# Characterization of a glycan-binding complex of minor pilins completes the analysis of Streptococcus sanguinis type 4 pili subunits

Meriam Shahin, Devon Sheppard, Claire Raynaud, Jamie-Lee Berry, Ishwori Gurung, Lisete Silva, Ten Feizi, Yan Liu, Vladimir Pelicic

► To cite this version:

Meriam Shahin, Devon Sheppard, Claire Raynaud, Jamie-Lee Berry, Ishwori Gurung, et al.. Characterization of a glycan-binding complex of minor pilins completes the analysis of Streptococcus sanguinis type 4 pili subunits. Proceedings of the National Academy of Sciences of the United States of America, 2023, 120 (3), pp.e2216237120. 10.1073/pnas.2216237120. hal-04009811

HAL Id: hal-04009811

https://hal.science/hal-04009811

Submitted on 1 Mar 2023

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
1 Characterisation of a glycan-binding complex of minor pilins completes the
2 analysis of *Streptococcus sanguinis* type 4 pili subunits

3
4 Meriam Shahin<sup>a,1</sup>, Devon Sheppard<sup>a,1</sup>, Claire Raynaud<sup>a</sup>, Jamie-Lee Berry<sup>a</sup>, Ishwori Gurung<sup>a</sup>, Lisete M. Silva<sup>b</sup>, Ten Feizi<sup>b</sup>, Yan Liu<sup>b</sup>, Vladimir Pelicic<sup>a,c,*</sup>

6
7 <sup>a</sup>MRC Centre for Molecular Bacteriology and Infection, Imperial College London,
London, United Kingdom

9
10 <sup>b</sup>Glycosciences Laboratory, Department of Metabolism, Digestion and Reproduction,
Imperial College London, London, United Kingdom

12
13 <sup>c</sup>Laboratoire de Chimie Bactérienne, UMR 7283 CNRS/Aix-Marseille Université,
14 Institut de Microbiologie de la Méditerranée, Marseille, France

15
16 <sup>1</sup>These authors contributed equally

17
18 *Corresponding author
19 email: vladimir.pelicic@inserm.fr

20
21 **Author Contributions:** Designed research (YL, VP), Performed research (MS, DS, CR, JLB, IG, LMS, YL), Contributed new reagents/analytic tools (TF), Analyzed data (MS, DS, JLB, YL, VP), Wrote the paper (MS, DS, LMS, YL, VP)

24
25 **Competing Interest Statement:** There is no competing interests

26
27 **Classification:** Biological Sciences. Microbiology

28
29 **Keywords:** type 4 pili, pilin, adhesion
30 Abstract

31 Type 4 filaments (T4F) – of which type 4 pili (T4P) are the archetype – are a
32 superfamily of nanomachines nearly ubiquitous in prokaryotes. T4F are polymers of
33 one major pilin, which also contain minor pilins whose roles are often poorly
34 understood. Here, we complete the structure/function analysis of the full set of T4P
35 pilins in the opportunistic bacterial pathogen *Streptococcus sanguinis*. We
36 determined the structure of the minor pilin PilA, which is unexpectedly similar to one
37 of the subunits of a tip-located complex of four minor pilins, widely conserved in T4F.

38 We found that PilA interacts and dramatically stabilises the minor pilin PilC. We
39 determined the structure of PilC, showing that it is a modular pilin with a lectin
40 module binding a subset of glycans prevalent in the human glycome, the host of S.
41 sanguinis. Altogether, our findings support a model whereby the minor pilins in S.
42 sanguinis T4P form a tip-located complex promoting adhesion to various host
43 receptors. This has general implications for T4F.
44 Significance Statement

Type 4 pili (T4P) – important in bacterial pathogens – are filaments composed of one
well-characterised major pilin, and several minor pilins whose roles are often poorly
understood. S. *sanguinis* T4P are composed of five pilins, which makes it a good
model to determine the role of each pilin subunit in detail. Here, we characterise PiA
and PiC, showing how they interact and how they function. Together with our
previous findings, this provides an integrated view of the role of the five pilin subunits
in S. *sanguinis* T4P. PiE1/PiE2 are the major pilins forming the backbone of the
filament, while the three minor pilins (PiA, PiB, PiC) form a tip-located complex
promoting adhesion to various host receptors.
## 54 Introduction

Type 4 filaments (T4F) – type 4 pili (T4P) and type 2 secretion systems (T2SS) being the best known – are a superfamily of nanomachines ubiquitous in prokaryotes (1, 2).

