Rgg protein structure–function and inhibition by cyclicpeptide compoundsVijay Parashara,1, Chaitanya Aggarwalb, Michael J. Federleb, and Matthew B. Neiditcha,2

aDepartment of Microbiology, Biochemistry, and Molecular Genetics, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark,NJ 07103; and bDepartment of Medicinal Chemistry and Pharmacognosy, Center for Pharmaceutical Biotechnology, University of Illinois at Chicago, Chicago,IL 60607

Edited by Bonnie L. Bassler, Howard Hughes Medical Institute, Princeton University, Princeton, NJ, and approved March 17, 2015 (received for review January7, 2015)

Peptide pheromone cell–cell signaling (quorum sensing) regulatesthe expression of diverse developmental phenotypes (including vir-ulence) in Firmicutes, which includes common human pathogens,e.g., Streptococcus pyogenes and Streptococcus pneumoniae. Cyto-plasmic transcription factors known as “Rgg proteins” are peptidepheromone receptors ubiquitous in Firmicutes. Here we presentX-ray crystal structures of a Streptococcus Rgg protein alone and incomplex with a tight-binding signaling antagonist, the cyclic un-decapeptide cyclosporin A. To our knowledge, these represent thefirst Rgg protein X-ray crystal structures. Based on the results ofextensive structure–function analysis, we reveal the peptide phero-mone-binding site and the mechanism by which cyclosporin A in-hibits activation of the peptide pheromone receptor. Guided by theRgg–cyclosporin A complex structure, we predicted that the non-immunosuppressive cyclosporin A analog valspodar would inhibitRgg activation. Indeed, we found that, like cyclosporin A, valspodarinhibits peptide pheromone activation of conserved Rgg proteins inmedically relevant Streptococcus species. Finally, the crystal struc-tures presented here revealed that the Rgg protein DNA-bindingdomains are covalently linked across their dimerization interfaceby a disulfide bond formed by a highly conserved cysteine. TheDNA-binding domain dimerization interface observed in our struc-tures is essentially identical to the interfaces previously describedfor other members of the XRE DNA-binding domain family, but thepresence of an intermolecular disulfide bond buried in this interfaceappears to be unique. We hypothesize that this disulfide bond may,under the right conditions, affect Rgg monomer–dimer equilibrium,stabilize Rgg conformation, or serve as a redox-sensitive switch.

quorum sensing | Rgg protein | SHP pheromone | cyclosporin A |Streptococcus

Gene expression in bacterial populations is coordinated bypheromone-regulated cell-to-cell signaling networks. This in-

tercellular communication, commonly referred to as “quorumsensing,” regulates diverse behaviors across the microbial world(1). Quorum sensing among Gram-positive bacteria is commonlymediated by peptide pheromones (reviewed in refs. 2 and 3). Thepheromones either are detected at the cell surface by membrane-bound receptors or are transported across the membrane byoligopeptide permeases, whereupon the pheromones engagecytoplasmic receptors (Fig. 1A). Gram-positive cytoplasmicpheromone receptors include Bacillus response regulator as-partate phosphatases (Rap), neutral protease regulator (NprR),and phosphatidylinositol-specific phospholipase C gene regula-tor (PlcR), Enterococcus pheromone-responsive transcriptionfactor (PrgX), and Streptococcus regulator gene of glucosyl-transferase (Rgg) (as well as the homologous MutR and GadR)(4–11). The Rap proteins are phosphatases and transcriptionalantiactivators, whereas NprR, PlcR, and PrgX are DNA-bindingtranscription factors. Structure–function studies revealed thatRap, NprR, PlcR, and PrgX (the RNPP family proteins) use astructurally similar C-terminal tetratricopeptide (TPR)-like re-peat domain to bind their cognate peptide pheromones (12–19).

The 3D structure of Rgg proteins was unknown; however, basedon their functional similarity to NprR, PrgX, and PlcR and theirremote sequence similarity to RNPP family proteins, Rgg pro-teins preliminarily were included in this group (4, 6, 7, 20).Rgg proteins are widespread in Firmicute species, including

but not limited to the Streptococcaceae, Lactobacillales, Listeriaceae,and Enterococcaceae (6). It also is common for organisms toexpress multiple paralogous Rgg proteins putatively servingnonredundant regulatory functions. For example, Streptococcuspyogenes, which contains a thoroughly studied Rgg regulatorysystem, expresses four Rgg paralogs: Rgg2Sp, Rgg3Sp, ComRSp,and RopBSp. Rgg2Sp, Rgg3Sp, and ComRSp are transcriptionfactors whose activity is regulated via interactions with phero-mones (4, 21, 22). RopB is a transcription factor as well, and itsrole in pathogenesis has been thoroughly documented; however,the exact identity of RopB’s cognate regulatory pheromone hasnot been determined (23–25). Thus far, there are two knownfamilies of Streptococcus peptide pheromones, the SigX-inducingpeptides (XIPs) and the short hydrophobic peptides (SHPs). InS. pyogenes, Rgg-XIP and Rgg-SHP pairs regulate diverse de-velopmental processes, including biofilm formation and in-duction of a cryptic competence regulon (4, 22).Rgg2Sp and Rgg3Sp are the most similar of the four S. pyogenes

