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. Author manuscript; available in PMC: 2011 Mar 14.
Published in final edited form as: Structure. 2010 Mar 14;18(4):537–547. doi: 10.1016/j.str.2010.02.007

Structure of a Virulence Regulatory Factor CvfB Reveals a Novel Winged-helix RNA Binding Module

Yasuhiko Matsumoto 1,+, Qingping Xu 2,3,+, Shinya Miyazaki 1, Chikara Kaito 1, Carol L Farr 2,4, Herbert L Axelrod 2,3, Hsiu-Ju Chiu 2,3, Heath E Klock 2,5, Mark W Knuth 2,5, Mitchell D Miller 2,3, Marc-André Elsliger 2,4, Ashley M Deacon 2,3, Adam Godzik 2,6,7, Scott A Lesley 2,4,5, Kazuhisa Sekimizu 1,*, Ian A Wilson 2,4,*
PMCID: PMC2858061  NIHMSID: NIHMS188132  PMID: 20399190

SUMMARY

CvfB is a conserved regulatory protein important for the virulence of Staphylococcus aureus. We show here that CvfB binds RNA. The crystal structure of the CvfB ortholog from Streptococcus pneumoniae at 1.4 Å resolution reveals a unique RNA binding protein that is formed from a concatenation of well-known structural modules that bind nucleic acids: three consecutive S1 RNA-binding domains and a winged-helix (WH) domain. The third S1 and the WH domains are required for cooperative RNA binding and form a continuous surface that likely contributes to the RNA interaction. The WH domain is critical to CvfB function and contains a unique structural motif. Thus CvfB represents a novel assembly of modules for binding RNA.

Keywords: CvfB, Winged-helix domain, S1 domain, RNA binding, virulence

INTRODUCTION

Pathogenic bacteria regulate the expression of many virulence factors, such as toxins and adhesion molecules that are involved in host infection via highly conserved virulence regulatory proteins. Staphylococcus aureus is an opportunistic human pathogen and secretes various toxins and defence factors to combat the host immune system, resulting in various diseases, such as septic arthritis, meningitis, and sepsis (Dinges et al., 2000; Foster, 2005; Lowy, 1998). Several virulence regulatory genes have been identified in S. aureus, such as the agr locus (Peng et al., 1988), sarA (Cheung et al., 1992), saeRS (Giraudo et al., 1997), srrAB (Yarwood et al., 2001), and arlRS (Fournier et al., 2001). We recently identified four novel virulence regulatory genes, cvfA, cvfB, cvfC, and sarZ, which are conserved among various pathogenic bacteria, using a silkworm infection model (Kaito et al., 2005; Kaito et al., 2006; Nagata et al., 2008). Among these genes, cvfB acts on the activation pathway of the agr locus, through activation of the hla gene encoding α-hemolysin and repression of the spa gene, encoding protein A, a cell wall protein in S. aureus (Matsumoto et al., 2007). CvfB also contributes to the production of a protease and nuclease via an agr-independent pathway (Matsumoto et al., 2007), but its biochemical function is unknown. Thus, cvfB might interact in concert with these other regulatory genes to form a network that controls the production of bacterial virulence factors (Bronner et al., 2004).

In this study, we report the biochemical characterization of CvfB from S. aureus and the high-resolution crystal structure of its ortholog from an equally important pathogen, Streptococcus pneumoniae. We show that S. aureus CvfB has RNA binding activity and the CvfB crystal structure reveals a unique combination of three S1 domains and a winged-helix (WH) domain that arrange in a tandem fashion to form an unusual assembly for an RNA binding protein. The highly conserved WH domain is required for RNA binding and hemolysin production in S. aureus, and therefore, represents a novel use for this nucleic acid binding module.

RESULTS

RNA Binding Activity of S. aureus CvfB

Sequence similarity searches indicated that CvfB contains S1 domains, which are common RNA-binding modules. Therefore, we tested whether S. aureus CvfB binds RNA. We purified recombinant CvfB, that was overexpressed in Escherichia coli, to >95% purity as indicated by a single band on polyacrylamide gel electrophoresis (SDS-PAGE) (Supplemental Fig. 1A), using nickel affinity and anion exchange (MonoQ) chromatography (Supplemental Table 1). Poly(U) binding experiments on the MonoQ chromatography fractions revealed that poly(U) binding activity co-migrated with CvfB (Fig. 1A). The binding of poly(U) to CvfB is saturable (Fig. 1B), and the apparent Kd value was 0.52 nM according to Scatchard analysis.

Figure 1.

Figure 1

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RNA binding activity of in S. aureus CvfB. (A) The elution profile using MonoQ chromatography. Filled circles indicate protein concentration. Open circles indicate poly(U) binding activity determined using a filter binding assay. The dotted line shows the NaCl concentration gradient. (B) Various concentrations of radio-labelled poly(U) and CvfB (3 pmol) were incubated on ice for 1 h. The amount of bound poly(U) was determined by a filter binding assay.

