Rgg Protein Structure–Function and Inhibition by Cyclic Peptide Compounds

Rgg protein structure–function and inhibition by cyclic peptide compounds

Vijay 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 January 7, 2015) Peptide pheromone cell–cell signaling (quorum sensing) regulates The 3D structure of Rgg proteins was unknown; however, based the expression of diverse developmental phenotypes (including vir- on their functional similarity to NprR, PrgX, and PlcR and their ulence) in Firmicutes, which includes common human pathogens, remote sequence similarity to RNPP family proteins, Rgg pro- e.g., Streptococcus pyogenes and Streptococcus pneumoniae.Cyto- teins preliminarily were included in this group (4, 6, 7, 20). plasmic transcription factors known as “Rgg proteins” are peptide Rgg proteins are widespread in Firmicute species, including pheromone receptors ubiquitous in Firmicutes. Here we present but not limited to the Streptococcaceae, Lactobacillales, Listeriaceae, X-ray crystal structures of a Streptococcus Rgg protein alone and in and Enterococcaceae (6). It also is common for organisms to complex with a tight-binding signaling antagonist, the cyclic un- express multiple paralogous Rgg proteins putatively serving decapeptide cyclosporin A. To our knowledge, these represent the nonredundant regulatory functions. For example, Streptococcus first Rgg protein X-ray crystal structures. Based on the results of pyogenes, which contains a thoroughly studied Rgg regulatory extensive structure–function analysis, we reveal the peptide phero- system, expresses four Rgg paralogs: Rgg2Sp, Rgg3Sp, ComRSp, mone-binding site and the mechanism by which cyclosporin A in- and RopBSp. Rgg2Sp, Rgg3Sp, and ComRSp are transcription hibits activation of the peptide pheromone receptor. Guided by the factors whose activity is regulated via interactions with phero- Rgg–cyclosporin A complex structure, we predicted that the non- mones (4, 21, 22). RopB is a transcription factor as well, and its role in pathogenesis has been thoroughly documented; however,

immunosuppressive cyclosporin A analog valspodar would inhibit MICROBIOLOGY the exact identity of RopB’s cognate regulatory pheromone has Rgg activation. Indeed, we found that, like cyclosporin A, valspodar – inhibits peptide pheromone activation of conserved Rgg proteins in not been determined (23 25). Thus far, there are two known Streptococcus medically relevant Streptococcus species. Finally, the crystal struc- families of peptide pheromones, the SigX-inducing peptides (XIPs) and the short hydrophobic peptides (SHPs). In tures presented here revealed that the Rgg protein DNA-binding S. pyogenes, Rgg-XIP and Rgg-SHP pairs regulate diverse de- domains are covalently linked across their dimerization interface velopmental processes, including biofilm formation and in- by a disulfide bond formed by a highly conserved cysteine. The duction of a cryptic competence regulon (4, 22). DNA-binding domain dimerization interface observed in our struc- Rgg2 and Rgg3 are the most similar of the four S. pyogenes tures is essentially identical to the interfaces previously described Sp Sp Rgg paralogs. In fact, Rgg2Sp and Rgg3Sp bind to the peptide for other members of the XRE DNA-binding domain family, but the presence of an intermolecular disulfide bond buried in this interface Significance appears 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. Peptide pheromones regulate developmental processes, including virulence, in Gram-positive bacteria. Immature propeptide phero- quorum sensing | Rgg protein | SHP pheromone | cyclosporin A | mones are synthesized, secreted, and undergo proteolytic matu- Streptococcus ration to serve as intercellular signals. The regulator gene of glucosyltransferase (Rgg) transcription factors are a large family of receptors that directly bind pheromones transported to ene expression in bacterial populations is coordinated by the cytosol. Here we report X-ray crystal structures of a Strep- Gpheromone-regulated cell-to-cell signaling networks. This in- tococcus Rgg protein alone and complexed with cyclosporin A, tercellular communication, commonly referred to as “quorum ” which is a potent inhibitor of pheromone signaling. Based on sensing, regulates diverse behaviors