Rgg paralogs. In fact, Rgg2Sp and Rgg3Sp bind to the peptide

Significance

Peptide pheromones regulate developmental processes, includingvirulence, in Gram-positive bacteria. Immature propeptide phero-mones are synthesized, secreted, and undergo proteolytic matu-ration to serve as intercellular signals. The regulator gene ofglucosyltransferase (Rgg) transcription factors are a largefamily of receptors that directly bind pheromones transported tothe cytosol. Here we report X-ray crystal structures of a Strep-tococcus Rgg protein alone and complexed with cyclosporin A,which is a potent inhibitor of pheromone signaling. Based onthese structures and extensive genetic and biochemical studies,we mapped the pheromone-binding site, discovered mechanisticaspects of Rgg regulation, and determined how cyclosporin Aand its nonimmunosuppressive analog valspodar function toinhibit pheromone-mediated receptor activation. We concludethat similar compounds targeting bacterial pheromone receptorshave potential for therapeutic applications.

Author contributions: V.P., C.A., M.J.F., and M.B.N. designed research, performed re-search, contributed new reagents/analytic tools, analyzed data, and wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: Crystallography, atomic coordinates, and structure factors for Rgg2Sdand Rgg2Sd–CsA have been deposited in the Protein Data Bank, www.pdb.org (PDB IDcodes 4YV6 and 4YV9).1Present address: Department of Oral Biology, School of Dental Medicine, Rutgers, TheState University of New Jersey, Newark, NJ 07103.

2To whom correspondence should be addressed. Email: matthew.neiditch@rutgers.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1500357112/-/DCSupplemental.

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pheromones SHP2 and SHP3 (Fig. 1B) with similar affinities,and Rgg2Sp and Rgg3Sp bind to identical DNA-regulatory ele-ments upstream of their target promoters (21, 26). The net re-sponse of S. pyogenes to SHP pheromone is the robust expressionof Rgg2/3-controlled promoters; however, Rgg2Sp and Rgg3Spaffect transcription by different mechanisms. More specifically,Rgg3Sp represses transcription in the absence of pheromone, andpheromone binding triggers derepression by dissociating Rgg3Spfrom DNA. Conversely, Rgg2Sp induces transcription only whenbound to an SHP pheromone (4, 27). Therefore, these regulatorswork systematically to down-regulate target gene transcription inthe absence of pheromone, and they up-regulate transcription inthe presence of pheromone. Although exceptions almost cer-tainly exist, Rgg proteins whose amino acid sequences are moresimilar to Rgg3Sp than to Rgg2Sp generally function as repressors,and Rgg proteins whose sequences are more similar to Rgg2Spthan to Rgg3Sp function as SHP-dependent activators (28, 29).Here we report X-ray crystal structures of Streptococcus dys-

galactiae Rgg2 (Rgg2Sd) alone and in complex with an inhibitor,the cyclic undecapeptide cyclosporin A (CsA). To our knowl-edge, these are the first Rgg protein X-ray crystal structuresreported. In addition to identifying the SHP-binding site, thestructural, genetic, and biochemical studies presented here en-abled us to show how CsA and its nonimmunosuppressive analogvalspodar function to inhibit SHP-mediated regulation of Rggactivity in both S. pyogenes and S. dysgalactiae. Based on theseresults, and because Rgg-SHP signaling systems regulate diversedevelopmental responses in Firmicutes, which includes wide-spread human and animal commensal and pathogenic bacteria,we conclude that Rgg-modulating compounds similar to thosedescribed here have potential for therapeutic application.