We next examined the binding specificity of CvfB to nucleic acids other than poly(U). Varying amounts (number of molecules) of poly(U), poly(G), poly(C), poly(A)poly(U), poly(C)poly(G) RNA, E. coli tRNA, sonicated salmon sperm DNA, or S. aureus tmRNA, were added as competitors in binding assays containing sufficient labeled poly(U) to saturate the binding sites in CvfB. The amount of labeled poly(U) bound to CvfB was measured using a filter binding assay. The amount of unlabeled poly(U) and poly(G) needed to inhibit labeled poly(U) binding by 50% was the same as that for labeled poly(U), whereas poly(A)poly(U) and poly(C)poly(G) required 4 times more, S. aureus tmRNA 25 times more, and poly(C), E. coli tRNA, and sonicated salmon sperm DNA at least 200 times more (Table 1). For poly(A), a direct binding assay was performed because poly(A) hybridizes with poly(U) (Supplemental Fig. 1B). The apparent Kd value of CvfB for poly(A) was 1.1 nM, only twice that of poly(U). Therefore, CvfB specifically binds poly(U), poly(A) and poly(G), but not poly(C), double-stranded RNA or some other RNA’s. Additionally, we measured the poly(U) binding activity of CvfB from S. pneumoniae, and confirmed that it binds RNA with an apparent Kd of 0.02nM and with weaker affinity for poly(U) (Supplemental Fig. 2).

Table 1.

Polynucleotide specificity for the binding activity of CvfB

Competitor Amount for 50% inhibition (competitior/labeled poly(U))
Poly(U) 1
Poly(G) 1
Poly(C) >250
Poly(A) poly(U) 4
Poly(C) poly(G) 4
E.coli tRNA >230
Sonicated salmon sperm DNA >200
tmRNA 25
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Structure Determination of the CvfB Ortholog from S. pneumoniae

In order to further investigate how CvfB functions, we undertook the structure determination of CvfB. We maximized our chances of obtaining a CvfB structure, by cloning and expressing 25 orthologs (in addition to S. aureus CvfB) for crystallization trials using the high-throughput structural genomics pipeline implemented at the Joint Center for Structural Genomics (JCSG) (Lesley et al., 2002). CvfB from S. pneumoniae was the only ortholog which resulted in diffraction quality crystals. A selenomethionine derivative of the full-length S. pneumoniae CvfB (residues 1–284) was expressed in E. coli with an N-terminal TEV cleavable His6-tag, and was purified by nickel affinity chromatography and crystallized. The diffraction data were indexed in space group P3221 and the structure solved at 1.4 Å resolution, with one molecule per asymmetric unit, using the SAD method (Rcryst=15.4/Rfree=17.5). The main-chain density was readily interpretable throughout the entire structure (Supplemental Fig. 3). The mean residual error of the coordinates was estimated at 0.05 Å by an Rfree-based diffraction-component precision index (DPI) method (Cruickshank, 1999). The CvfB structure displayed good geometry with an all-atom clash score of 3.63, and the Ramachandran plot produced by MolProbity (Davis et al., 2004) showed that all residues were in allowed regions, with 98.2 % in favored regions. The final model contained 285 residues including the residual purification tag (Gly0), one chloride ion, 10 ethylene glycol molecules from the cryoprotectant solution, and 646 waters. 12.6% of the residues were modeled in multiple conformations. Data collection, refinement and model statistics are summarized in Table 2.

Table 2.

Data collection and refinement statistics

Data collection λ1 SADSe
Space group P3221
Unit Cell (Å) a=62.56, b=62.56, c=160.20
Wavelength (Å) 0.9784
Resolution range (Å) 29.14-1.40
Number of observations 773,419
Number of unique reflections 72,475
Completeness (%) 99.8 (99.2)a
Mean I/σ (I) 16.8 (2.7)a
Rsym on I 0.10 (0.96)a
Model and refinement statistics
Resolution range (Å) 29.14-1.40
No. of reflections (total) 72,406
No. of reflections (test) 3,649
Completeness (% total) 99.8
Cutoff criteria |F|>0
Rcryst 0.154
Rfree 0.175
Stereochemical parameters
Restraints (RMS observed)
 Bond angle (°) 0.013
 Bond length (Å) 1.42
Average isotropic B-valueb (Å2) 16.6
ESU based on Rfree (Å) 0.054
Protein residues/atoms 385/2,540
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a

Highest resolution shell (1.48-1.40) in parentheses. The high resolution cutoff was chosen such that the mean I/σ(I) in the highest resolution shell is around 2.

b

This value represents the total B that includes TLS and residual B components, and bound solvent molecules. The average B-values for the whole protein, S1A, S1B, S1C and WH domain are 13.5, 15.7, 12.7, 12.8, 13.3 Å2 respectively.

ESU = Estimated overall coordinate error (Cruickshank, 1999).

Rsym = Σ|Ii−<Ii>|/Σ|Ii|, where Ii is the scaled intensity of the ith measurement and <Ii> is the mean intensity for that reflection.

Rcryst = Σ||Fobs|−|Fcalc||/Σ|Fobs|, where Fcalc and Fobs are the calculated and observed structure factor amplitudes, respectively.

Rfree = as for Rcryst, but for 5.0% of the total reflections chosen at random and omitted from refinement.

Overall Structural Description

The crystal structure revealed that CvfB consists of three consecutive S1 domains (S1A, residues 1–63; S1B, residues 64–134; S1C, residues 135–216), connected to a winged-helix domain (WH, residues 217–284) at the C-terminus (Fig. 2A). The four domains are arranged sequentially in an “L”-shape with dimensions of 89 Å × 51 Å × 37 Å, such that only neighboring domains interact with each other (Fig. 2B). The two arms (S1A/S1B and S1C/WH) of the “L” subtend an angle of ~109°. The three S1 domains form a tightly integrated unit, whereas the WH domain is more loosely connected to S1C through a short linker (residues 215–221).

Figure 2.