across the microbial world these structures and extensive genetic and biochemical studies, (1). Quorum sensing among Gram-positive bacteria is commonly we mapped the pheromone-binding site, discovered mechanistic mediated by peptide pheromones (reviewed in refs. 2 and 3). The aspects of Rgg regulation, and determined how cyclosporin A pheromones either are detected at the cell surface by membrane- and its nonimmunosuppressive analog valspodar function to bound receptors or are transported across the membrane by inhibit pheromone-mediated receptor activation. We conclude oligopeptide permeases, whereupon the pheromones engage that similar compounds targeting bacterial pheromone receptors cytoplasmic receptors (Fig. 1A). Gram-positive cytoplasmic have potential for therapeutic applications. pheromone receptors include Bacillus response regulator aspartate phosphatases (Rap), neutral protease regulator (NprR), Author contributions: V.P., C.A., M.J.F., and M.B.N. designed research, performed re- and phosphatidylinositol-specific phospholipase C gene regula- search, contributed new reagents/analytic tools, analyzed data, and wrote the paper. tor (PlcR), Enterococcus pheromone-responsive transcription The authors declare no conflict of interest. factor (PrgX), and Streptococcus regulator gene of glucosyl- This article is a PNAS Direct Submission. transferase (Rgg) (as well as the homologous MutR and GadR) Data deposition: Crystallography, atomic coordinates, and structure factors for Rgg2Sd – – and Rgg2Sd CsA have been deposited in the Protein Data Bank, www.pdb.org (PDB ID (4 11). The Rap proteins are phosphatases and transcriptional codes 4YV6 and 4YV9). antiactivators, whereas NprR, PlcR, and PrgX are DNA-binding 1 Present address: Department of Oral Biology, School of Dental Medicine, Rutgers, The transcription factors. Structure–function studies revealed that State University of New Jersey, Newark, NJ 07103. Rap, NprR, PlcR, and PrgX (the RNPP family proteins) use a 2To whom correspondence should be addressed. Email: [email protected]. structurally similar C-terminal tetratricopeptide (TPR)-like re- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. peat domain to bind their cognate peptide pheromones (12–19). 1073/pnas.1500357112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1500357112 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 Results Rgg2 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 (protomers A, B, C, and D) in the crystallographic asymmetric unit (Fig. S1). Gel filtration analysis established that Rgg2Sd forms homodimers in solution (Fig. S2), and the noncrystallographic dimer interface (crystallographic interfaces A–B and C–D) is large, burying more than 5,600 Å2 of surface area (Fig. S1). Rgg2Sd monomers within a homodimer are related by approximate twofold noncrystallographic symmetry, and the monomers are domain swapped (Fig. 2). More specifically, each monomer consists of an N-terminal DNA-binding domain (DBD) (residues 1–65) connected by a short linker region (residues 66–70) to a large C-terminal repeat domain (residues 71–284). Both the N- and C-terminal domains mediate contacts across the dimer interface, and the N- and C-terminal domains are swapped around the approximate twofold axis. Comparison of the Rgg2Sd DBD structure with all previously determined structures in the Protein Data Bank (PDB) database Fig. 1. Peptide pheromone signaling. (A) Intercellular vs. extracellular showed that the Rgg2Sd DBD structurally is most similar to detection of peptide pheromones. Members of the RRNPP protein family members of the XRE family of helix-turn-helix (HTH) DBDs (30, 31). Members of this family of DBDs contain five α-helices (Fig. modulate gene expression in response to direct binding of specific peptide α α pheromones that are translocated to the cytoplasm. Rgg proteins such as 2A and Fig. S3) in which helices 2and 3 and the linker con- NprR, PrgX, and PlcR are DNA-binding transcription factors. Rap proteins necting them form the DBD HTH fold. Helix α3 is the principal govern gene expression indirectly through protein–protein interactions DNA-binding helix, and, as detailed below, residues in helices α4, with other regulators, e.g., Spo0F (11) and ComA (5). The RRNPP proteins α5, and the loop