ResultsRgg2 X-Ray Crystal Structure. The X-ray crystal structure of full-length Rgg2Sd alone was determined to a resolution of 2.05 Å(Fig. 2 and Table S1). There are four Rgg2Sd protomers (pro-tomers A, B, C, and D) in the crystallographic asymmetric unit(Fig. S1). Gel filtration analysis established that Rgg2Sd formshomodimers in solution (Fig. S2), and the noncrystallographicdimer interface (crystallographic interfaces A–B and C–D) islarge, burying more than 5,600 Å2 of surface area (Fig. S1).Rgg2Sd monomers within a homodimer are related by approx-

imate twofold noncrystallographic symmetry, and the monomersare domain swapped (Fig. 2). More specifically, each monomerconsists of an N-terminal DNA-binding domain (DBD) (residues1–65) connected by a short linker region (residues 66–70) to alarge C-terminal repeat domain (residues 71–284). Both theN- and C-terminal domains mediate contacts across the dimerinterface, and the N- and C-terminal domains are swapped aroundthe approximate twofold axis.Comparison of the Rgg2Sd DBD structure with all previously

determined structures in the Protein Data Bank (PDB) databaseshowed that the Rgg2Sd DBD structurally is most similar tomembers of the XRE family of helix-turn-helix (HTH) DBDs (30,31). Members of this family of DBDs contain five α-helices (Fig.2A and Fig. S3) in which helices α2 and α3 and the linker con-necting them form the DBD HTH fold. Helix α3 is the principalDNA-binding helix, and, as detailed below, residues in helices α4,α5, and the loop connecting α3 and α4 commonly mediate DBDhomodimerization (32, 33). The Rgg2Sd DBD dimerization in-terface observed in the Rgg2Sd crystal structure is essentiallyidentical to those observed in many prototypical members of theXRE family, such as Bacillus subtilis SinR (Fig. S3 A–C) (33) andthe RNPP proteins PlcR and PrgX (Fig. S3 D–F) (15, 34); how-ever, in the Rgg2Sd structure (and the RggSd–CsA structure de-scribed below) the Rgg2Sd DBDs are covalently linked across thedimerization interface by a disulfide bond between the α4 helices(Fig. 2A and Fig. S3 A, C, and F). Cys45 forms the disulfide bond,and this cysteine is absolutely conserved among more than 120Rgg2 and Rgg3 orthologs from 27 different species. In contrast,this cysteine is absent from the more than 400 proteins thatcomprise the other Rgg subfamilies, including the group II, groupIII, ComR, and RopB proteins (6).The Rgg2Sd C-terminal repeat domain contains five HTH

folds and a capping helix that together form a right-handed su-perhelical structure with a concave inner surface and convexouter surface (Fig. 2). This structure resembles a TPR domainsuperhelix, but the primary amino acid sequence does not

Fig. 1. Peptide pheromone signaling. (A) Intercellular vs. extracellulardetection of peptide pheromones. Members of the RRNPP protein familymodulate gene expression in response to direct binding of specific peptidepheromones that are translocated to the cytoplasm. Rgg proteins such asNprR, PrgX, and PlcR are DNA-binding transcription factors. Rap proteinsgovern gene expression indirectly through protein–protein interactionswith other regulators, e.g., Spo0F (11) and ComA (5). The RRNPP proteinsare depicted here as dimers, but Rap proteins were shown to be monomericin solution (16, 44), and PrgX likely forms tetramers (18). In contrast to theRRNPP systems, extracellular pheromone detection occurs by two-compo-nent signal transduction (TCST) pathways that control transcriptionthrough phosphorylation of a response regulator. The different peptidecolors highlight the fact that multiple pheromone types can be producedby single or multiple species. (B) SHP2Sd, SHP2Sp, and SHP3Sp amino acidsequences.

Fig. 2. Rgg2Sd crystal structure. (A) The Rgg2Sd dimer formed by protomers A(Rgg2A) and B (Rgg2B). The α-helices are depicted as cylinders. The repeatdomain α-helices are labeled according to the following convention: R1A is theA helix of HTH repeat 1, and R1B is the B helix of the HTH repeat 1. For sim-plicity, the protomer A repeat domain α-helices are not labeled. (B) Surfacerepresentation of the Rgg2Sd dimer. To obtain this view of the SHP- (and CsA)-binding surface, the RggSd dimer depicted in Awas rotated as indicated by thearrow. The protomer B residues highlighted in green displayed reduced SHP2-dependent activity in vivo. The corresponding residues in protomer A are notvisible in this orientation.