Figure 2

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Crystal structure of CvfB from S. pneumoniae. (A) Ribbon diagram of the CvfB monomer. Each domain is shown in a different color: S1A (residues 0–64) in green; S1B (residues 65–134) in red; S1C (residues 135–216) in blue; and the winged-helix (WH) domain (residues 217–284) in yellow. (B) Surface representation of CvfB monomer, in the same orientation as (A). (C) Sequence alignment between CvfBs from S. pneumoniae and S. aureus. Secondary structure elements (colored by domain as in (A) and the numbering of S. pneumoniae CvfB is shown at the top.

S1 and WH domains are well-characterized structural modules that are often involved in binding nucleic acids (Aravind et al., 2005; Arcus, 2002). However, the DALI structural similarity server (Holm and Sander, 1995) did not detect any overall resemblance to any known protein for the complete CvfB structure, nor for any combination of two or more adjacent domains. Therefore, the CvfB structure is a novel combination of these structural modules.

The CvfB protein family consists of more than 300 highly conserved bacterial members. The majority of CvfBs are present in firmicutes and proteobacteria, including many pathogens. S. pneumoniae CvfB shares 32% sequence identity with S. aureus CvfB (Fig. 2C), and 36% to the YitL protein of Bacillus subtilis. An alignment of homologous sequences indicates that S1C and WH are more conserved compared to S1A and S1B, with S1A being the least conserved. Mapping sequence conservation onto the structure of CvfB indicates that the most highly conserved residues can be assigned into two main groups. The first group are located in the hydrophobic core of each module and near the interfaces between the S1 domains. These residues likely maintain the structural integrity of these modules. The second group consist of residues that primarily reside on the surfaces of the S1C and WH domains and are more likely to be involved in function (Supplemental Fig. 4). Thus, CvfB is likely to be structurally and functionally conserved in different bacteria.

S1C Contains a Canonical RNA Binding Surface

The S1 domains of CvfB adopt the typical S1 fold that consists of a five-stranded, antiparallel β-sheet (β1A–β5A in S1A, β1B–β5B in S1B, and β1C–β5C in S1C) that forms a β-barrel. S1B has two 310-helices between its third and fourth strands, compared to only one 310-helix in S1A, S1C and other typical S1 domains. The three S1 domains share a highly conserved structural core and can be superimposed with an average rmsd of 1.96 Å for 58 equivalent Cα atoms in each domain (S1A/S1B: rmsd of 1.62 Å for 59 Cα, 14% sequence identity; S1B/S1C: rmsd of 2.4 Å for 61 Cα, 21% seq id, S1A/S1C: rmsd of 2.1 Å for 60 Cα, 10% seq id; Figs. 3A–C). The OB-fold (oligonucleotide/oligosaccharide-binding fold) in nucleic acid binding proteins, of which the S1 domain is a member (Bycroft et al., 1997; Frazao et al., 2006), generally possess a conserved site for nucleic acid interaction (Arcus, 2002). This site consists of a groove of positive electrostatic potential, which extends across the second and third strands, with additional contributions from the surrounding loops. These three S1 domains in CvfB likely have evolved from a common ancestor, as they all contain an equivalent phenylalanine on the second β-strand (Phe22 on S1A, Phe86 on S1B and Phe168 on S1C, Fig. 3C), which is highly conserved among S1 domains (see below), although the role of each S1 domain may differ.

Figure 3.

Figure 3

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S1 domains and domain interfaces. (A) Structure of the S1C domain. Structural comparisons of S1 domains, including S1A, S1B, S1C, and the domain I of archaeal initiation factor 2 α-subunit (aIF2α, PDB 1yz6). The conserved core of all four S1 domains mapped onto the S1C domain is colored red. The five β-strands are numbered from 1 to 5. The highly conserved residues on the putative RNA binding surface are shown as cyan spheres. The conserved polarity of the RNA in OB-fold nucleic acid binding proteins (Theobald et al., 2003) (5′-end to 3′-end of the RNA runs from β-strand 5 to β-strand 2) is indicated by a dash line. (B) Superimposition of Cα traces of S1A (green), S1B (red), S1C (blue) and aIF2α (gray) shown in stereo view. (C) A structure-based sequence alignment of the S1 domains, S1A, S1B, S1C, and the domain I of aIF2α. S1C residues that are likely involved in RNA binding are highlighted by orange dots at the bottom row. The secondary structures and numbering of S1C are shown at the top row. (D–E) Domain interfaces between the S1 domains in CvfB (S1A in green, S1B in red, S1C in blue). Approximate 2-fold axes relating two S1 domains are shown at the top. The residues contributing to the interfaces are shown in sticks (Phe22, Phe86 and Phe168 are also shown in sticks to highlight the locations of potential nucleic acid binding sites). Selected hydrogen bonds are shown in dashed lines. (D) Interaction between S1A and S1B. (E) Interaction between S1B and S1C.