connecting α3andα4 commonly mediate DBD are depicted here as dimers, but Rap proteins were shown to be monomeric homodimerization (32, 33). The Rgg2Sd DBD dimerization in- in solution (16, 44), and PrgX likely forms tetramers (18). In contrast to the terface observed in the Rgg2Sd crystal structure is essentially RRNPP systems, extracellular pheromone detection occurs by two-compo- identical to those observed in many prototypical members of the nent signal transduction (TCST) pathways that control transcription XRE family, such as Bacillus subtilis SinR (Fig. S3 A–C) (33) and through phosphorylation of a response regulator. The different peptide the RNPP proteins PlcR and PrgX (Fig. S3 D–F) (15, 34); how- colors highlight the fact that multiple pheromone types can be produced ever, in the Rgg2Sd structure (and the RggSd–CsA structure de- by single or multiple species. (B)SHP2Sd,SHP2Sp,andSHP3Sp amino acid scribed below) the Rgg2Sd DBDs are covalently linked across the sequences. dimerization interface by a disulfide bond between the α4helices (Fig. 2A and Fig. S3 A, C,andF). Cys45 forms the disulfide bond, and this cysteine is absolutely conserved among more than 120 pheromones SHP2 and SHP3 (Fig. 1B) with similar affinities, Rgg2 and Rgg3 orthologs from 27 different species. In contrast, and Rgg2Sp and Rgg3Sp bind to identical DNA-regulatory ele- this cysteine is absent from the more than 400 proteins that ments upstream of their target promoters (21, 26). The net re- comprise the other Rgg subfamilies, including the group II, group sponse of S. pyogenes to SHP pheromone is the robust expression III, ComR, and RopB proteins (6). of Rgg2/3-controlled promoters; however, Rgg2 and Rgg3 The Rgg2Sd C-terminal repeat domain contains five HTH Sp Sp folds and a capping helix that together form a right-handed su- affect transcription by different mechanisms. More specifically, perhelical structure with a concave inner surface and convex Rgg3Sp represses transcription in the absence of pheromone, and outer surface (Fig. 2). This structure resembles a TPR domain pheromone binding triggers derepression by dissociating Rgg3Sp superhelix, but the primary amino acid sequence does not from DNA. Conversely, Rgg2Sp induces transcription only when bound to an SHP pheromone (4, 27). Therefore, these regulators work systematically to down-regulate target gene transcription in the absence of pheromone, and they up-regulate transcription in the presence of pheromone. Although exceptions almost cer- tainly exist, Rgg proteins whose amino acid sequences are more similar to Rgg3Sp than to Rgg2Sp generally function as repressors, and Rgg proteins whose sequences are more similar to Rgg2Sp than to Rgg3Sp function as SHP-dependent activators (28, 29). Here we report X-ray crystal structures of Streptococcus dysgalactiae Rgg2 (Rgg2Sd) alone and in complex with an inhibitor, the cyclic undecapeptide cyclosporin A (CsA). To our knowledge, these are the first Rgg protein X-ray crystal structures reported. In addition to identifying the SHP-binding site, the structural, genetic, and biochemical studies presented here en- Fig. 2. Rgg2Sd crystal structure. (A)TheRgg2Sd dimer formed by protomers A abled us to show how CsA and its nonimmunosuppressive analog (Rgg2A)andB(Rgg2B). The α-helices are depicted as cylinders. The repeat valspodar function to inhibit SHP-mediated regulation of Rgg domain α-helices are labeled according to the following convention: R1A is the activity in both S. pyogenes and S. dysgalactiae. Based on these A helix of HTH repeat 1, and R1B is the B helix of the HTH repeat 1. For sim- results, and because Rgg-SHP signaling systems regulate diverse plicity, the protomer A repeat domain α-helices are not labeled. (B)Surface developmental responses in Firmicutes, which includes wide- representation of the Rgg2Sd dimer. To obtain this view of the SHP- (and CsA)- binding surface, the RggSd dimer depicted in A was rotated as indicated by the spread human and animal commensal and pathogenic bacteria, arrow. The protomer B residues highlighted in green displayed reduced SHP2- we conclude that Rgg-modulating compounds similar to those dependent activity in vivo. The corresponding residues in protomer A are not described here have potential for therapeutic application. visible in this orientation.