagenesis to mediate discrimination between PGRP-S mutant retains the capacity to recognize TCT, with L-Lys and Dap at position 3 of the PGN peptide stem and Ϸ2-fold higher affinity than wild-type PGRP-S or PGRP-I␣C. between crosslinked and noncrosslinked PGN. Group I PGRPs, These results demonstrate that a single site in the PGN-binding cleft like PGRP-I␣C, contain Asn-Phe at positions 236 and 237 (or of PGRPs, corresponding to residues 236 and 237 in PGRP-I␣C the homologous combinations Asp-Phe, Asn-Tyr, Asn-Trp, Gln- (Fig. 4A), mediates discrimination between L-Lys and Dap at Tyr, or Gln-Tyr). These PGRPs are likely to exhibit a specificity peptide position 3 and between crosslinked and noncrosslinked profile similar to that of PGRP-I␣C (i.e., recognition of MPP Lys-type PGN. and CL-PGN but not MPP-Dap). Group II PGRPs, like PGRP-S Further support for this conclusion is provided by mutagenesis of and PGRP-LCx, contain Gly-Trp (or Gly-Tyr or Gly-Phe) and Drosophila PGRPs. The double-mutant PGRP-LCx (Gly393Asn likely bind MPP-Dap better than MPP and do not bind CL-PGN. and Trp394Phe), which bears the same substitutions as the PGRP-S Group III PGRPs, which thus far has only one member (PGRP- mutant, follows the expected pattern of Lys-type versus Dap-type LCa), contain Gln-Lys and bind only MPP. Group IV PGRPs, 3 Ϫ1 discrimination by recognizing MPP (Kb ϭ 24 ϫ 10 M ;Fig.3G whose specificity we cannot currently assign, contain other and H), but not MPP-Dap (Table 1), a pattern inverse from that of sequences at these positions. It is notable that Ϸ65% (31 of 46) wild type. However, the mutations do not confer on PGRP-LCx the of the known PGRPs can be classified into Groups I or II. same ability to bind CL-PGN we observed for PGRP-I␣C and The overall validity of this classification is supported by PGRP-S (Gly68Asn and Trp69Phe), presumably due to additional biological data. Thus, Drosophila PGRP-SA, which we predict structural differences between the Drosophila and human proteins. should recognize Lys-type but not Dap-type PGN, is, in fact, the As a result, PGRP-LCx (Gly393Asn and Trp394Phe) closely re- main recognition element for Gram-positive bacteria in insects sembles PGRP-LCa in its specificity profile. By contrast, PGRP- (2). Similarly, our classification predicts that Drosophila PGRP- LCa (Gln412Asn and Lys413Phe), a highly promiscuous variant, LE, like PGRP-LCx, should bind Dap-type PGN. Indeed, it has has acquired the ability to bind both CL-PGN (83 ϫ 103 MϪ1) and been demonstrated that PGRP-LE functions synergistically with MPP-Dap (72 ϫ 103 MϪ1) while retaining MPP and TCT recog- PGRP-LC in conferring resistance to E. coli and other bacteria nition (Table 1). Taken together, these results underscore the having Dap-type PGN (46). plasticity of the PGN-binding site of PGRPs. Adaptive immunity relies on highly diverse receptors (antibodies The structures of the PGRP-I␣C–MTP complex (22) and and T cell receptors) generated through somatic gene recombina- PGRP-S (35) provide an explanation for the effect of the mutations tion, whereas innate immunity is mediated by germline-encoded, on recognition of Lys- versus Dap-type PGN. In the PGRP-I␣C– nonrearranging receptors of restricted diversity (1, 2). Although MTP complex (Fig. 4A), the side chain of Asn-236 protrudes from more structurally conserved than other microbial components (e.g., the wall of the binding groove, making van der Waals contacts with proteins), ligands such as PGN and LPS present sufficient heter- the side chain of L-Lys, especially atoms C␧ and N␨. Attachment of ogeneity to pose a significant challenge to specific recognition by a carboxy group to the C␧ atom, which distinguishes Dap from pattern recognition receptors. Moreover, the innate immune sys- L-Lys, would create steric clashes with Asn-236, decreasing