Based on sequence analysis, S1C is more closely related to canonical S1 domains compared with S1A and S1B (see Supplemental data). The surface features of S1C also suggest that it contains a canonical RNA binding site. This potential binding site contains Phe168, Phe179, His181 at the center, and Arg161, Lys 163, Ser165, Ser183, Glu184, Arg198, Ile200, Arg203, Asn210, and Ser212 at the perimeter; most of these residues are highly conserved among CvfB homologs. S1C is structurally most similar to the RNA-binding domain I of the archaeal initiation factor 2 α-subunit (aIF2α) with an rmsd of 2.3 Å for 71 aligned Cα atoms (28% seq id, Figs. 3A–C) (Yatime et al., 2005). The RNA binding surfaces of CvfB (S1C) and aIF2α (domain I) are very similar, with 11 residues conserved out of the 13 possibly involved in RNA binding (Fig. 3C). Among these residues, Phe168 and His181 are also observed in the S1 domains of Tex and PnPase (Bycroft et al., 1997; Johnson et al., 2008). Thus, it is likely that S1C interacts with RNA in a similar manner to other S1 domains (Fig. 3A). One unique structural feature of CvfB is a short helix (α1) that occupies the lower part of the β-sheet surface of S1C and could influence RNA interactions by restricting the potential nucleic acid interface (Fig. 3A).

Domain interface between S1 domains

Multiple consecutive S1 domains have been observed in many RNA binding proteins, most notably in the E. coli ribosomal protein S1 (Subramanian, 1983), a translation initiation factor with six S1 domains. CvfB represents the first structure with more than two consecutive S1 domains. S1A/S1B and S1B/S1C display two different modes of dimerization interaction (Figs. 3D–E). S1A interacts with the L23 (loop between β-strand 2 and β-strand 3) and L45 loops of S1B through its N-terminus and L45 loop in a tail-to-tail manner (Fig. 3D). These secondary structures near the interfaces are conserved. For example, the L45 hairpin turn of S1B is stabilized by hydrogen bonds between the highly conserved Asp124 and Arg128. The interface between S1A and S1B is complementary in shape and buries 736 Å2 of surface area per domain. The interaction between S1A/S1B involves hydrophobic contacts and 7 hydrogen bonds.

S1B and S1C are juxtaposed together with a pseudo two-fold symmetry along the direction of the β-barrel (Fig. 3E). The relative orientation of the two S1 domains in CvfB resembles that of BRCA2 (OB1 and OB2, OB-fold domains) (Yang et al., 2002). S1B is connected to S1C through a linker (residue 135–152) that consists of a short helix (α1). The domain interface, which buries 632 Å2 per domain, is also stabilized by conserved hydrophobic contacts and hydrogen bonding interactions. The secondary structural elements contributing to inter-domain interfaces include the L34 loop (residues 102–118) of S1B, β1, L34 of S1C, and the linker. The primary interactions are centered at two points of contact points between the L34 loops of S1C and S1B, and the α1 linker area. The interface is further stabilized by hydrogen bonds that include two between the strictly conserved Asp115 and Arg190, three between main-chain atoms, and additional water-mediated hydrogen bond network. These conserved and extensive interfaces between the S1 domains support the notion that the three S1 domains likely function as a rigid unit.

OB-fold modules support packing of homologous domains in a variety of ways. Inspection of structures with two or more repeats of OB-fold domains, such as Replication protein A (RPA70) (Bochkarev et al., 1997), BRCA2 (OB2-OB3) (Yang et al., 2002), yeast exosome core Rrp44 (Lorentzen et al., 2008), RNase II (Frazao et al., 2006), and the elongation factor P (Hanawa-Suetsugu et al., 2004), indicates that two OB-fold domains are often related by pseudo-translation in either side-by-side or head-to-tail fashion. These arrangements produce collinear alignment of multiple DNA/RNA binding sites that interact with a extended ssDNA/RNA targets. The packing of S1 domains in CvfB generates an interesting arrangement of potential RNA binding sites. The corresponding sites of S1A and S1B are on the same side of the molecule, but separated by a ridge formed the L45 loop of S1A and the L23 loop of S1B (Fig. 3D). The potential nucleic acid binding site of S1C is on opposite side of that of S1B (Fig. 3E).

An Extended Surface across WH and S1C

The inner sides of both arms of the CvfB molecule have negative electrostatic potential, while positive electrostatic patches are present on the outer side and lateral surfaces (Fig. 4A). The potential RNA binding residues on S1C, as indentified above, forms a curved groove that leads to a highly conserved surface on the WH domain. This region lies parallel to the “recognition helix”, α4, which contains highly conserved, solvent-exposed residues (Ser257, Lys258, Lys262, Lys263, Gly266, Met269, and Lys270). Additional residues contributing to this surface are Asp243 and Lys244, Pro247, Asp248 and Lys251 (Fig. 4B). Thus, these residues define a potential nucleic acid binding surface on WH and the two adjacent binding surfaces on S1C and WH likely constitute an extended binding interface on the same side of the molecule.

Figure 4.

Figure 4

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Potential nucleic acid binding sites in CvfB. (A) Electrostatic potential of CvfB. The potential nucleic acid binding site for each domain is marked by 1, 2, 3 and 4 from S1A to WH domains. Crude models of RNA fragments (yellow) passing through the potential nucleic acid binding sites on S1 domains are shown, assuming a conserved mode of RNA recognition by S1 domains. (B) Putative RNA binding sites of S1C and WH. Potential residues involved in RNA binding are highlighted (positively charged: blue, polar: magenta, hydrophobic: yellow, and acidic: red). (C) A conserved binding interface on S1B (red) and its interface with S1A (green). The black dotted lines indicate directions that are approximately perpendicular to the β-sheet surfaces. Hydrogen bonds are denoted by dashed sticks.