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1500357112 Parashar et al. Downloaded by guest on October 2, 2021 contain TPR sequence motifs as determined using TPRpred and C). We refer to this region (residues 241–274) as the “cap (35). Ten additional C-terminal amino acids (residues 275–284) subdomain.” The cap subdomains of protomers A and C are follow the capping helix, but there was insufficient electron positioned proximal to the concave surface of the repeat do- density to model these residues. main, whereas the cap subdomains of protomers B and D are Structural alignment of the full-length Rgg2Sd protomers positioned distal to the concave surface of the repeat domain (rmsd for modeled Cα carbons = 0.48–1.195 Å for all pair- (Fig. S4 B and C). As detailed below, the conformational dif- wise comparisons) revealed only very subtle rigid-body differences ferences in the cap subdomains result from flexibility in the loop in the positions of the essentially identical DBDs relative to the connecting repeat 5 α-helices A and B (Fig. 2A and Fig. S4 B C-terminal repeat domains (Fig. S4A). The linker region connect- and C), and the position of the cap subdomain has important ing the DNA-binding and repeat domain is flexible, and clear implications for ligand binding to the concave surface of the electron density corresponding to this entire region existed for repeat domain. only one of the four modeled protomers (protomer B) in the crystallographic asymmetric unit (Fig. 2A and Fig. S1). The Identification of the SHP2-Binding Site. A search (30) of the PDB conformational flexibility in the linker region could facilitate database for proteins structurally similar to Rgg2Sd identified rigid-body movements of the DBDs relative to the repeat do- PrgX (PDB ID code 2AXZ, Z score = 19.2) (18), PlcR (PDB ID mains (Fig. S4A). code 3U3W, Z score = 13.1) (15), NprR (PDB ID code 4GPK, Structural alignment of the C-terminal repeat domains revealed Z score = 11.7) (19), and RapI (PDB ID code 4I1A, Z score = conformational differences near the C-terminal portion of the 10.7) (17). In previous studies, the concave surface of the repeat protomers in a subdomain consisting of repeat 5 α-helix B (resi- domain of peptide pheromone receptors was identified as the dues 241–257), the capping helix (residues 260–274), and the loop pheromone-binding site (Fig. S5 A–D) (13, 14, 16–18). We hy- (residues 258–259) connecting these helices (Fig. 2A and Fig. S4 B pothesized that the Streptococcus Rgg receptors similarly used the concave surface of their repeat domain as the SHP-binding site. To test this hypothesis, we developed a test-bed assay using a Δrgg2 Δshp strain of S. pyogenes (BNL200) that is unable to produce or respond to SHP pheromones (Fig. 3A). When S. pyogenes rgg2 or S. dysgalactiae rgg2 were transferred to the test-bed strain, response to synthetic SHP peptide was restored, as indicated by luminescence activity produced by the integrated

Pshp-luxAB reporter (Fig. 3A). The response was specific for MICROBIOLOGY SHP2, because the negative control peptide Rev-SHP2 did not trigger light expression above that of the Δrgg2 Δshp strain. To begin to map the SHP2-binding site, Rgg2Sd mutants containing targeted alanine substitutions in surface-exposed residues of the concave surface of the repeat domain were expressed in the test-bed strain and assessed for their response to synthetic SHP2 pheromone (Fig. 3B). In comparison with the wild-type Rgg2Sd control, Rgg2Sd-N150A, Rgg2Sd-R153A, Rgg2Sd-N190A, and Rgg2Sd-Y222A were insensitive to pheromone, and, to different degrees, Rgg2Sd-R81A, Rgg2Sd-I146A, Rgg2Sd-K178A, Rgg2Sd- L183A, Rgg2Sd-L187A, Rgg2Sd-D217A, Rgg2Sd-L219A, and Rgg2Sd-L262A displayed reduced SHP2-dependent activity (Fig. 3B). We used EMSA to measure the DNA-binding activity of the mutants that were insensitive to pheromone (Fig. S6). Rgg2Sd- Y222A displayed wild-type–level activity, whereas Rgg2Sd- N150A, Rgg2Sd-R153A, and Rgg2Sd-N190A displayed a partial loss 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). Based on these data and additional evidence outlined in Discussion,we conclude that SHP2 activates Rgg2Sd by binding to the concave surface of the Rgg2Sd C-terminal repeat domain.

