Structure and function of accessory Sec proteins involved in the adhesin export pathway of Streptococcus gordonii

Many pathogenic bacteria, including Streptococcus gordonii, possess a pathway for the export of a single serine-rich-repeat protein that mediates the adhesion of bacteria to host cells and the extracellular matrix. These adhesins are O-glycosylated by several cytosolic glycosyltransferases and require three accessory Sec proteins (Asp1-3) for export, but how the adhesins are processed for secretion is not well defined. Here, we show that O-glycosylation of S. gordonii adhesin GspB occurs in a sequential manner by three enzymes (GtfA/B, Nss, Gly) that attach N-acetylglucosamine and glucose to Ser/Thr residues. The modified substrate is subsequently transferred from the last glycosyltransferase to the Asp1/2/3 complex. Crystal structures show that both Asp1 and Asp3 are related to carbohydrate binding proteins. Asp1 also has an affinity for phospholipids, which is attenuated by Asp2. These results suggest a mechanism for the modification of adhesin in the cytosol and its subsequent targeting to the export machinery.


INTRODUCTION
Adhesion proteins are instrumental for the pathogenicity of bacteria (1). Streptococci and staphylococci bacteria express serine-rich repeat (SRR) adhesins that are exported from the cell, but remain associated with the cell wall and allow the bacteria to attach to the host cells and their extracellular matrix (2,3). In addition, these adhesins may also mediate interactions between bacteria, facilitating biofilm formation and bacterial colonization (4). The biosynthesis of SRR adhesins is a promising target of novel antibiotics that could be used to treat diseases caused by streptococci and staphylococci, such as infective endocarditis, pneumococcal pneumonia, neonatal sepsis, and meningitis (3).
SRR adhesins use a dedicated pathway for their export from the cytosol, called the accessory Sec system (5,6); most other proteins are exported from the bacterial cell by the canonical Sec pathway (7). In the canonical pathway, proteins are moved by the SecA ATPase through the protein-conducting SecY channel. In the accessory Sec pathway, export is mediated by distinct SecA and SecY proteins (SecA2 and SecY2).
These components are encoded in an operon that also includes the adhesin substrate as well as several glycosyltransferases and accessory Sec system proteins (Asps) (5,6). The glycosyltransferases attach sugar residues to adhesin before its export from the cytosol (2,8), but the exact roles of the glycosyltransferases and Asps in the export pathway is not well defined.
The SRR adhesins are initially modified with N-acetylglucosamine (GlcNAc) at multiple Ser/Thr residues by the heterodimeric GtfA/B glycosyltransferase (9)(10)(11)(12)(13)(14). The deletion of GtfA or GtfB results in non-glycosylated adhesins that are prone to degradation (11,14,15). Glycosylation is physiologically important as the deletion of GtfA also reduces the adhesion of bacteria to host cells (15,16). Recent results show that GtfA is the catalytic subunit, while GtfB is involved in substrate binding (10). Most SRR adhesins are further modified by additional glycosyltransferases that are also encoded by the same operon (5,6). In S. parasanguinis and S. pneumoniae, they modify adhesins in a sequential manner (17,18). In S. gordonii, there are two such glycosyltransferases, Nss and Gly (5). Deletion of either enzyme results in compromised modification of the SRR adhesin GspB (9). Nss from related streptococcal species adds glucose to GlcNAc attached to Ser/Thr-containing peptides (19)(20)(21). It is unclear how Gly modifies the adhesin GspB, and whether Nss and Gly act sequentially or have redundant functions.
S. gordonii encode three Asps (Asp1,2,3), which are conserved among different bacterial species that express SRR adhesins (5,6). Deletion of any of the Asps blocks the export of the adhesin GspB and results in its intracellular accumulation (15,22). An essential role for the Asps in the biogenesis of SRR adhesins has also been observed in other species (16,(23)(24)(25). Interactions among the Asps and of the Asps with substrate and SecA2 have been reported for both S. gordonii and S. parasanguinis (where the Asps are called Gaps) (22)(23)(24)(26)(27)(28), but it remains unclear how the Asps function in GspB export.
Here, we show that the modification of the adhesin GspB of S. gordonii by the glycosyltransferases occurs in a sequential manner. First, GlcNAc residues are attached to Ser/Thr residues in the SRR domains of GspB. Next, Nss adds glucose to GlcNAc, and finally, Gly adds glucose to previously attached glucose residues.
Interestingly, Gly remains bound to the modified substrate. Release of modified GspB from Gly is caused by the complex of the three Asps (Asp complex). Crystal structures show that indeed both Asp1 and Asp3 are carbohydrate-binding proteins. Asp1 is a catalytically inactive member of the GT-B family of glycosyltransferases and Asp3 contains a carbohydrate-binding module also found in several glycosidases. Our results also show that Asp1 has an affinity for negatively charged phospholipids, which may facilitate substrate delivery to the membrane. Taken together, our results suggest a model for the pathway by which the adhesin is modified and targeted to the export machinery.