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contain TPR sequence motifs as determined using TPRpred(35). Ten additional C-terminal amino acids (residues 275–284)follow the capping helix, but there was insufficient electrondensity to model these residues.Structural alignment of the full-length Rgg2Sd protomers

(rmsd for modeled Cα carbons = 0.48–1.195 Å for all pair-wise comparisons) revealed only very subtle rigid-body differencesin the positions of the essentially identical DBDs relative to theC-terminal repeat domains (Fig. S4A). The linker region connect-ing the DNA-binding and repeat domain is flexible, and clearelectron density corresponding to this entire region existed foronly one of the four modeled protomers (protomer B) in thecrystallographic asymmetric unit (Fig. 2A and Fig. S1). Theconformational flexibility in the linker region could facilitaterigid-body movements of the DBDs relative to the repeat do-mains (Fig. S4A).Structural alignment of the C-terminal repeat domains revealed

conformational differences near the C-terminal portion of theprotomers in a subdomain consisting of repeat 5 α-helix B (resi-dues 241–257), the capping helix (residues 260–274), and the loop(residues 258–259) connecting these helices (Fig. 2A and Fig. S4 B

and C). We refer to this region (residues 241–274) as the “capsubdomain.” The cap subdomains of protomers A and C arepositioned proximal to the concave surface of the repeat do-main, whereas the cap subdomains of protomers B and D arepositioned distal to the concave surface of the repeat domain(Fig. S4 B and C). As detailed below, the conformational dif-ferences in the cap subdomains result from flexibility in the loopconnecting repeat 5 α-helices A and B (Fig. 2A and Fig. S4 Band C), and the position of the cap subdomain has importantimplications for ligand binding to the concave surface of therepeat domain.

Identification of the SHP2-Binding Site. A search (30) of the PDBdatabase for proteins structurally similar to Rgg2Sd identifiedPrgX (PDB ID code 2AXZ, Z score = 19.2) (18), PlcR (PDB IDcode 3U3W, Z score = 13.1) (15), NprR (PDB ID code 4GPK,Z score = 11.7) (19), and RapI (PDB ID code 4I1A, Z score =10.7) (17). In previous studies, the concave surface of the repeatdomain of peptide pheromone receptors was identified as thepheromone-binding site (Fig. S5 A–D) (13, 14, 16–18). We hy-pothesized that the Streptococcus Rgg receptors similarly usedthe concave surface of their repeat domain as the SHP-bindingsite. To test this hypothesis, we developed a test-bed assay usinga Δrgg2 Δshp strain of S. pyogenes (BNL200) that is unable toproduce or respond to SHP pheromones (Fig. 3A). WhenS. pyogenes rgg2 or S. dysgalactiae rgg2 were transferred to thetest-bed strain, response to synthetic SHP peptide was restored,as indicated by luminescence activity produced by the integratedPshp-luxAB reporter (Fig. 3A). The response was specific forSHP2, because the negative control peptide Rev-SHP2 did nottrigger light expression above that of the Δrgg2 Δshp strain.To begin to map the SHP2-binding site, Rgg2Sd mutants con-

taining targeted alanine substitutions in surface-exposed residuesof the concave surface of the repeat domain were expressed in thetest-bed strain and assessed for their response to synthetic SHP2pheromone (Fig. 3B). In comparison with the wild-type Rgg2Sdcontrol, Rgg2Sd-N150A, Rgg2Sd-R153A, Rgg2Sd-N190A, andRgg2Sd-Y222A were insensitive to pheromone, and, to differentdegrees, Rgg2Sd-R81A, Rgg2Sd-I146A, Rgg2Sd-K178A, Rgg2Sd-L183A, Rgg2Sd-L187A, Rgg2Sd-D217A, Rgg2Sd-L219A, andRgg2Sd-L262A displayed reduced SHP2-dependent activity (Fig.3B). We used EMSA to measure the DNA-binding activity of themutants that were insensitive to pheromone (Fig. S6). Rgg2Sd-Y222A displayed wild-type–level activity, whereas Rgg2Sd-N150A, Rgg2Sd-R153A, and Rgg2Sd-N190A displayed a partialloss of function. Only one of the substitution mutations in-troduced into the concave surface of the repeat domain, Y84A,had no effect on SHP2-triggered Rgg2Sd activity (Fig. 3B). Basedon these data and additional evidence outlined in Discussion, weconclude that SHP2 activates Rgg2Sd by binding to the concavesurface of the Rgg2Sd C-terminal repeat domain.

The Cyclic Undecapeptide CsA Is a Potent Inhibitor of Rgg Function inVivo and in Vitro. To identify inhibitors of Rgg function, we de-veloped a fluorescence polarization (FP) assay whereby drug anddruglike compounds (Prestwick Chemical) were screened fortheir ability to disrupt the binding of fluorescent-labeled syn-thetic SHP peptides (FITC-SHP) to RggSp (26) or Rgg2Sd (Fig. 3C–D). Using this assay, we determined that the cyclic undeca-peptide CsA is a potent inhibitor of SHP2 binding to Rgg2Sp andRggSd in vitro (IC50 = 0.4 μM) (Fig. 3D). Finally, using the invivo SHP bioassay described above, we found that CsA inhibitsboth Rgg2Sd and Rgg2Sp activity in vivo (Fig. 3A).