The WH domain makes multiple contacts made with symmetry-related molecules in the crystal lattice. Thus, the conformation of the WH domain could be influenced by crystal packing. The length of linker (residue 215–221) between WH and S1C is conserved. The N-terminus of the linker is tethered to S1C domain with multiple hydrogen bonds between the linker residues (Pro215 and Arg216) and residues from S1C (Arg198, Asn152, and Asn154). The C-terminus of the linker (Leu221) is stabilized by hydrophobic interaction with CvfB-WH. Thus, only flexibility between S1C and WH may be possible due to some potential freedom of the main-chain dihedral angles of residues 217–220. However, this flexibility is likely reduced by a hydrogen bond between the side chains of Glu216 and Ser217.

As discussed above, the potential nucleic acid binding surfaces on the S1 domains are not continuous (Figs. 4A, C). As a result, the potential RNA binding site on S1B appears to be disconnected from the proposed RNA binding surface on S1C-WH. An additional electropositive patch located near the interface of S1B and S1C of S. pneumoniae CvfB is not likely a general feature of CvfBs since the underlying residues are not conserved.

Dissecting RNA Binding Activity of CvfB Domains

Next, we attempted to identify the domains of CvfB required for RNA binding activity using the S. aureus protein. We engineered several CvfB protein constructs that contained various domain segments (Fig. 5A). Each protein construct was purified by nickel affinity chromatography to >70% purity (Fig. 5B). We then determined the amount of each construct needed for 50% saturation of the binding (i.e. K value) to evaluate their affinity for poly(U) (Fig. 5C). The K values of CvfB-1 (1–62, S1A) and CvfB-2 (1–149, S1A-S1B) were 14-fold higher (i.e. poorer binding) than that of wild-type CvfB. On the other hand, the K values of CvfB-4 (63–300, S1B-S1C-WH) and CvfB-5 (150–300, S1C-WH) were similar to those of wild-type CvfB whereas CvfB-6 (WH) was 4-fold higher than CvfB-5 (S1C-WH). Moreover, the K value of CvfB-3 (1–225, S1A- S1B-S1C) was 7-fold higher than that wild-type CvfB. These results suggest that the S1C domain and the WH domain jointly contribute to most of the RNA binding activity and neither S1A nor S1B are essential for RNA binding activity, in good agreement with the analysis of the CvfB structure.

Figure 5.

Figure 5

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Mapping of the RNA binding region of S. aureus CvfB. (A) The domain constructs are indicated as black bars. The K values of CvfB mutant proteins for poly(U) are indicated. (B) Analysis by SDS-PAGE of the CvfB protein constructs purified by nickel affinity chromatography. (C) Titration of CvfB constructs using a fixed amount of poly(U). Labelled poly(U) (50 pM) and various amounts of the individual CvfB protein constructs were incubated on ice for 1h. The amount of residual labelled poly(U) was determined using a filter binding assay. (D) Model of a potential interaction between CvfB and mRNA. (E) SDS-PAGE analysis of the site-directed CvfB mutants: F175A, K249A, G271E and K249A/K267A/K275A. (F) Poly(U) binding activity of these mutants is shown in (E), as determined using the filter binding assay.

It is well-known that RNA binding proteins are modular, consisting of multiple repeats of a few basic units (Lunde et al., 2007). The length and rigidity of the linkers between these modules can significantly affect RNA binding affinity, with shorter linkers resulting in a dramatic increase in RNA binding affinity (Shamoo et al., 1995). The short length of the linker between S1C and WH, coupled with the close proximity of their potential nucleic acid binding surfaces, suggests that they bind RNA cooperatively (Fig. 5D), consistent with the experimental evidence presented above. The concatenation of S1 and WH domains represents a novel combination of RNA binding modules.

Furthermore, we constructed additional site-directed mutations of several conserved residues on the potential nucleic acid binding surfaces identified above, F175A, K249A, G271E and K249A/K267A/K275A on S1C and WH, respectively (Figs. 5E–F). Phe175 (Phe168 of S. pneumoniae CvfB) is highly conserved among S1 domains. Lys249 (Lys244 of S. pneumoniae CvfB) is located on a loop that is unique to the CvfB WH domain (DK-loop, see below). Gly271, Lys267 and Lys275 are all located on the “recognition helix” of the WH domain. G271E and K249A/K267A/K275A mutants were designed to significantly alter the electrostatic property of the nucleic acid binding surface by either introducing a negative charge into a surface patch with positive electrostatic potential or by eradicating positive charges directly. All of the mutants characterized were soluble, indicating that they were likely to be properly folded. The poly(U) binding affinity of the first two mutants decreased by half compared to that of the wild type. As expected, the triple lysine mutant had a more dramatic effect on the RNA binding affinity. The G271E mutation had a similar effect as that of the ΔWH domain mutant. These results clearly support our proposal that the surfaces defined by conserved residues on S1C and WH are involved in RNA binding (Fig. 4B).

A Novel WH Domain in CvfB

As we have shown, the small and compact WH domain located at the C-terminus of CvfB contributes significantly to RNA binding. WH domains, a variant of general Helix-Turn-Helix (HTH) DNA binding proteins, are widely distributed in transcription factors. The electrostatic surface properties of the CvfB-WH domain are similar to the canonical WH domains that use a “recognition helix” to bind the major groove of double-stranded DNA (Brennan, 1993).