The Cyclic Undecapeptide CsA Is a Potent Inhibitor of Rgg Function in Vivo and in Vitro. To identify inhibitors of Rgg function, we developed a fluorescence polarization (FP) assay whereby drug and druglike compounds (Prestwick Chemical) were screened for their ability to disrupt the binding of fluorescent-labeled synthetic SHP peptides (FITC-SHP) to RggSp (26) or Rgg2Sd (Fig. 3 C–D). Using this assay, we determined that the cyclic undecapeptide CsA is a potent inhibitor of SHP2 binding to Rgg2Sp and Fig. 3. In vivo and in vitro functional analysis. (A) Relative luminescence activity RggSd in vitro (IC50 = 0.4 μM) (Fig. 3D). Finally, using the in of the Pshp2-luxAB reporter in response to exogenous SHP2. Rgg2 variants were vivo SHP bioassay described above, we found that CsA inhibits expressed from a plasmid under their native promoters in group A Streptococcus both Rgg2Sd and Rgg2Sp activity in vivo (Fig. 3A). (GAS) strain BNL200 (Δrgg2 Δshp2Δshp3, attP::Pshp2-luxAB); empty vector (blue), RggSp (red) and RggSd (green). (B) Luciferase response of Rgg2Sd mutants in – GAS test bed in response to 10 nM SHP (red), 10 nM SHP + 10 μMCsA Rgg2 CsA Complex X-Ray Crystal Structure. To determine how CsA (green), or vehicle (blue). (C) Direct FP of 10 nM FITC-SHP2 synthetic peptide functions to inhibit SHP-triggered Rgg2 transcriptional activity, we determined the X-ray crystal structure of Rgg2 in complex titrated with purified RggSd.(D) CsA competes directly with FITC-SHP2 for Sd binding to 500 nM RggSd in the FP assay. Plots indicate the means of at with CsA (Fig. 4 and Table S1). The single-wavelength anoma- least three independent experiments. Kd values were determined by lous diffraction (SAD) method was used to obtain experimental applying linear-regression on dose–response curves using GraphPad phases, and the Rgg2Sd–CsA structure ultimately was refined to Prism (version 6.01). 1.95-Å resolution (Table S1). There are four Rgg2Sd protomers

Parashar et al. PNAS Early Edition | 3of6 Downloaded by guest on October 2, 2021 in the Rgg2Sd–CsA asymmetric unit, and each protomer binds to protomer A, and Rgg2Sd-Tyr222, which forms H-bonds with CsA CsA (Fig. 4 and Figs. S4D and S7). As detailed below, CsA only in protomers B, C, and D (Fig. S7). makes extensive interactions throughout the concave surface of How can CsA adopt two conformations in the Rgg2Sd–CsA the Rgg2Sd repeat domain and a few contacts across the dimer cocrystal structure? In brief, these two conformations are possible interface (Fig. 4 and Fig. S7). because the Rgg protomers make nonidentical crystal contacts CsA adopts a nearly identical conformation (and makes sim- (the contacts between the crystallographic asymmetric units), and ilar receptor contacts) in complex with Rgg2Sd protomers B, C, some of the Rgg2Sd–CsA protomers adopt different confor- and D (Fig. 4A and Fig. S7); however, CsA adopts a distinct mations (Fig. S4 D and E). More specifically, Rgg2Sd–CsA conformation in complex with protomer A (Fig. 4B and Fig. protomers B, C, and D are in a conformation most similar to S7B). Although CsA adopts two different conformations, the Rgg2Sd protomers B and D, whereas Rgg2Sd–CsA protomer A is CsA-binding site in all the protomers is similar (Fig. S7). That in a conformation most similar to Rgg2Sd protomers A and C is, the great majority of the Rgg protomer A residues that (compare Fig. S4 B–E). It appears that CsA drives the confor- contact CsA in one conformation are also used by protomers mational change in protomer C observed in the Rgg2Sd–CsA B, C, and D to contact CsA in the other conformation; how- structure, which, as discussed above, is enabled by both the ever, important conformational differences in the Rgg2Sd pro- flexibility in the loop connecting the C terminus of repeat 5 tomers discussed below explain how they accommodate two CsA α-helix B to the N terminus of the cap subdomain (Fig. S4E) and conformations. – the nonrestrictive (largely solvent-mediated) crystal contacts The Rgg2Sd CsA interface consists largely of hydrophobic near the protomer C cap subdomain. Because CsA binding drove interactions and a few H-bonds (Fig. S7). One conformational the conformationally flexible protomer C into a conformation difference between the protomer A and the protomer B, C, and similar to protomers