T4F, which mediate a wide variety of functions (1), are composed of type 4 pilins, assembled by conserved multi-protein machineries spanning the cell envelope (3).

T4F have been studied for decades because they are virulence factors in many human pathogens.

Our current understanding of T4F is as follows. T4F are polymers of type 4 pilins: one predominant (major) and several low abundance (minor) subunits (4).

These subunits are synthesised as prepilins with a distinctive N-terminal sequence motif – the class 3 signal peptide (SP) (5) – consisting of a short hydrophilic leader peptide ending with a Gly or Ala, followed by a hydrophobic tract of 21 residues, which anchors prepilins in the cytoplasmic membrane (CM). Pilins display a characteristic "lollipop" structure where the hydrophobic tract of the SP constitutes the N-terminal portion (α1N) of an α-helix of approx. 50 residues (α1) that supports a globular head (4). However, larger minor pilins – displaying extra domains "grafted" onto a pilin moiety and hence defined as modular (6) – are not uncommon, which partly explains the extreme functional versatility of T4F. Pilins are assembled in filaments at the CM (3) in two steps. The leader peptide in prepilins is first cleaved by a dedicated prepilin peptidase (PPase) (7-9), before the pilins are polymerised by a machinery centred on a platform protein and a hexameric ATPase (10). All T4F are helical polymers where pilins are held together by extensive interactions between their α1-helices, which pack within the core of the filament, with often a portion of α1N "melted" (11). However, different helical symmetry parameters are seen in different T4F (12-16).

While the role of major pilins is clear, i.e., they constitute the backbone of T4F, the role and precise localisation of the minor pilins remain poorly defined (4). Typically, minor pilins are divided into two categories: widely conserved and non-
conserved (or system-specific) (4). Non-conserved minor pilins are highly variable, and their roles cannot be predicted. In some T4F, these pilins have been well studied, such as for example ComP, PilV, PilX in *Neisseria meningitidis* T4P. These three pilins are dispensable for filament assembly but are key for different T4P-mediated functions. ComP binds DNA to promote its uptake during natural transformation (17, 18), PilV promotes bacterial adhesion to human cells (19), and PilX supports the formation of bacterial aggregates (20). In contrast, a group of four minor pilins – often labelled with the letters H, I, J, K – are widely conserved in different T4F and essential for filament assembly (21-23). Their role and mechanism of action are likely to be the same since HIJK sets from different T4F are functionally interchangeable in promoting filament assembly (24). These pilins interact to form a quasihelical complex (25, 26), which is capped by the bulky K subunit. This suggested a localisation at the pilus tip (25, 26), which was recently confirmed by cryo-electron tomography (cryo-ET) of *Myxococcus xanthus* T4P (27). Since T4F are assembled from tip to base (3), the HIJK complex is therefore thought to initiate filament assembly (28, 29).

Recently, the study of T4F in monoderm bacteria opened new research avenues (30), with *Streptococcus sanguinis* becoming a cutting edge model (31). This commensal of the human oral cavity (32) is an opportunistic pathogen frequently causing infective endocarditis (IE) (33). It uses an elementary machinery to assemble T4P composed of two major (PilE1, PilE2) and three minor (PilA, PilB, PilC) pilins (34, 35). This makes it possible to characterise the role of all pilin subunits in *S. sanguinis* T4P in detail. Since we previously characterised PilE1, PilE2 and PilB (6, 34, 35), here we focused on PilA and PilC. We report the detailed structure/function analysis of these two minor pilins, showing how they interact and how they function. This provides an integrated view of the role of all the pilin subunits in a defined T4F, which has implications for this widespread superfamily of nanomachines.
109 **Results**

110

111 **PilA and PilC minor pilins have remarkably different characteristics**

112 Previously, we showed that all the genes involved in T4P biology in *S. sanguinis*
113 2908 strain cluster together in a 22 kb locus named *pil* (34). This locus encodes five
114 pilins – *PilE1*, *PilE2*, *PilA*, *PilB*, *PilC* (Fig. 1A) – which are essential for piliation (34).
115 These proteins are processed by the PPase *PilD* and become subunits of T4P (34,
116 35). *PilE1* and *PilE2* are the major pilins, whereas *PilA*, *PilB* and *PilC* are minor pilins
117 (35). We previously characterised *PilE1*, *PilE2* and *PilB* (6, 35). Here, we complete
118 the analysis of the role of pilins in *S. sanguinis* T4P by focusing on *PilA* and *PilC*.