CvfB-WH contains a stretch of highly conserved residues (241–270, Fig. 6A), where residues 258–270 correspond to the “recognition helix”. However, the region between β16 and α4 (residues 241–257) differs significantly from other WH domains (Fig. 6B). This region is stabilized by extensive interactions. One short β-strand (β16, residues 238–239) anchors its N-terminus to the β-hairpin (β17–β18) of WH. The β16-α3 turn (DK-loop, residues 241–245) is stabilized by hydrophobic contacts and hydrogen bonds involving highly conserved residues (Ser245, Lys258, and Gln276). The DK-loop packs against the recognition helix α4, which would pose a significant “steric barrier” if α4 were to insert into the major groove of DNA (Fig. 6C). Other types of recognition of dsDNA in WH domains through the wing are also sterically unfavorable due to the short two-residue wing in CvfB (W1) (Gajiwala and Burley, 2000). Thus, the structure of CvfB-WH clearly can not accommodate the binding to dsDNA or dsRNA by any currently known mechanisms, again consistent with our binding studies.

Figure 6.

Figure 6

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The winged-helix (WH) domain of CvfB. (A) Sequence conservation of CvfB-WH domain represented by a sequence logo, generated using WEBLOGO (Crooks et al., 2004) using 321 CvfB orthologs. The residue number and secondary structures are shown at the bottom. A ribbon representation of the WH domain with conserved surface residues shown in sticks. The substructure unique to the CvfB-WH domain is shown in red. (B) Structural comparison of CvfB-WH with the WH domain of methicillin-resistance regulating transcriptional repressor MecI (PDB 1sax, residues 3–73, cyan) (Garcia-Castellanos et al., 2004). The wing in the WH domain is labelled as W1. (C) A steric barrier created by the DK-loop region of CvfB-WH suggests that it cannot interact with dsDNA (in a canonical mode). (D–H) Structural comparisons between CvfB-WH with other known RNA binding WH modules and the KH domain. RNA binding residues are colored red. Nucleic acids are shown in yellow. (D) CvfB-WH. (E) SelB (PDB 1wsu). (F) La protein WH (PDB 1zh5). (G) Zα domain (PDB 2gxb). (H) KH domain (PDB 2asb).

WH modules have, nevertheless, been identified in a few RNA binding proteins, such as SelB (Yoshizawa et al., 2005), La motif (Teplova et al., 2006) and Zα domains (Placido et al., 2007). SelB achieves high binding specificity through base-specific interactions by the WH motif, and interactions with the RNA backbone through shape and charge complementarity of the protein surface (Fig. 6E). The La protein binds RNA that has a UUU(OH) tail with the backside of its WH motif (Fig. 6F), mainly involving conserved aromatic amino acids. Zα domains recognize RNA/DNA backbones with the recognition helix and the W1 wing (Fig. 6G). The conformations of the RNA recognized by the WH motifs differ greatly among these proteins. Thus, WH motifs can support different modes of RNA, as well as DNA interaction. The unusual conformation in the region before the recognition helix (β16-α4 region) in CvfB is not observed in other WH modules. Additionally, the distribution of conserved residues in CvfB is also unique. Thus, we conclude that the CvfB-WH likely represents a novel RNA binding module.

Most RNA binding modules make use of a β-sheet surface for RNA recognition, as found in S1 domains (Auweter et al., 2006; Theobald et al., 2003). KH domains are a common RNA-binding structural motif whose RNA binding interface involves a helix-turn-helix (Fig. 5H) (Auweter et al., 2006; Beuth et al., 2005). Interestingly, the arrangement of the conserved secondary structural elements in CvfB-WH is similar to that of the RNA binding surface in the KH domain despite their different overall folds (Figs. 6D,H). The predicted polarity of the RNA on the CvfB-WH also appears to be similar to KH.

CvfB-WH is Necessary for Hemolysin Production in S. aureus

We previously reported that cvfB is needed for transcription of the hla gene, which encodes α-hemolysin in S. aureus (Matsumoto et al., 2007). Here, we investigated whether the WH domain of CvfB is also essential for hemolysin production in S. aureus. Thus, we measured the hemolytic activity of cvfB deletion mutants expressing CvfB-3 (S1A-S1B-S1C) or CvfB-4 (S1B-S1C-WH). The decreased hemolytic activity of the cvfB deletion mutant of the S. aureus RN4220 strain was restored by wild-type CvfB expression, whereas restoration did not occur with CvfB-3 (S1A-S1B-S1C), which has little poly(U) binding activity (Fig. 6A). On the other hand, hemolytic activity was restored by expression of CvfB-4 (S1B-S1C-WH), which has the same poly(U) binding activity as wild-type CvfB (Fig. 7A). Further, Western blot analysis using an anti-CvfB antibody demonstrated that the CvfB mutant proteins were expressed in the cvfB deletion mutant (Supplemental Fig. 5). We repeated the above analysis using the agr-null mutant of strain NCTC8325-4, in which the disruption of cvfB caused a decrease in hemolysin production (Matsumoto et al., 2007). The amount of activity restored is similar to that of RN4220 strain (Fig. 7B). These results suggest that CvfB-WH is required for hemolysin production in S. aureus.

Figure 7.

Figure 7

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The WH domain of CvfB regulates hemolysin production in S. aureus. RN4220 (A) or CK1844 (B) were used as the parent strain. Hemolytic activities of culture supernatants of the cvfB deletion mutants [M1223 (A) or CK12231 (B)] transformed with a vector for expressing wild-type or CvfB mutant proteins (CvfB-3, CvfB-4) were measured with sheep erythrocytes. Average values of duplicates are shown. Hemolytic activities of the parent strain [RN4220 (A) or CK1844 (B)] were 6.1 ± 0.1 or 16.8 ± 1.8, respectively. Student t test P values between CvfB-4 and other CvfB mutants were <0.05, respectively.