Rgg2–CsA Complex X-Ray Crystal Structure. To determine how CsAfunctions to inhibit SHP-triggered Rgg2 transcriptional activity,we determined the X-ray crystal structure of Rgg2Sd in complexwith CsA (Fig. 4 and Table S1). The single-wavelength anoma-lous diffraction (SAD) method was used to obtain experimentalphases, and the Rgg2Sd–CsA structure ultimately was refined to1.95-Å resolution (Table S1). There are four Rgg2Sd protomers

Fig. 3. In vivo and in vitro functional analysis. (A) Relative luminescence activityof the Pshp2-luxAB reporter in response to exogenous SHP2. Rgg2 variants wereexpressed from a plasmid under their native promoters in group A Streptococcus(GAS) strain BNL200 (Δrgg2 Δshp2Δshp3, attP::Pshp2-luxAB); empty vector (blue),RggSp (red) and RggSd (green). (B) Luciferase response of Rgg2Sd mutants inGAS test bed in response to 10 nM SHP (red), 10 nM SHP + 10 μM CsA(green), or vehicle (blue). (C) Direct FP of 10 nM FITC-SHP2 synthetic peptidetitrated with purified RggSd. (D) CsA competes directly with FITC-SHP2 forbinding to 500 nM RggSd in the FP assay. Plots indicate the means of atleast three independent experiments. Kd values were determined byapplying linear-regression on dose–response curves using GraphPadPrism (version 6.01).

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in the Rgg2Sd–CsA asymmetric unit, and each protomer binds toCsA (Fig. 4 and Figs. S4D and S7). As detailed below, CsAmakes extensive interactions throughout the concave surface ofthe Rgg2Sd repeat domain and a few contacts across the dimerinterface (Fig. 4 and Fig. S7).CsA adopts a nearly identical conformation (and makes sim-

ilar receptor contacts) in complex with Rgg2Sd protomers B, C,and D (Fig. 4A and Fig. S7); however, CsA adopts a distinctconformation in complex with protomer A (Fig. 4B and Fig.S7B). Although CsA adopts two different conformations, theCsA-binding site in all the protomers is similar (Fig. S7). Thatis, the great majority of the Rgg protomer A residues thatcontact CsA in one conformation are also used by protomersB, C, and D to contact CsA in the other conformation; how-ever, important conformational differences in the Rgg2Sd pro-tomers discussed below explain how they accommodate two CsAconformations.The Rgg2Sd–CsA interface consists largely of hydrophobic

interactions and a few H-bonds (Fig. S7). One conformationaldifference between the protomer A and the protomer B, C, andD CsA-binding interfaces is in the position of the cap subdomain(Fig. S4 E and F). The Rgg2Sd protomer A cap subdomain ispositioned proximal to the CsA-binding site, and the protomerB, C, and D cap subdomains are positioned distal to the CsA-binding site. It is important to note that electron density inprotomer D was insufficient to model the loop (residues 258 and259) connecting the C terminus of repeat 5 α-helix B to the Nterminus of the cap subdomain (Fig. S4E). This finding is con-sistent with the idea that the loop is a flexible hinge allowing thecap subdomain to undergo rigid-body conformational changes.Other notable conformational differences include Rgg2Sd-Arg153,which mediates numerous H-bonds to CsA exclusively in

protomer A, and Rgg2Sd-Tyr222, which forms H-bonds with CsAonly in protomers B, C, and D (Fig. S7).How can CsA adopt two conformations in the Rgg2Sd–CsA

cocrystal structure? In brief, these two conformations are possiblebecause the Rgg protomers make nonidentical crystal contacts(the contacts between the crystallographic asymmetric units), andsome of the Rgg2Sd–CsA protomers adopt different confor-mations (Fig. S4 D and E). More specifically, Rgg2Sd–CsAprotomers B, C, and D are in a conformation most similar toRgg2Sd protomers B and D, whereas Rgg2Sd–CsA protomer A isin a conformation most similar to Rgg2Sd protomers A and C(compare Fig. S4 B–E). It appears that CsA drives the confor-mational change in protomer C observed in the Rgg2Sd–CsAstructure, which, as discussed above, is enabled by both theflexibility in the loop connecting the C terminus of repeat 5α-helix B to the N terminus of the cap subdomain (Fig. S4E) andthe nonrestrictive (largely solvent-mediated) crystal contactsnear the protomer C cap subdomain. Because CsA binding drovethe conformationally flexible protomer C into a conformationsimilar to protomers B and D of Rgg2Sd and Rgg2Sd–CsA,we propose that in solution the receptor-bound CsA conforma-tional equilibrium is toward that of Rgg2Sd–CsA protomers B, C,and D.