119 Although it has a typical pilin size – 17 kDa for the processed protein – PilA is
120 an unusual pilin. Its class 3 SP, which was identified by visual inspection (34), is
121 degenerate (Fig. 1B) and could not be identified by any of the available bioinformatic
122 tools, including PilFind dedicated to this purpose (36). This is mainly due to the
123 absence of the typical Glu residue in the fifth position of the processed protein (Glu5)
124 (23) and the presence of an uncommon Pro4 (Fig. 1B). PilA has no sequence
125 homologs outside of S. sanguinis, which makes it impossible to predict its role.

126 Compared to PilA, PilC has radically different features. It has a canonical class
127 3 SP (Fig. 1C), with a typical Glu5 in the processed protein. Processed PilC is
128 unusually large for a pilin, with a theoretical molecular mass of 52.8 kDa. As recently
129 reported for PilB (6), the large size of PilC is due to its predicted modular
130 architecture, with a domain grafted onto what appears to be an unusually short pilin
131 moiety (Fig. 1C). In PilC, this extra module, readily identifiable by sequence-based
132 bioinformatics, belongs to the concanavalin A-like lectin/glucanase domain
133 superfamily (SSF49899), which is composed of carbohydrate-binding proteins in the
134 three domains of life with different ligand specificities. These observations raise the
135 possibility that PilC is a modular pilin with a lectin module promoting T4P-mediated
136 adhesion of S. sanguinis to glycans.
137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

161

162

163

PilA is structurally similar to a widely conserved subunit from a complex of minor pilins found at the tip of many T4F

To better understand the role of PilA, we solved its 3D structure by X-ray crystallography. We produced a 15.7 kDa recombinant protein in *Escherichia coli*, in which the N-terminal 24 residues of the mature pilin (Fig. 1B) were replaced by a hexahistidine tag (6His). This usually promotes pilin solubility without structurally impacting the rest of the protein (37). We purified soluble 6His-PilA using a combination of affinity and size-exclusion chromatography (SEC), noticing that it purifies as a dimer. The protein crystallised readily. After optimising the best diffracting crystals and using protein produced in the presence of seleno-methionine (SeMet) for phase determination, we solved a 1.77 Å resolution structure (Table S1).

As can be seen in Fig. 2A, PilA – which looks like an "ice axe" – exhibits a typical pilin architecture, with an N-terminal α1-helix packed against a β-sheet composed of five anti-parallel β-strands. Modelling the full-length PilA protein using AlphaFold (38) reveals a canonical lollipop shape (4) (Fig. S1). The almost perfect superposition of our structure with the AlphaFold prediction (Fig. S1) – with a mere 0.58 Å root-mean-square deviation (RMSD) – validates the accuracy of both AlphaFold (38) and our structure.

A closer inspection of the crystal packing showed that PilA forms head-to-toe dimers (Fig. 2B), stabilised by a series of hydrogen bonds between residues in α1-helix and the last two β-strands of the β-sheet (β4 and β5) (Fig. S2). This explains why 6His-PilA purifies as a dimer. Strikingly, while PilA exhibits no sequence homology to other pilins, our structure reveals extensive structural similarity to the I subunit of HIJK tip-located complex of conserved minor pilins (25, 26) in a variety of T4F. For example, PilA superposes with an RMSD of 1.52 Å with Gspl (PDB 3CI0) from enterotoxigenic *E. coli* T2SS (Fig. 2C). Critically, since I interacts with J and K
164 (25, 26), this suggested to us that PilA might be interacting with another minor pilin
165 (PilB and/or PilC) to form a complex capping S. sanguinis T4P.

166

167 PilA interacts with PilC

168 To identify interactions between PilA and other pilins in S. sanguinis T4P, we
169 performed pull-down assays with proteins purified using two different affinity tags:
170 6His and Strep-tag II. In brief, (i) purified proteins corresponding to the soluble
171 portion of the pilins were mixed in solution, (ii) the 6His-tagged bait was selectively
172 captured using cobalt-based magnetic beads, and (iii) pull-down of the Strep-tagged
173 prey was assessed by immunoblotting using specific anti-Pil antibodies. We verified
174 beforehand that Strep-tagged proteins were not captured using these beads
175 (although PilB exhibited very limited binding). Interestingly, while the bait proteins
176 were always captured, prey proteins were pulled down only for the PilA/PilC pair (Fig.
177 3A), both by 6His-PilA and 6His-PilC.