DISCUSSION

CvfB likely regulates the expression of virulence genes through its RNA binding activity. RNA binding proteins have been previously implicated in bacterial pathogenicity, as for example, PNPase and RNase II (Bycroft et al., 1997) and PNPases of Yersinia and Salmonella (Rosenzweig et al., 2005). The regulatory mechanism of virulence gene expression by these RNA binding proteins has not yet been elucidated. RNA-binding proteins can regulate gene expression in bacteria through their interactions with RNAs in a variety of ways, such as preventing RNA decay by RNases, mediating of secondary structures of the mRNA to regulate translation initiation, and transcription termination/antitermination. Understanding how binding of CvfB to RNA modulates virulence gene expression may help to define a novel regulatory mechanism of virulence gene expression by RNA binding proteins.

Possible Secondary RNA Binding Sites

The roles of S1A and S1B in CvfB are less clear compared to those of the S1C and WH domains. Many residues on the β-sheet surface of S1B are also highly conserved (Leu83, Asp88, Phe86, Lys94, Glu95 and Val97) and form a cleft on S1B which span β5 to β2 (Fig. 4C). In the absence of the S1C domain, CvfB-2 (S1A-S1B) has a higher affinity for poly(U) than CvfB-1 (S1A) alone (Fig. 5C). Therefore, S1B domain may also have some RNA binding capacity. At least two possibilities can be considered for the potential roles of S1B and S1A. First, the cleft on S1B may be an extension of the S1C-WH binding surface. However, this seems less likely since these binding surfaces are not contiguous. In order for a single ssRNA molecule to make contacts with all the potential sites, it would have to curve and make non-specific and unfavourable contacts with significant portions of the intervening protein surface (Fig. 4A). Alternatively, the site on S1B (and S1A) may represent a secondary binding site for another RNA molecule (or protein). Indeed, RNA binding molecules often contain auxiliary domains which mediate protein-protein interactions or subcellular targeting, which allows the bound mRNA to be processed by an mRNA processing machinery, such as the ribosome, basal transcription or the degradation complex (Siomi and Dreyfuss, 1997). For example, the first two S1 domains of the ribosomal protein S1 are involved in binding ribosome whereas the last four binds mRNAs (Subramanian, 1983).

CvfB-WH Domain May be Important for RNA Specificity

We demonstrated here that CvfB has poly(U) binding activity. Analysis of various CvfB constructs revealed that the RNA binding activity of CvfB is necessary for the expression of hemolysin in S. aureus. However, the exact nature of the RNA molecules with which CvfB interacts in vivo remains unclear. The specificity of CvfB for RNA is different from that of other RNA binding proteins that contain S1 RNA binding domains. For example, chloroplast S1-like ribosomal protein (CS1) from Spinacia oleracea has a higher affinity for poly(U) when compared to poly(C), poly(G), and poly(A) (Shteiman-Kotler and Schuster, 2000) while CvfB has a similar affinity for poly(U), poly(A), and poly(G). The affinity of E. coli S1 ribosomal protein for tmRNA is 100-fold higher than that for poly(U) (McGinness and Sauer, 2004; Wower et al., 2000). Our findings indicate that the affinity of CvfB for tmRNA is much less than that for poly(U).

The S1C RNA binding surface is similar to a conventional S1 domain, which often lacks sequence specificity for RNA. It remains possible that additional secondary structures (such as α1) near the binding site may alter the specificity of S1C. The WH domain is lysine rich, indicating that electrostatic interactions are important for the binding of RNA by CvfB-WH. The distribution of strictly conserved residues on CvfB-WH provides an intriguing clue that the WH domain might possess specificity for the bases. The sequence motif, 262Kx3Gx3K270, is arranged on the recognition helix surface, such that a glycine residue (Gly266) is sandwiched between Lys262 and Met269-Lys270 (269 is a conserved hydrophobic residue). A cavity created by the absence of a side chain at the glycine position could be occupied by a base. The neighboring phosphoryl groups defining the positions of this base could then be tethered by the two lysines (Lys262 and Lys270), likely with an additional contribution from the nearby invariant Lys258. The selection of a base could be achieved by the strictly conserved Asp243, which is near Lys262, and the size/shape of the cavity (Fig. 8). This model is consistent with the results from site-directed mutagenesis (Figs. 5E–F).

Figure 8.

Figure 8

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A hypothetical model of CvfB-WH and RNA recognition. A potential base-specific interaction by the conserved structural motif within CvfB-WH (residues for S. aureus CvfB are shown in parenthesis). The phosphoryl groups are shown as yellow spheres and labelled as P1 and P2.

In summary, we propose a following model for RNA binding by CvfB. The two C-terminal domains (S1C-WH) of CvfB likely bind mRNA (Fig. 5D). The polarity of the RNA can be inferred from the S1C domain as the polarity of the RNA in OB-fold nucleic acid-binding proteins, that runs 5′ to 3′ perpendicular to the β-sheet surface from β5 to β2, is high conserved (Theobald et al., 2003). As a result, the 5′-end of the RNA bound to the S1C domain would point towards the WH domain. The RNA model would extend on the WH domain approximately parallel to the “recognition helix”. S1C may interact with RNA similar to typical OB-fold proteins, such that aromatic hydrophobic residues in the centre of the face (e.g. Phe168, Phe179 and His181) stack against the nucleotide bases and positively charged residues at the periphery (e.g. Arg161, Arg203) bind to the phosphate backbone (Arcus, 2002). Assuming that the RNA is in an extended conformation, we can estimate that each module could recognize ~4 nucleotides and overall 10–12 nucleotides across the two domains. The WH domain may play an important role in base specificity, mediated by the “recognition helix” and the unique structural motif preceding it. The hinge between S1C and WH domains may help to accommodate any flexibility in recognition of different mRNA molecules.