B and D of Rgg2Sd and Rgg2Sd–CsA, D CsA-binding interfaces is in the position of the cap subdomain we propose that in solution the receptor-bound CsA conforma- (Fig. S4 E and F). The Rgg2Sd protomer A cap subdomain is tional equilibrium is toward that of Rgg2Sd–CsA protomers B, C, positioned proximal to the CsA-binding site, and the protomer and D. B, C, and D cap subdomains are positioned distal to the CsA- binding site. It is important to note that electron density in Functional Analysis of the Rgg2–CsA Interface in Vivo. To begin to protomer D was insufficient to model the loop (residues 258 and determine which of the receptor–ligand interactions observed 259) connecting the C terminus of repeat 5 α-helix B to the N in the Rgg2 –CsA complex crystal structure are functionally terminus of the cap subdomain (Fig. S4E). This finding is con- Sd important, we measured the ability of CsA to inhibit SHP- sistent with the idea that the loop is a flexible hinge allowing the cap subdomain to undergo rigid-body conformational changes. induced activity of Rgg2Sd mutants containing single alanine substitutions in Rgg2Sd–CsA interfacial positions (Fig. 3 A and Other notable conformational differences include Rgg2Sd-Arg153, which mediates numerous H-bonds to CsA exclusively in B and Fig. S7). The alanine substitutions were engineered at positions where CsA contacts Rgg2Sd in the concave surface of the repeat domain or at the dimer interface (Fig. S7). Our biochemical, genetic, and structural data suggested that the SHP2- and CsA-binding sites overlap significantly, and, in ac- cordance with these results, we found that many of the Rgg2Sd residues that interact with CsA also are required for SHP2 to activate Rgg2Sd (e.g., Asn150, Arg153, Asn190, and Tyr222) (Fig. 3B). However, we also identified a number of CsA-binding residues (e.g., Tyr84, Lys178, Leu183, Leu187, Asp219, and Leu262) that are not absolutely critical for SHP2-mediated Rgg2Sd activation, and alanine substitution mutations in these positions desensitized Rgg2Sd to CsA-mediated inhibition of SHP-induced Rgg2 activity (Fig. 3B). These residues mediate critically 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 that contact CsA (Fig. S7) are identically conserved in Rgg2Sp. The one nonidentical 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 surfaces of the Rgg2Sd and Rgg2Sp repeat domains also is consistent with our observation that the S. dysgalactiae SHP2 and S. pyogenes SHP2 sequences are identical (Fig. 1B).

Fig. 4. Rgg2Sd–CsA crystal structure. Surface representation of the Rgg2Sd– The Nonimmunosuppressive CsA Analog Valspodar Antagonizes Rgg2Sd CsA dimer formed by protomers A (Rgg2A) and B (Rgg2B). (A, Left)Inthis and Rgg2Sp Activity in Vivo. To begin to assess the importance of the orientation, CsA (ball and stick model) bound to Rgg2B is visible. The con- CsA structural features (namely its side chains) to Rgg inhibitory formation of CsA bound to Rgg2B also represents the conformation of CsA function, we tested the activity of the CsA analog SDZ PSC 833 bound to protomers C and D. (Right) An expanded view of the area enclosed (also known as “PSC 833” or “valspodar”) (36, 37) in vivo. Val- by the black dashed lines in the left panel. (B, Left) In this orientation, CsA spodar is a nonimmunosuppressive CsA analog that substitutes (ball and stick model) bound to Rgg2A is visible. (Right) An expanded view of 3′-keto-MeBmt and valine in place of CsA 4-methyl-4-[(E)-2- the area enclosed by the black dashed lines in the left panel. In all panels, the butenyl]-4,N-methyl-threonine (MeBmt) and γ-aminobutanoic Rgg2Sd surface that interacts with CsA is colored orange or green. The green surface highlights the positions where alanine substitution mutations de- acid, respectively. Modeling these substitutions showed that they sensitized Rgg2 to CsA-mediated inhibition of SHP-induced Rgg2 activity. were accommodated without significant van der Walls overlap in Sd – Abu, γ-amino-butanoic acid; Ala, alanine; Dal, D-alanine; MeBmt, 4-methyl- all protomers of the Rgg2Sd CsA structure, and, like CsA, val- 4-[(E)-2-butenyl]-4,N-methyl-threonine; Mle, N-methyl-L-leucine; Mva, spodar completely inhibited SHP-dependent Rgg2Sp and Rgg2Sd N-methylvaline; Sar, sarcosine; Val, valine. activity in vivo (Fig. 3A).