Glycosyltransferases act in a sequential manner
To test the role of the glycosyltransferases in adhesin modification, we produced a fragment of the GspB substrate by in vitro translation in reticulocyte lysate in the presence of 35 S-methionine. The GspB fragment (GspB-F; Figure S1) contains residues 91 to 736, including the first Ser/Thr-rich domain (SRR1), an intervening sequence that normally binds to host cells (binding region; BR), and the N-terminal part of the second Ser/Thr-rich domain (SRR2N). It lacks the N-terminal signal sequence. GspB-F with the signal sequence is glycosylated in S. gordonii cells and secreted with the same efficiency as full-length adhesin (29). In vitro translation of GspB-F generated nonglycosylated protein that could be visualized as a single band after SDS-PAGE and autoradiography ( Figure 1A; lane 1). As described previously, when a purified complex of GtfA and GftB and UDP-GlcNAc were added after translation, a size shift was observed, caused by modification of GspB-F with GlcNAc residues (G1 species; lane 2). Finally, yet a larger species was generated when purified Gly was introduced (G3 species; Figure 1A; lane 4). Nss did not attach Glc residues to unmodified GspB-F ( Figure 1C; lane 1 versus 3), indicating that it can only modify substrate after GtfA/B has added GlcNAc residues. The same is true for Gly ( Figure 1D; lane 1). Finally, modification by Gly was dependent on the prior action of Nss ( Figure 1E; lane 3 versus 4). Taken together, these results indicate that GtfA/B, Nss, and Gly function in a defined order; GtfA adds GlcNAc residues to Ser/Thr residues in the SRR domains, which are then further modified with Glc residues by the sequential action of Nss and Gly.
To test whether the modification of GspB with multiple sugars occurs in vivo, we purified GspB-F secreted from S. gordonii and used mass spectrometry to analyze sugars released by β-elimination ( Figure S2A). The results show that the protein indeed contains one N-acetyl hexose (HexNAc) and either zero, one, or two hexoses.
Modification with three sugar residues was also seen when GspB-F was expressed in E. coli together with GtfA/B, Nss, and Gly ( Figure S2B). Although identification of modified GspB-F peptides by mass spectrometry was challenging, we identified with confidence a SRR1 peptide that contained a Ser modified by one GlcNAc and two hexoses ( Figure S2C). Taken together, these results are consistent with the idea that Nss and Gly add glucose residues to GlcNAc attached by GtfA/B to Ser/Thr of GspB-F.
Nss consists of a single domain that has a typical GT-B glycosyltransferase fold (20,21). Gly consists of three domains ( Figure 1F; left panel). The first two domains are predicted to have GT-A and GT-B glycosyltransferase folds, respectively, and the third domain has a recently identified GT-D fold (30). Of note, the isolated GT-D domain from a Gly homolog of S. parasanguinis has enzymatic activity for its substrate (30). We found that the isolated GT-D of S. gordonii Gly was capable of adding Glc residues to GspB-F pre-modified with GtfA/B and Nss ( Figure 1F; right panel, lane 6), whereas the isolated N-terminal fragment containing the GT-A and GT-B folds was inactive (lane 5).
Thus, despite the fact that the N-terminal domains are sequence-related to glycosyltransferases, they seem to lack enzymatic activity.