EXPERIMENTAL PROCEDURES

Protein Production for Crystallization (S. pneumoniae)

The selenomethionine derivative of full length CvfB was cloned and expressed in E. coli with an N-terminal TEV cleavable His-tag, and purified by nickel affinity chromatography. Details can be found in Supplemental Data.

Oligomeric State of CvfB

The oligomeric state of CvfB was determined using a 0.8 × 30cm2 Shodex Protein KW-803 column (Thomson Instruments) pre-calibrated with gel filtration standards (Bio-Rad). CvfB is likely a monomer in solution, supported by crystal packing analysis and analytical size exclusion chromatography.

Crystallization

CvfB was crystallized at 4°C using the nanodroplet vapor diffusion method (Santarsiero et al., 2002) with standard JCSG crystallization protocols (Lesley et al., 2002). The crystallization reagent consisted of 0.364 M ammonium dihydrogen phosphate (NH4·H2PO4). The plate-shaped crystal used for structural solution was 0.2 × 0.1 × 0.02 mm3 in size. Ethylene glycol was added as a cryoprotectant to a final concentration of 20% (v/v).

Data Collection, Structure Determination and Refinement

Single-wavelength anomalous diffraction (SAD) data corresponding to the peak energy of a selenium MAD experiment were collected at SSRL beamline 11-1. Data processing and structure solution were carried using an automatic structure solution pipeline XSOLVE developed at JCSG. Details can be found in Supplemental data.

Filter Binding Assay (S. aureus)

Cloning, expression, and purification of recombinant CvfB and mutants from S. aureus are described in Supplemental data (Supplemental Tables 2–3). Poly(U), poly(C), poly(G), poly(A)poly(U), poly(C)poly(G), and E. coli tRNA were purchased from Sigma (St. Louis, MO). Sonicated salmon sperm DNA was purchased from Stratagene (La Jolla, CA). S. aureus tmRNA was synthesized using T7 RiboMAX Express Large Scale RNA Production System (Promega Co., Madison, WI) from the DNA fragment amplified using primers FSAtmRNA01 and RSAtmRNA01 with RN4220 genomic DNA as a template (Wower et al., 2000).

Poly(U) or poly(A) fragments (Sigma) were labeled with [γ32P]-ATP using T4-polynucleotide kinase. For estimating Kd values from Scatchard plots, the CvfB protein (3 pmol) was incubated with various concentrations of labelled polynucleotide (0.015–12 nM) in 100 μl binding buffer A (10 mM Tris-HCl pH 7.6, 100 mM NH4Cl, 20 mM magnesium acetate, 1 mM dithiothreitol) at 4°C for 1 h. The samples were filtered through a HAWP filter (Millipore, Bedford, MA) treated with alkaline (Smolarsky and Tal, 1970), and washed 3 times with 5 ml binding buffer A. The amount of radioactivity on the filter was measured using a liquid scintillation counter. For estimating the K value, labelled poly(U) (50 pM) was incubated with various concentrations of protein in 100 μl binding buffer B (10 mM Tris-HCl pH7.6, 100 mM NH4Cl, 20 mM magnesium acetate, 1 mM dithiothreitol, 0.1 mg/ml BSA(bovine serum albumin)) at 4°C for 1 h (Riggs et al., 1970). After incubation, the amount of binding complex was measured using the method described above. BSA did not bind RNA under above assay condition (data not shown).

Hemolytic Activity Assay

Hemolytic activity was measured using the previously described method (Vandenesch et al., 1991). Briefly, a supernatant of the culture at 12 h after inoculation was incubated with sheep red blood cells at 37°C for 1 h. The reaction mixture was centrifuged (1 krpm, 5 min) and the increase in the OD450 of the supernatant was determined. The activity was expressed by hemolytic unit (HU) corresponding to the reciprocal of the dilution of supernatant that yielded 50% lysis of the erythrocytes.

Supplementary Material

01

Acknowledgments

We thank all the members of the JCSG for their general contributions to the protein production and structural work. We also thank Dr. Keiichi Hiramatsu (Juntendo University, Tokyo, Japan) and Dr. Naotake Ogasawara (Nara Institute of Science and Technology, Nara, Japan) for reagents. The project is sponsored by US National Institutes of Health, National Institute of General Medical Sciences Protein Structure Initiative (U54 GM074898 to the JCSG) and by a Grant-in-Aid for Scientific Research (Japan) (21022015, 20790057, 20390021) and also in part by the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO, Japan) and Genome Pharmaceuticals Institute Co., Ltd. (Tokyo, Japan). Portions of this research were carried out at the Stanford Synchrotron Radiation Lightsource (SSRL). SSRL is a national user facility operated by Stanford University on behalf of the U.S. DOE, OBES. The content is solely the responsibility of the authors and does not necessarily represent the official views of NIGMS or NIH.

Footnotes

Accession Code

Coordinates and structure factors have been deposited in the Protein Data Bank (PDB, http://www.wwPDB.org/) under accession code 3go5.

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SUPPLEMENTAL DATA

Supplemental Data include Supplemental Experimental Procedures, five figures, and three tables and can be found with this article online at http://www.....

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