Functional Analysis of the Rgg2–CsA Interface in Vivo. To begin todetermine which of the receptor–ligand interactions observedin the Rgg2Sd–CsA complex crystal structure are functionallyimportant, we measured the ability of CsA to inhibit SHP-induced activity of Rgg2Sd mutants containing single alaninesubstitutions in Rgg2Sd–CsA interfacial positions (Fig. 3 A andB and Fig. S7). The alanine substitutions were engineered atpositions where CsA contacts Rgg2Sd in the concave surface ofthe repeat domain or at the dimer interface (Fig. S7). Ourbiochemical, genetic, and structural data suggested that theSHP2- and CsA-binding sites overlap significantly, and, in ac-cordance with these results, we found that many of the Rgg2Sdresidues that interact with CsA also are required for SHP2 toactivate Rgg2Sd (e.g., Asn150, Arg153, Asn190, and Tyr222)(Fig. 3B). However, we also identified a number of CsA-bindingresidues (e.g., Tyr84, Lys178, Leu183, Leu187, Asp219, andLeu262) that are not absolutely critical for SHP2-mediatedRgg2Sd activation, and alanine substitution mutations in thesepositions desensitized Rgg2Sd to CsA-mediated inhibition ofSHP-induced Rgg2 activity (Fig. 3B). These residues mediatecritically important interactions with CsA but appear to con-tribute less to the SHP2-binding energy than Asn150, Arg153,Asn190, and Tyr222.Consistent with CsA antagonizing SHP binding to both Rgg2Sd

and Rgg2Sp in vitro and in vivo, 23 of the 24 Rgg2Sd residues thatcontact CsA (Fig. S7) are identically conserved in Rgg2Sp. The onenonidentical CsA-binding site residue, Rgg2Sd-A261 (Rgg2Sp-S261)contacts CsA only in the Rgg2Sd promoter A conformation (Fig.S7B). The great similarity of residues within the concave surfacesof the Rgg2Sd and Rgg2Sp repeat domains also is consistent withour observation that the S. dysgalactiae SHP2 and S. pyogenesSHP2 sequences are identical (Fig. 1B).

The Nonimmunosuppressive CsA Analog Valspodar Antagonizes Rgg2Sdand Rgg2Sp Activity in Vivo. To begin to assess the importance of theCsA structural features (namely its side chains) to Rgg inhibitoryfunction, we tested the activity of the CsA analog SDZ PSC 833(also known as “PSC 833” or “valspodar”) (36, 37) in vivo. Val-spodar is a nonimmunosuppressive CsA analog that substitutes3′-keto-MeBmt and valine in place of CsA 4-methyl-4-[(E)-2-butenyl]-4,N-methyl-threonine (MeBmt) and γ-aminobutanoicacid, respectively. Modeling these substitutions showed that theywere accommodated without significant van der Walls overlap inall protomers of the Rgg2Sd–CsA structure, and, like CsA, val-spodar completely inhibited SHP-dependent Rgg2Sp and Rgg2Sdactivity in vivo (Fig. 3A).

Fig. 4. Rgg2Sd–CsA crystal structure. Surface representation of the Rgg2Sd–CsA dimer formed by protomers A (Rgg2A) and B (Rgg2B). (A, Left) In thisorientation, CsA (ball and stick model) bound to Rgg2B is visible. The con-formation of CsA bound to Rgg2B also represents the conformation of CsAbound to protomers C and D. (Right) An expanded view of the area enclosedby the black dashed lines in the left panel. (B, Left) In this orientation, CsA(ball and stick model) bound to Rgg2A is visible. (Right) An expanded view ofthe area enclosed by the black dashed lines in the left panel. In all panels, theRgg2Sd surface that interacts with CsA is colored orange or green. The greensurface highlights the positions where alanine substitution mutations de-sensitized Rgg2Sd to CsA-mediated inhibition of SHP-induced Rgg2 activity.Abu, γ-amino-butanoic acid; Ala, alanine; Dal, D-alanine; MeBmt, 4-methyl-4-[(E)-2-butenyl]-4,N-methyl-threonine; Mle, N-methyl-L-leucine; Mva,N-methylvaline; Sar, sarcosine; Val, valine.