Substrate binds to Gly and is released by the Asp complex
To our surprise, we noticed that fully modified GspB-F remained associated with Gly, the last glycosyltransferase; essentially all G3 species could be recovered with a fusion of Gly with glutathione S-transferase (Gly-GST), followed by binding to a glutathione resin ( Figure 2A Figure 2C; lane 1). This experiment also shows that the binding of Gly to its enzymatic product is separable from the modification reaction per se. In our system, product binding by the N-terminal GT-A/B domains does not interfere with the enzymatic activity of the C-terminal GT-D domain, because Gly is in large excess over substrate and the N-terminal domains binds reversibly to the product.
Next, we tested the role of the Asps. Neither of the three Asps had an effect on the glycosylation reactions catalyzed by GtfA/B, Nss, or Gly ( Figure S3). However, the complex of the three Asps released the fully glycosylated G3 species from Gly-GST ( Figure 2D; lane 4). Asp1 alone or a complex of Asp1 and Asp3 (Asp1/3) was inactive in the release reaction (lanes 2, 3). These results suggest that the complex of all three Asp proteins may be involved in the transfer of glycosylated substrate from the last glycosyltransferase to the next step in the export pathway.

Structures of the Asps
Since our data suggest that the Asp complex can accept fully glycosylated substrate from Gly, we suspected that it can interact with carbohydrates. To test this possibility, we determined the crystal structures of Asp1 alone (resolution of 2.77Å) and of an Asp1/3 complex (resolution of 3.11Å) ( Table S1). The structure of Asp1 is similar to that of GtfA and GtfB ( Figure 3A,B,F). Like GtfA or GtfB, Asp1 has two Rossmann-like folds (R-folds I and II), which are typical for the GT-B family of glycosyltransferases ( Figure   3A). In addition, it has the typical extended β-sheet domain (EBD). Together, these domains form a U-shaped structure. As in the enzymatically inactive GtfB protein, the cleft between R-folds I and II is negatively charged ( Figure 3B). In contrast, GtfA and other enzymatically active GT-B family members have a positively charged cleft that is required to bind UDP-sugars ( Figure 3B). Like GtfB, Asp1 lacks two positively charged residues in the active site and has a Gln residue at position 438 in place of an essential Glu residue ( Figure 3B, lower panels). The structure thus supports the idea that Asp1, like GtfB, is a carbohydrate binding protein, rather than an active glycosyltransferase.
Consistent with the postulated substrate binding site, when two conserved Asp residues in the cleft between the R-folds were mutated to Arg, secretion of GspB-F from S. gordonii cells was abolished ( Figures 4A, B; sequence alignment shown in Figure S4).
Asp1 forms a stable 1:1 complex with Asp3 ( Figure S5A). Asp3 could not be stably isolated on its own, suggesting that it has Asp1 as an obligatory partner. Consistent with this observation, deletion of the Asp1 homolog Gap1 in S. parasanguinis results in the degradation of the Asp3 homolog Gap3 (28). The Asp1/3 structure shows that Asp3 consists of two anti-parallel β-sheets (β-sandwich) ( Figure 3C). Asp3 uses two different regions to bind to Asp1 (interfaces I and II). Interface I binds to the EBD of Asp1, and interface II to both the EBD and the cleft between the R-folds ( Figure 3C). Asp3 is structurally related to carbohydrate binding modules (CBM) in glycosidases (31,32) ( Figure 3D). Interestingly, different CBMs bind their sugar ligands with different surfaces, some with the concave surface of the β-sandwich and others with the tips of the β-strands (33, 34) ( Figure 3D). In the case of Asp3, the latter binding site seems to be more important, as mutations of conserved residues in this area had a drastic effect Asp1, 2, and 3 co-migrated in gel filtration ( Figure S5B), but light scattering experiments indicated that there was a mixture of monomeric and dimeric complexes that contain one copy each of Asp1, 2, and 3 ( Figure S5C). This heterogeneity is likely the explanation for why these complexes did not crystallize. We therefore attempted to obtain structural information by other means. Addition of trypsin to the Asp1/3 complex generated one Asp1 and one Asp3 peptide, which were not observed with the Asp1/2/3 complex ( Figure 5A; indicated by stars). The cleavage sites protected by Asp2 are R430 of Asp1 and R23 of Asp3 ( Figure 5B; R23 is in an unstructured region, so the figure shows flanking residues). Thus, Asp2 seems to bind to both Asp1 and Asp3 at the open end of the U-shaped Asp1/3 complex. Next, we used negative-stain electron microscopy (EM) to analyze the Asp1/3 and Asp1/2/3 complexes ( Figure 5C, Figure   S6). To better locate the individual proteins in the images, we fused the maltose-binding protein (MBP) to Asp1, Asp2, or both. Complexes containing the MBP fusions were monomeric, indicating that the MBP domain interfered with dimerization ( Figure S5C).
The results confirm that Asp2 sits at the open end of the Asp1/3 complex ( Figure 5C, Figure S6). Negative-stain EM also confirmed that without MBP, the Asp1/2/3 complex consisted of a mixture of monomers and dimers. In the dimer, the Asp1/2/3 monomers associate in an anti-parallel fashion ( Figure 5C, fourth panel from the left). It is unclear which form of the Asp complex is physiologically relevant.