178 To characterise the PilAC complex, we used SEC coupled with multi-angle light
179 scattering (SEC-MALS), which allows for absolute characterisation of molecular
180 mass and stoichiometry of proteins in solution. SEC-MALS was first used with 6His-
181 PilA and 6His-PilC separately. Both proteins eluted as single, monodisperse peaks
182 with estimated molecular masses (in kDa) of 31.7 ± 0.35, and 57.7 ± 0.63,
183 respectively (Fig. 3B). For 6His-PilA, this value is significantly higher than the
184 theoretical molecular mass (15.7 kDa), confirming that the purified protein is a dimer
185 in solution. When SEC-MALS was performed after mixing equimolar amounts of
186 purified 6His-PilA and 6His-PilC, we observed a single, monodisperse peak of 70.8 ±
187 0.07 kDa (Fig. 3B), indicating that the PilAC complex has a 1:1 stoichiometry. The
188 disappearance of the PilA-PilA homodimer shows that PilC is the preferred binding
189 partner of PilA. Using isothermal titration calorimetry (ITC), we showed that PilA has
190 high affinity for PilC (Fig. 3C), with a dissociation constant (K_D) of 23.8 ± 7.9 nM.
We then assessed how PilA and PilC interact. We first performed pull-down assays using a purified 6His-PilC<sub>Δpilin</sub> protein lacking the pilin moiety (Fig. 1C). In contrast to 6His-PilC (Fig. 3B), 6His-PilC<sub>Δpilin</sub> was unable to pull down its Strep-PilA partner (Fig. 4A). This suggests that PilA interacts with the pilin moiety of PilC. Next, we used multidimensional nuclear magnetic resonance (NMR) to identify the portion of PilA binding to PilC. First, we performed a partial NMR assignment of the resonances in 6His-PilA (Fig. S3), highlighting the assigned residues on our crystal structure (Fig. 4B). Then, we identified the chemical shift perturbations occurring in PilA when PilC was added (Fig. S3). Nine chemical shifts perturbations concerned PilA residues not involved in the formation of PilA dimers, which are therefore likely to be involved in the PilA-PilC interaction (Fig. 4B). The PilAC interaction interface involves the α1 and α2 helices and the first 2 β-strands (β1 and β2) of PilA (Fig. 4B). Curiously, although this interface is distinct from the one involved in the formation of PilA homodimers (Fig. S2), the formation of a PilAC complex led to the disappearance of the PilA dimer. We probed this interaction interface by mutagenesis of Ala<sub>68</sub>, Lys<sub>85</sub> and Thr<sub>87</sub> in PilA, and quantification of the ability of these mutant proteins to interact with PilC using ITC (Fig. 4C). This showed that PilA<sub>T87A</sub> displayed a significantly reduced affinity for PilC, corresponding to 38.3 ± 5.9% of the wild-type (WT) binding.

Taken together, these experiments show that PilA and PilC interact, confirming our hypothesis that PilA is part of a complex of minor pilins in *S. sanguinis* T4P.

212

## PilA stabilises PilC

We noticed that purified 6His-PilC was unstable, which prompted us to perform long-term stability tests. We kept the purified protein at 4°C, took samples once a week, which we analysed by SDS-PAGE/Coomassie. This revealed that 6His-PilC was completely degraded by week two (Fig. 5A), yielding two degradation products. Edman sequencing showed that proteolysis occurred after Ala<sub>101</sub> close to the end of

191

192

193

194

195

196

197

198

199

200

201

202

203

204

205

206

207

208

209

210

211

212

213

214

215

216

217

218
219 the pilin moiety (Fig. 1C), hence the two degradation products were named PilC<sub>Nter</sub>
220 (smaller N-terminal product) and PilC<sub>Cter</sub> (larger C-terminal product). PilC<sub>Nter</sub> is highly
221 unstable as it was no longer detectable by week two. In the same conditions, 6His-
222 PilA remained unchanged over four weeks, confirming that it is a very stable protein
223 (Fig. 5A). Interestingly, when the PilAC complex was analysed in the same way, PilC
224 degradation was significantly delayed. In the presence of PilA, PilC degradation
225 began only at week three, and even at week four the majority of PilC was still intact
226 (Fig. 5A).