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1500357112 Parashar et al. Downloaded by guest on October 2, 2021 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-C45S were 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.

A Disulfide Bond in the Rgg2 DBD Dimerization Interface. Although binding and the basis of their interaction specificity. Studies are the DBD dimerization interface observed in both the Rgg2Sd and underway in our laboratory to determine the X-ray crystal – RggSd CsA structures is essentially identical to those pre- structures of Rgg proteins bound to SHP peptides and/or DNA. viously described for members of the XRE family (Fig. S3), the Finally, because the great majority—but not all—of the alanine presence of an intermolecular disulfide bond buried in this substitutions targeted to the concave surface of the repeat interface is, at least to our knowledge, unique. As detailed domain disrupted SHP2 and CsA binding, we conclude that above, Cys45, which forms the disulfide bond, is highly con- SHP2 and CsA bind to largely overlapping, nonidentical re- served in Rgg2 and Rgg3 orthologs, and the disulfide bond is gions of the concave surface. present in both dimers in the crystallographic asymmetric unit How do the cyclic undecapeptides CsA and valspodar antago- in the CsA-bound and CsA-free structures (Fig. 2A and Fig. S3 A, C,andF). nize SHP signaling? It was shown previously that a C-terminal portion of PrgX rearranges upon pheromone (cCF10) binding To begin to explore a possible role for the disulfide bond in MICROBIOLOGY the regulation of Rgg2 , we measured the SHP2-dependent and that the PrgX C terminus and cCF10 interact to form a small Sd β activity of the Rgg -C45S mutant in vivo (Fig. 5A). In com- -sheet (18). The Rgg2Sd C-terminal cap subdomain is con- Sd – parison with wild-type Rgg2Sd, Rgg2Sd-C45S exhibited only a formationally flexible (Fig. S4 B F), as is the Rgg2Sd C-terminal very slightly reduced response to SHP2 (Fig. 5A). In addition, we tail, which was disordered in the crystal structure and could not grew the group A Streptococcus strain BNL178 in both oxidizing be modeled. We propose that, like PrgX-cCF10, the SHP pep- and reducing conditions (Fig. 5B). The oxidizing reagent para- tide and Rgg C-terminal tail can form a β-sheet. Furthermore, quat and the reducing agents DTT and N-acetyl cysteine had we propose that the large undecapeptide CsA (and valspodar) little or no effect on SHP2-triggered Rgg2Sd activity in vivo. In alone can occupy the same 3D space that would be occupied contrast, after denaturation in the absence of DTT in vitro, by both the linear SHP peptide and the Rgg C-terminal tail. Cys45 was required for the formation of a species consistent with CsA and valspodar may function not only by competitively the dimer form of Rgg2Sd (Fig. 5C). inhibiting SHP binding but also by blocking the Rgg2Sd C-terminal tail from adopting an active (SHP-bound) confor- Discussion mation and, in turn, a potentially important allosteric conforma- TheRgg,Rap,NprR,PlcR,andPrgXproteinsarecytoplasmic tional change in the C-terminal domain that could be required receptors regulated by peptide pheromones. As a result of the for receptor activation. Based on the structural similarity of RggSd– and RggSd–CsA crystal structures, we now know that the Rgg proteins have domain architectures identical to those the Rgg and RNPP proteins and their similar modes of phero- of the PrgX, NprR, and PlcR proteins, i.e., an N-terminal DBD mone binding, we speculate that cyclic peptides could serve as and a C-terminal repeat domain. Based on the structural similarity antagonists not of only Rgg proteins but also of other RRNPP family proteins. of Rgg2Sd and the RNPP proteins, and because Rgg and RNPP proteins are peptide pheromone receptors, the Rgg proteins now Finally, perhaps one of the most interesting mechanistic ques- can be considered bona fide members of the RNPP family. Rgg tions to address going forward pertains to what in vivo role, if any, proteins are, in fact, the largest constituents of this family, and, is played by the Rgg2Sd intermolecular disulfide bond observed in as previously proposed, the family can be renamed “RRNPP” to the Rgg2Sd– and Rgg2Sd–CsA crystal structures and in solution include these proteins (4, 20). (Fig. 5). The disulfide bond forms at the core of the DBD di- A longstanding goal has been to identify inhibitors of merization