Substrate targeting to membranes by Asp1/3
After being released from Gly by the Asp complex, the glycosylated substrate needs to be targeted to the membrane, a process that might be mediated by the Asps. We therefore tested whether the Asps have an affinity for membranes. To this end, purified Asps were incubated with liposomes of different phospholipid composition. The samples were then subjected to flotation in a Nycodenz gradient and fractions were analyzed by SDS-PAGE and Coomassie staining ( Figure 6A). With liposomes containing a high percentage of negatively charged lipids (dioleoylphosphatidylglycerol; DOPG), Asp1 alone or Asp1/3 floated to the second fraction from the top ( Figure 6B), which is also the peak position of the lipids ( Figure S7). The Asp1/2/3 complex also bound to the liposomes, but it peaked at fraction 3, suggesting that Asp2 weakens the interaction with the liposomes. Indeed, when the percentage of negatively charged lipids was decreased, the binding of Asp1/2/3 was selectively reduced (Figures 6C, D). These results indicate that Asp1 and Asp1/3 have an affinity for negatively charged lipids.
Given that Asp1 and Asp3 are always in a complex, Asp1 is likely responsible for membrane targeting of both proteins. Asp2 inhibits membrane interaction, suggesting that lipid head groups and Asp2 may compete for interaction with the Asp1/3 complex.
No interaction of Asp1 and Asp1/3 with polar lipids from E. coli was observed ( Figure   6E), consistent with the fact that E. coli contains a much lower percentage of negatively charged lipids than do streptococci (35)(36)(37).
To test whether a glycosylated substrate can be targeted to the membrane by the Asps, we incubated glycosylated GspB-F with either Asp1, Asp1/3, or Asp1/2/3, followed by