affinity. tem must be adaptive enough to counter new microbial challenges. The corresponding Gly-68 of PGRP-S (Fig. 4B), because it lacks a In the case of PGRPs, we have shown that two amino acid side chain, does not interfere with binding. Accordingly, mutation mutations suffice to alter binding specificity from Lys-type to of Gly-68 (or Gly-393 of PGRP-LCx) to Asn should reduce, or Dap-type PGN (and vice versa) or from noncrosslinked to abolish, recognition of Dap-type PGN ligands, as observed for crosslinked forms of this cell wall component. Because these MPP-Dap (Table 1). Less evident is the structural basis for the variables together account for most of the known diversity in PGN effects of mutations on discrimination between crosslinked and structure (8, 9), it appears the binding sites of individual PGRPs are noncrosslinked Lys-type PGN, which will require crystallographic poised to facilitate rapid evolution of new specificities to respond to analysis of the interaction of PGRPs with the crossbridge. changes in the microbial environment while using existing signaling or effector pathways. Indeed, the relative ease with which the Discussion PGN-binding characteristics of PGRPs can be manipulated raises The binding of PGN derivatives to PGRP receptors, although the intriguing possibility of rewiring the innate immune system by, 5 Ϫ1 highly selective, is of low affinity (Kb Ͻ 10 M ). Such low- for instance, genetically reprogramming the Drosophila PGRP-LC– affinity, high-specificity recognition systems are gaining increas- Imd and PGRP-SA–Toll pathways to respond to Gram-positive and ing importance in serving key biological functions, including T Gram-negative bacteria, respectively, thereby inverting the existing cell receptor recognition of peptide͞MHC ligands (36–38), cell activation pattern. In addition, detailed knowledge of the interac- surface carbohydrate–protein interactions (39–41), and cell–cell tion of innate immune receptors with microbial ligands should adhesion (42). Moreover, it may be that even seemingly small provide a rational basis for the use of microbial cell wall molecules (Ͻ10-fold) differences in the affinity of PGRPs for monovalent as adjuvants for vaccines and modulators of inflammation (3). PGN ligands, such as we measured in several cases, are amplified by multiple PGRP–PGN interactions to establish specificity Methods effects at the cellular level, as described for carbohydrate- PGRP Production. Procedures for expressing human PGRP-S binding proteins (43, 44). Indeed, the polymeric nature of natural (residues 1–175) and the C-terminal PGN-binding domain of PGN should facilitate multivalent binding of PGRPs. On the human PGRP-I␣ (PGRP-I␣C; residues 177–341) by in vitro protein side, dimerization or oligomerization of PGRPs, as folding from E. coli inclusion bodies have been described in refs. observed for PGRP-LC (15, 24, 25) and PGRP-I␣C (45), may 22 and 35. The PGN-binding domains of Drosophila PGRP-LCx further enhance specificity at the cell surface or in solution. In (residues 325–500) and PGRP-LCa (residues 343–520) were this regard, cadherins have been shown to mediate highly specific prepared similarly to the human proteins (see Supporting Mate- intercellular adhesion through amplification of small affinity rials and Methods, which is published as supporting information differences between low-affinity cadherin dimers as a result of on the PNAS web site). Folded proteins were purified by using multiple interactions (42). a MonoS (PGRP-S, -LCx, and -LCa) or MonoQ (PGRP-I␣C) Our results provide a basis for predicting the PGN-binding ion exchange column (Amersham Pharmacia Biosciences) fol- specificity of PGRPs that have not been characterized experi- lowed by a Superdex 75 gel-filtration column (Amersham Phar- mentally. Fig. 4C presents a structure-based sequence alignment macia Biosciences). Site-directed mutagenesis of human