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A Disulfide Bond in the Rgg2 DBD Dimerization Interface. Althoughthe DBD dimerization interface observed in both the Rgg2Sd andRggSd–CsA structures is essentially identical to those pre-viously described for members of the XRE family (Fig. S3), thepresence of an intermolecular disulfide bond buried in thisinterface is, at least to our knowledge, unique. As detailedabove, Cys45, which forms the disulfide bond, is highly con-served in Rgg2 and Rgg3 orthologs, and the disulfide bond ispresent in both dimers in the crystallographic asymmetric unitin the CsA-bound and CsA-free structures (Fig. 2A and Fig. S3A, C, and F).To begin to explore a possible role for the disulfide bond in

the regulation of Rgg2Sd, we measured the SHP2-dependentactivity of the RggSd-C45S mutant in vivo (Fig. 5A). In com-parison with wild-type Rgg2Sd, Rgg2Sd-C45S exhibited only avery slightly reduced response to SHP2 (Fig. 5A). In addition, wegrew the group A Streptococcus strain BNL178 in both oxidizingand reducing conditions (Fig. 5B). The oxidizing reagent para-quat and the reducing agents DTT and N-acetyl cysteine hadlittle or no effect on SHP2-triggered Rgg2Sd activity in vivo. Incontrast, after denaturation in the absence of DTT in vitro,Cys45 was required for the formation of a species consistent withthe dimer form of Rgg2Sd (Fig. 5C).

DiscussionThe Rgg, Rap, NprR, PlcR, and PrgX proteins are cytoplasmicreceptors regulated by peptide pheromones. As a result of theRggSd– and RggSd–CsA crystal structures, we now know thatthe Rgg proteins have domain architectures identical to thoseof the PrgX, NprR, and PlcR proteins, i.e., an N-terminal DBDand a C-terminal repeat domain. Based on the structural similarityof Rgg2Sd and the RNPP proteins, and because Rgg and RNPPproteins are peptide pheromone receptors, the Rgg proteins nowcan be considered bona fide members of the RNPP family. Rggproteins are, in fact, the largest constituents of this family, and,as previously proposed, the family can be renamed “RRNPP” toinclude these proteins (4, 20).A longstanding goal has been to identify inhibitors of

RRNPP proteins, which commonly regulate the expression ofcritical developmental phenotypes. These inhibitors could dis-rupt cell–cell signaling and potentially function as antibiotics orantiinfectives. The Rgg2Sd–CsA X-ray crystal structure showedthat CsA binds to the concave surface of the Rgg2Sd repeatdomain. Based on our genetic analysis of Rgg2Sd and on studiesfrom our laboratories and others that identified the concavesurface of RNPP proteins as the pheromone-binding site, weconclude that SHP peptides bind the concave surface of Rggprotein repeat domains. Additional support for this idea comesfrom our computational docking studies showing that theconcave surface of the Rgg2Sd repeat domain can physicallyaccommodate the binding of SHP2 peptides (Fig. S5E). X-raycrystal structures of Rgg2–SHP2 and Rgg3–SHP3 cocomplexesare required to reveal the functionally relevant SHP-bindingmode and to determine the atomic-level details of Rgg–SHP

binding and the basis of their interaction specificity. Studies areunderway in our laboratory to determine the X-ray crystalstructures of Rgg proteins bound to SHP peptides and/or DNA.Finally, because the great majority—but not all—of the alaninesubstitutions targeted to the concave surface of the repeatdomain disrupted SHP2 and CsA binding, we conclude thatSHP2 and CsA bind to largely overlapping, nonidentical re-gions of the concave surface.How do the cyclic undecapeptides CsA and valspodar antago-

nize SHP signaling? It was shown previously that a C-terminalportion of PrgX rearranges upon pheromone (cCF10) bindingand that the PrgX C terminus and cCF10 interact to form a smallβ-sheet (18). The Rgg2Sd C-terminal cap subdomain is con-formationally flexible (Fig. S4 B–F), as is the Rgg2Sd C-terminaltail, which was disordered in the crystal structure and could notbe modeled. We propose that, like PrgX-cCF10, the SHP pep-tide and Rgg C-terminal tail can form a β-sheet. Furthermore,we propose that the large undecapeptide CsA (and valspodar)alone can occupy the same 3D space that would be occupiedby both the linear SHP peptide and the Rgg C-terminal tail.CsA and valspodar may function not only by competitivelyinhibiting SHP binding but also by blocking the Rgg2SdC-terminal tail from adopting an active (SHP-bound) confor-mation and, in turn, a potentially important allosteric conforma-tional change in the C-terminal domain that could be requiredfor receptor activation. Based on the structural similarity ofthe Rgg and RNPP proteins and their similar modes of phero-mone binding, we speculate that cyclic peptides could serve asantagonists not of only Rgg proteins but also of other RRNPPfamily proteins.Finally, perhaps one of the most interesting mechanistic ques-