DISCUSSION
Our results suggest a model for the first steps in the export of an SRR adhesin from the pathogenic bacterium S. gordonii. The adhesin (GspB) is first made as an unmodified protein. It is then sequentially glycosylated by three glycosyltransferases (see model in Figure 8, box 1). The first enzyme, GftA/B adds GlcNAc residues to Ser/Thr residues in SRR domains (G1 species). Next, Nss adds Glc to the GlcNAc residues (G2 species), and finally, Gly adds further Glc residues to those attached by Nss (G3 species). The fully glycosylated substrate is then transferred to the Asp1/2/3 complex (box 2). In the next step, the Asp1 protein would mediate the interaction of the Asp1/2/3 complex with the lipid bilayer (box 2). The Asp1/2/3 complex has a relatively low affinity for the membrane, so we assume that it continuously cycles between the cytosol and membrane, with the majority staying in the cytosol. Membrane binding of the Asp complex probably requires a conformational change to expose a lipid interaction domain on Asp1, an interface that seems to be fully available for membrane interaction in Asp1 or the Asp1/3 complex. Once at the membrane, the substrate would be delivered to SecA2 and SecY2 for translocation across the membrane (box 3). This is consistent with previous findings that the Asp1/2/3 localizes near SecA2 at the membrane (38).
Our data show that the glycosyltransferases act in a strictly sequential manner. GtfA/B only modifies Ser/Thr residues and has specificity for GlcNAc, while Nss and Gly recognize GlcNAc and Glc residues, respectively. Gly is an unusual enzyme, as it has affinity for the product of the reaction it catalyzes. This is explained by the fact that the presence of carbohydrate-binding motifs in Asp1 and Asp3 strongly supports the idea that they bind glycosylated adhesin. Indeed, our mutagenesis data provide evidence for substrate interaction with Asp3. Asp1 also seems to bind substrate, as it allows coflotation with liposomes. The interactions of Asp1 and Asp3 with substrate are weak, as they do not survive in pull-down or gel filtration experiments (data not shown). However, substrate release from Gly (Fig. 2) and our co-flotation experiments (Fig. 7) indicate that the Asps do interact with substrate. The low binding affinity is in fact typical for carbohydrate-binding proteins (39). Our results do not exclude the possibility that, under certain conditions, the Asps can also interact with non-glycosylated adhesin. In fact, Asp1 has a similar structure as GtfB ( Figure 3B), which can bind non-glycosylated substrate (10), and Asp2 and Asp3 have been shown to bind non-glycosylated GspB (40). Such an interaction would explain why GspB is secreted in an Asp-dependent manner in glycosylation-defective S. gordonii strains, although in this situation, much of the substrate is degraded (22). It should also be noted that some bacterial species export SRR adhesins in an Asp-dependent manner although they lack Gly and Nss (5).
Asp1 also has an affinity for the lipid bilayer, which facilitates the recruitment of substrate to the membrane. Given that binding of Asp1 requires negatively charged phospholipids and is enhanced at higher salt concentrations (compare Figure 6B  GspB accumulates in the cytosol as soluble protein (15).
How the substrate is delivered to SecA2 and SecY2 remains to be clarified. The signal sequence of adhesin and the adjacent "accessory Sec transport" (AST) domain are required to target the precursor to the accessory Sec system and initiate translocation (41). Although not very hydrophobic (5), the signal sequence could still facilitate the interaction with the lipid bilayer. Once the substrate is bound to the membrane, it could associate with SecA2, a transfer that may be facilitated by an interaction between the Asp complex and SecA2 (22,38). Based on a homology model, SecA2 has a pronounced positively charged surface patch ( Figure S8A, B), which could mediate its interaction with negatively charged phospholipids in the membrane. No such basic surface patch is seen in a homology model for S. gordonii SecA1 ( Figure S8C). Since we were unsuccessful in purifying soluble SecA2, even in the presence of detergent, we speculate that SecA2 requires a lipid environment to maintain its native conformation and that it is permanently bound to the membrane, in contrast to SecA1, which cycles between the cytosol and membrane (7). According to the model, SecA2 would rely on the Asps to deliver substrate to the membrane where SecA2 could engage both the AST domain and the remainder of the mature domain, whereas in the canonical secretion system, SecA1 would do the job, with the chaperone SecB acting upstream for some substrates and bacteria (7). Once substrate has been recruited to the SecA2/SecY2 complex, it is likely translocated across the membrane by a mechanism similar to that of the canonical system.

EXPERIMENTAL PROCEDURES
Details are given in the Supporting Information.