227 The stabilising effect of PilA on PilC was further characterised by performing
228 trypsin sensitivity assays. In brief, we incubated proteins with trypsin on ice for 60
229 min, took samples at specific timepoints, which we analysed by SDS-
230 PAGE/Coomassie (Fig. 5B). As above, 6His-PilA was intact over the course of the
231 assay, while 6His-PilC the protein was entirely converted into PilC<sub>Nter</sub> and PilC<sub>Cter</sub> by
232 20 min. As above, the pilin moiety PilC<sub>Nter</sub> was unstable and no longer detectable
233 after 40 min. In contrast, when it was part of a complex with 6His-PilA, 6His-PilC
234 degradation was delayed (Fig. 5B). In addition, PilC<sub>Nter</sub> was also stabilised as it was
235 still detectable after 60 min.

236 Taken together, these findings show that the functional consequence of the
237 PilAC interaction is that PilA stabilises PilC, especially its pilin moiety.

238
239
**Crystal structures of PilC reveal a modular pilin**

240 To confirm the prediction that PilC might be a modular pilin, we solved its 3D
241 structure by X-ray crystallography. Due to the instability of its pilin moiety, we
242 produced a 41.4 kDa recombinant 6His-PilC<sub>Δpilin</sub> protein in <i>E. coli</i> lacking the pilin
243 moiety (Fig. 1C). This protein crystallised readily. After optimising the best diffracting
244 crystals and using SeMet protein for phase determination, we solved a high-
245 resolution structure at 1.45 Å (Table S1). Strikingly, while bioinformatics only
246 predicted that the C-terminus of PilC corresponds to a lectin domain, our structure
247 reveals two distinct modules (Fig. 6A). The first module adopts an Ig-like fold (PilC<sub>lg</sub>),
248 consisting of two β-sheets packed against each other (Fig. 6B). This is a widespread
249 domain found in proteins of different functions (39), which is thought to be involved in
250 protein-protein interactions. PilC<sub>lg</sub> thus shows significant structural similarity to
251 multiple structures, including the N-acetylglucosamine-binding protein GbpA from
252 Vibrio cholerae (40) (PDB 2XWX). The Ig-like domains in these two proteins, which
253 display no sequence homology, superpose with an RMSD of 2.17 Å (Fig. 6B).

254 The second module that corresponds to the lectin (PilC<sub>lectin</sub>), is a β-sandwich
255 characterised by two opposing antiparallel β-sheets of six strands (Fig. 6C), with
256 concave and convex sides. PilC<sub>lectin</sub> shows significant structural similarity to multiple
257 carbohydrate-binding proteins, including galectins such as human Gal-7 (PDB 2GAL)
258 PilC<sub>lectin</sub> and Gal-7, which display no sequence homology, superpose with an
259 RMSD of 1.95 Å (Fig. 6C). In the Gal-7 structure (Fig. 6C), the protein interacts with
260 galactose via a carbohydrate-recognition domain (CRD) forming a patch on the
261 concave side of the protein (41). Since the two proteins share good structural
262 homology in that region (Fig. 6C), we speculate that the CRD might be on the
263 concave side of PilC<sub>lectin</sub>.

264 To try to determine a PilC structure encompassing the pilin moiety, we
265 expressed and crystallised PilC from another S. sanguinis strain SK36 (PilC<sup>SK36</sup>), in
266 which the N-terminal 24 residues of the processed protein were replaced by 6His.
267 After collecting a complete dataset, we solved a 1.60 Å resolution structure (Table
268 S1). Surprisingly, although the purified protein contained the pilin moiety, this portion
269 was missing in the crystals. It must therefore have been degraded during prolonged
270 incubation in the crystallisation trays due to its inherent instability. When we
271 compared our two PilC structures, we found that they are essentially identical,
272 superposing onto each other with an RMSD of only 0.79 Å (Fig. 6D). This suggests
273 that these proteins – showing only 57 % sequence identity (Fig. S4) – are likely to
274 bind similar glycan ligands. Finally, we used AlphaFold to model the full-length PilC,
275 revealing a lollipop with a bulky domain grafted (Fig. S5A), which superposes nicely
276 onto our crystal structure (1.03 Å RMSD). Interestingly, the globular head of PilC is
277 strikingly different from known pilins. Instead of the usual β-sheet with several anti-
278 parallel β-strands, PilC displays a highly distorted β-sheet composed of three β-
279 strands, where β1 and β3 are orthogonal to each other, and linked by β2 with a 90°
280 kink (Fig. S5B).
281