tions to address going forward pertains to what in vivo role, if any,is played by the Rgg2Sd intermolecular disulfide bond observed inthe Rgg2Sd– and Rgg2Sd–CsA crystal structures and in solution(Fig. 5). The disulfide bond forms at the core of the DBD di-merization interface, which is structurally identical to other XREprotein dimer interfaces (Fig. S3). Furthermore, as discussedabove, the disulfide is formed by Cys45, which is remarkably wellconserved in more than 120 Rgg2 and Rgg3 orthologs from 27different species. We also note that the Rgg2Sd and Rgg2Sd–CsAcrystals were grown in the presence of 5 mM DTT and soaked incryoprotection solutions containing 5 mM DTT immediately be-fore data collection. Therefore, although the disulfide bond canbe reduced by boiling the protein in DTT, the bond is sufficientlyburied in the natively folded protein to resist chemical reduction.This degree of shielding may enable the disulfide bond to persistin the reducing environment of the bacterial cytoplasm. Underthe right conditions, which we have not yet identified, the con-served cysteine may play a regulatory role, perhaps affecting Rggmonomer–dimer equilibrium, stabilizing Rgg conformation, orfunctioning as a redox-sensitive switch.

Fig. 5. Functional analysis of the Rgg2Sd disulfide bond. (A) Luciferase response of Rgg2Sd–C45S [BNL200(pCA128)] compared with WT Rgg2Sd [BNL2000(pCA113)] and empty vector [NL200(pLZ12-Sp)]. (B) Luciferase response of WT Rgg2 in presence of reducing agents (10 mM DTT or 15 mM N-acetyl cysteine,NAC) or oxidizing agent (10 mM paraquat). (C) In vitro formation of the Rgg2Sd disulfide bond requires Cys45. Samples of 10 μg Rgg2Sd (WT) or Rgg2Sd-C45Swere boiled in the presence (+) or absence (−) of 5 mM DTT before analysis by SDS/PAGE. M, molecular weight standards; Rgg21, Rgg2Sd monomer; Rgg22,Rgg2Sd dimer. The asterisk marks the His-SUMO-Rgg2Sd contaminant.

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MethodsPrimers used in this study are listed in Table S2. Bacterial strains and plasmidsused in this study are listed in Table S3.

The Rgg2–CsA crystal structure was determined by the SAD methodusing crystals of selenomethionyl Rgg2–CsA. PHENIX (AutoSol) was used tolocate heavy atom positions, calculate phases, and generate an initialmodel at 1.95-Å resolution (38). The final model was generated throughiterative cycles of building in COOT (39) and refinement in PHENIX. TheRgg2 and CsA models were built de novo into the SAD-phased map. Theearliest rounds of refinement in PHENIX used simulated annealing, in-dividual atomic coordinates, and individual B-factor refinement. The laterrounds of refinement in PHENIX used individual atomic coordinates, in-dividual B-factor refinement, and a TLS model whose initial parameterswere guided by the TLS Motion Determination (TLSMD) server (40). Duringthe final rounds of refinement in PHENIX, the ADP weights were opti-mized, i.e., the weights yielding the lowest Rfree value were used for re-finement. CsA was modeled only after the Rgg2 models were nearlycomplete. CsA residues were numbered according to the convention used

in the PDB database, which also is in agreement with the proposed CsAbiosynthetic reaction mechanism (41). Water and sulfate molecules werebuilt into clear electron density during the final stages of refinement. Thestructure of Rgg2 alone was determined by molecular replacement usingprotomer A from the Rgg2–CsA structure as a search model. Ramachandranstatistics were calculated in MolProbity (42). Molecular graphics were pro-duced with PyMOL (43).

ACKNOWLEDGMENTS. We thank Glenn Capodagli, Guozhou Chen, AtulKhataokar, and Evan Waldron for critical review of the manuscript; BreahLaSarre for construction of BNL200; and Phil Jeffrey for advice and discus-sions. X-ray diffraction data were collected at the National SynchrotronLight Source beamline X29A. Support for this work was provided byNational Institutes of Health Grants R01 AI081736 and R03 AI101669 (toM.B.N.) and R01 AI091779 (to M.J.F.); by the Burroughs Wellcome Fund In-vestigators of Pathogenesis of Infectious Diseases (M.J.F.); by the ChicagoBiomedical Consortium with support from the Searle Funds at the ChicagoCommunity Trust (C.A. and M.J.F.); and by the New Jersey Health Founda-tion (V.P.).

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