interface, which is structurally identical to other XRE RRNPP proteins, which commonly regulate the expression of protein dimer interfaces (Fig. S3). Furthermore, as discussed critical developmental phenotypes. These inhibitors could dis- above, the disulfide is formed by Cys45, which is remarkably well rupt cell–cell signaling and potentially function as antibiotics or conserved in more than 120 Rgg2 and Rgg3 orthologs from 27 antiinfectives. The Rgg2 –CsA X-ray crystal structure showed Sd different species. We also note that the Rgg2Sd and Rgg2Sd–CsA that CsA binds to the concave surface of the Rgg2Sd repeat crystals were grown in the presence of 5 mM DTT and soaked in domain. Based on our genetic analysis of Rgg2Sd andonstudies cryoprotection solutions containing 5 mM DTT immediately be- from our laboratories and others that identified the concave fore data collection. Therefore, although the disulfide bond can surface of RNPP proteins as the pheromone-binding site, we conclude that SHP peptides bind the concave surface of Rgg be reduced by boiling the protein in DTT, the bond is sufficiently protein repeat domains. Additional support for this idea comes buried in the natively folded protein to resist chemical reduction. from our computational docking studies showing that the This degree of shielding may enable the disulfide bond to persist in the reducing environment of the bacterial cytoplasm. Under concave surface of the Rgg2Sd repeat domain can physically accommodate the binding of SHP2 peptides (Fig. S5E). X-ray the right conditions, which we have not yet identified, the con- crystal structures of Rgg2–SHP2 and Rgg3–SHP3 cocomplexes served cysteine may play a regulatory role, perhaps affecting Rgg are required to reveal the functionally relevant SHP-binding monomer–dimer equilibrium, stabilizing Rgg conformation, or mode and to determine the atomic-level details of Rgg–SHP functioning as a redox-sensitive switch.

Parashar et al. PNAS Early Edition | 5of6 Downloaded by guest on October 2, 2021 Methods in the PDB database, which also is in agreement with the proposed CsA Primers used in this study are listed in Table S2. Bacterial strains and plasmids biosynthetic reaction mechanism (41). Water and sulfate molecules were used in this study are listed in Table S3. built into clear electron density during the final stages of refinement. The The Rgg2–CsA crystal structure was determined by the SAD method structure of Rgg2 alone was determined by molecular replacement using using crystals of selenomethionyl Rgg2–CsA. PHENIX (AutoSol) was used to protomer A from the Rgg2–CsA structure as a search model. Ramachandran locate heavy atom positions, calculate phases, and generate an initial statistics were calculated in MolProbity (42). Molecular graphics were pro- model at 1.95-Å resolution (38). The final model was generated through duced with PyMOL (43). iterative cycles of building in COOT (39) and refinement in PHENIX. The Rgg2 and CsA models were built de novo into the SAD-phased map. The ACKNOWLEDGMENTS. We thank Glenn Capodagli, Guozhou Chen, Atul earliest rounds of refinement in PHENIX used simulated annealing, in- Khataokar, and Evan Waldron for critical review of the manuscript; Breah dividual atomic coordinates, and individual B-factor refinement. The later LaSarre for construction of BNL200; and Phil Jeffrey for advice and discus- rounds of refinement in PHENIX used individual atomic coordinates, in- sions. X-ray diffraction data were collected at the National Synchrotron dividual B-factor refinement, and a TLS model whose initial parameters Light Source beamline X29A. Support for this work was provided by National Institutes of Health Grants R01 AI081736 and R03 AI101669 (to were guided by the TLS Motion Determination (TLSMD) server (40). During M.B.N.) and R01 AI091779 (to M.J.F.); by the Burroughs Wellcome Fund In- the final rounds of refinement in PHENIX, the ADP weights were opti- vestigators of Pathogenesis of Infectious Diseases (M.J.F.); by the Chicago mized, i.e., the weights yielding the lowest Rfree valuewereusedforre- Biomedical Consortium with support from the Searle Funds at the Chicago finement. CsA was modeled only after the Rgg2 models were nearly Community Trust (C.A. and M.J.F.); and by the New Jersey Health Founda- complete. CsA residues were numbered according to the convention used tion (V.P.).

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