688 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0507656103 Swaminathan et al. Downloaded by guest on October 1, 2021 PGRP-S and Drosophila PGRP-LCx was performed by using a haustively, and PGN derivative solutions were prepared in the final QuikChange mutagenesis kit (Stratagene). Mutant proteins dialysate. Concentrations of PGN derivatives were based on dry were expressed and purified similarly to wild type. All purified weights. Protein concentrations were determined by absorbance at PGRPs were Ͼ95% pure by SDS͞PAGE and behaved as mono- 280 nm with molar extinction coefficients (␧,MϪ1 cmϪ1) of 28,800 mers in size exclusion chromatography. N-terminal sequencing (PGRP-I␣C), 39,800 (PGRP-S), 34,300 [PGRP-S (G68N, W69F)], and MALDI mass spectrometry confirmed the identity of 43,560 (PGRP-LCx), 38,060 [PGRP-LCx (G393N, W394F)], 32,560 wild-type and mutant proteins. (PGRP-LCa), and 32,560 [PGRP-LCa (Q412N, K413F)] (48). Solutions were prepared in 1 mM sodium phosphate buffer (pH PGN Derivatives. The target branched glycopeptide 3 (Fig. 1C) was 7.2 Ϯ 0.1) (PGRP-I␣C and PGRP-S), 1 mM Mes (pH 5.8 Ϯ 0.1) assembled by polymer-supported synthesis with a hyperacid sensi- (PGRP-I␣C, PGRP-S, PGRP-LCx, and PGRP-LCa), or 1 mM Ϯ tive Sieber Amide resin (Supporting Information; see also Fig. 6, Hepes (pH 6.8 0.1) [PGRP-S (G68N, W69F), PGRP-LCx which is published as supporting information on the PNAS web (G393N, W394F), and PGRP-LCa (Q412N, K413F)]. Buffer con- site). Briefly, the resin-bound compound 6 was obtained through a ditions were chosen for maintaining solubility at high protein ␣ series of steps by using standard coupling chemistry, Fmoc- concentrations necessary for ITC; for PGRP-I C, the binding of PGN derivatives is pH-independent between pH 5.8 and 7.2. protected amino acids, and a suitably protected MurNAc derivative. ␮ Upon removal of the ivDde protecting group on the lysine side Aliquots (1–10 l) of the PGN derivative solution (0.85–32.4 mM) were added to 1.41 ml of PGRP solution (0.022–0.264 mM) via a chain at position three of 6, the pentaglycine bridge of the branched ␮ glycopeptide 8 was added. After incorporation of the second 250- l microsyringe stirrer at 310 rpm. Titration data were analyzed by using a single-site fitting model. peptide stem, the partially deprotected 9 was cleaved from the solid A computerized nonlinear least square fitting method was used to support and the anomeric allyl moiety removed from MurNAc. determine the change in enthalpy (⌬H° ), equilibrium binding Finally, purification by size exclusion resulted in the target com- b constant (K ), and molar stoichiometry (n). The shape of the pound 3 (Fig. 1C)asamixtureof␣͞␤ anomers. Compounds 1 and b titration curve is determined by the unitless quantity c, defined as 2 (Fig. 1B) were prepared by a similar approach with either the product of the initial macromolecule concentration ([PGRP]0) Fmoc-L-Lys(Mtt) or Fmoc-Dap(Boc, tBu) (47). GlcNAc-MurNAc- and Kb, c ϭ Kb[PGRP]0. The c values for the present titrations (0.6 Ͻ L-Ala-D-Ala and Dap were purchased from Sigma. Tracheal cyto- c Ͻ 11.9) were within the permitted range (0.001 Ͻ c) for accurate toxin was purified from Bordetella purtussis culture supernatants as Kb determinations (20, 21). Data acquisition and analysis were described in ref. 31. performed by using the software package ORIGIN.

ITC Measurements and Analysis. Thermodynamic parameters for the This work was supported by National Institutes of Health Grants binding of PGRPs to PGN derivatives were determined by using a AI060025 (to N.S.), GM61761 and GM065248 (to G.-J.B.), and AI47990 Microcal VP-ITC titration calorimeter. PGRPs were dialyzed ex- and AI065612 (to R.A.M.).

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