Purification of proteins
All proteins were expressed in E. coli. GtfA/B complex was prepared as previously described (10). S. gordonii Nss, Gly, Gly N-domain (residue 1-411), and Gly C-domain (residue 412 -682) were purified utilizing a His-tag. Nss and Gly were also expressed with a C-terminal glutathione S-transferase (GST) tag. Asp1 was expressed with a Cterminal GST tag, either alone or together with Asp3. Purification was performed with glutathione sepharose 4B beads, the GST portion was cleaved off, and the proteins were further purified by ion exchange and gel filtration chromatography. Mutations were introduced into Asp1 or Asp3 by QuikChange mutagenesis. For electron microscopy, untagged Asp1 and Asp3 were co-expressed with Asp2 fused its N-terminus with either maltose binding protein or GST. After purification on either amylose or glutathione resin, the proteins were further purified by ion exchange chromatography and gel filtration. To generate the Asp1/2/3 complex without a tag, the GST tag was cleaved from Asp2 by thrombin protease, and subsequently removed by gel filtration.

Pull-down experiments
In vitro-synthesized substrate binding of Nss and Gly was tested with GST-tagged enzymes and magnetic glutathione resin. Where indicated, Asp proteins, Gly, or Gly-C domain were added prior to the resin. Bound and unbound fractions were analyzed by SDS-PAGE and autoradiography.

Crystallization and structure determination
Crystallization of Asp1 and Asp1/3 complex was performed by the hanging-drop vapordiffusion method at 22 °C. Both native and selenium SAD data sets were collected at beamline 24ID-C at the Argonne National Laboratory and processed with XDS (42) and autoPROC (43). The positions of Se atoms were determined and phases were calculated using AutoSol Wizard in PHENIX (44). A complete model of Asp1/3 was built in Coot (45). The structure was refined with Phenix.refine (46). The structure of Asp1 was determined by molecular replacement using PHASER in PHENIX (44), with the Asp1 lacking the R-fold II as the initial search model. The model was modified in Coot (45) and refined with Phenix.refine (46).
GspB-F secretion was determined by immunoblotting with anti-FLAG monoclonal antibodies (Sigma) as described before (29).
Secreted GspB-F was purified from a S. gordonii carrying signal sequence-containing GspB-F in place of wild type GspB. GspB-F was enriched from the medium by ammonium sulfate precipitation. After dialysis, glycosylated the protein was purified with a resin containing succinylated wheat germ agglutinin (sWGA), followed by gel filtration.

Negative stain electron microscopy
Negatively stained specimens were prepared as descibed (49). Grids were imaged on a Tecnai T12 electron microscope (FEI) operated at 120 kV at a nominal magnification of 67,000x using a 4k x 4k CCD camera (UltraScan 4000, Gatan), corresponding to a calibrated pixel size of 1.68 Å on the specimen level. The images were processed as described (50).

Limited trypsin proteolysis
Asp1/3 or Asp1/2/3 (5.6 μg) were incubated with 6.6, 2. Glycosylated GspB-F was isolated from S. gordonii cells as described before (10) and incubated with Asps prior to the addition of liposomes. The Nycodenz gradient was prepared in 20 mM Tris/HCl, pH 7.5, 300 mM NaCl. After centrifugation, fractions were subjected to SDS-PAGE and immunoblotting using Flag antibodies (Sigma).        glycosyltransferases. The first enzyme is GtfA/B, a tetramer that adds GlcNAc residues to Ser/Thr residues (G1 species). Then, Nss adds Glc to GlcNAc (G2 species), and finally Gly adds Glc to Glc residues (G3 species). Gly remains bound to the G3 species, until it is transferred to the Asp1/2/3 complex. Box 2: Although the Asp complex has a low affinity for the lipid bilayer, it probably continuously cycles between the cytosol and membrane. The Asp complex likely undergoes a conformational change, in which Asp2 moves away from a lipid binding surface, allowing the Asp complex to deliver the substrate GspB to the membrane. Box 3: GspB engages SecA2 and the SecY2 channel for its translocation across the membrane and the Asp complex returns to the cytosol.