282

PilC binds two types of glycans prevalent in the human glycome

283 The presence of a lectin domain suggests that PilC might be an adhesin recognising
284 host glycans. We therefore investigated the glycan binding ability and specificity of
285 PilC using a glycan microarray designed using the neoglycolipid (NGL) technology
286 (42). 6His-PilC bound to a range of glycan probes – results are shown for 672
287 sequence-defined oligosaccharide probes (Dataset S1) – corroborating its lectin
288 activity (Fig. 7A). Two major types of negatively charged glycans were bound:
289 sialylated glycans and sulphated glycosaminoglycans (GAG). Most of the sialylated
290 ligands display a α2-3-sialyl linkage and terminate with 3'-sialyllactose (3'-SL) or 3'-
291 sialyl-N-acetyllactosamine (3'-SLN). Also well recognised (Fig. 7A) were ganglioside
292 GD1a and four probes terminating with the Sdª antigen, which is known to be present
293 in human secretions including saliva (43, 44). A few probes displaying other sialyl
294 linkages – including α2-9-polysialic acid – were also bound (Fig. 7A). The sulphated
295 GAG ligands are mainly oligosaccharide probes derived from heparin, representative
296 of the highly sulphated domains of heparan sulphate, which is ubiquitously expressed
297 on the cell surface and in the extracellular matrix. Both sialylated glycans and GAGs
298 are prevalent in the human glycome (45). We observed a similar binding profile for
299 PilC<sup>SK36</sup> (Fig. S6), showing that glycan-binding ability is conserved in PilC orthologs.
300

301 We tested whether the lectin activity of PilC is affected by its pilin moiety, by
302 probing the glycan microarray with 6His-PilC<sub>Δpilin</sub>. PilC<sub>Δpilin</sub> bound the same type of
303 glycans as PilC, suggesting that glycan binding is largely due to the lectin module.
However, the pilin moiety affects the binding (Fig. S7). When compared to PilC, PilC<sub>Δpilin</sub> binds strongly to heparin oligosaccharide probes and weakly to a more restricted range of α2-3-linked sialyl glycans (Fig. S7). Next, using ITC, we quantified the affinity of PilC<sub>Δpilin</sub> for 3'-SL, 3'-SLN and their constituent subunits (sialic acid and lactose) (Fig. 7B). While no binding was measured with lactose or sialic acid, PilC bound both 3'-SL and 3'-SLN in accord with the microarray findings. There was a slight preference for 3'-SL over 3'-SLN, with K<sub>D</sub> of 79.1 ± 0.36 µM and 143.73 ± 7.36 µM, respectively (Fig. 7B).

Finally, we probed the glycan-binding activity of PilC by mutagenesis of residues on the concave side of PilC<sub>lectin</sub> where the CRD is predicted to be (Fig. 6C). We made a series of PilC<sub>Δpilin</sub> derivatives in which surface-exposed residues in the putative CRD were changed by site-directed mutagenesis (Fig. 8A). We then purified these mutant proteins and quantified their affinity for 3'-SL using ITC, before comparing it to WT PilC (Fig. 8B). This revealed that PilC<sub>T347A</sub> and PilC<sub>K349A</sub> displayed significantly reduced affinity for 3'-SL, which was particularly pronounced for PilC<sub>K349A</sub>, with 39.3 ± 1.4 % of the WT binding. This finding indicates that the concave side of its lectin module is involved in the ability of PilC to bind specific glycans.

**Modelling predicts that the PilAC complex is located at the tip of S. sanguinis T4P, together with PilB**

Since the instability of PilC pilin moiety precluded us from determining a crystal structure of the PilAC complex, we modelled this complex using AlphaFold (38). In the PilAC complex (Fig. 9A), the two proteins are staggered, PilA is on the top and the extra modules in PilC are on the side. PilA extends the β-sheet of PilC (Fig. S8), which is likely to explain its stabilising effect on PilC. The complex is stabilised by a series of 14 hydrogen bonds and five salt bridges (Fig. S8). The predicted PilAC interaction interface is in strikingly good agreement with that identified by NMR (Fig.
330 4B), including the α1 and α2 helices, and the β1 and β2 strands of PilA. This
331 significantly strengthens the validity of our model.

332 S. sanguinis T4P contain another modular pilin, PilB, which was previously
333 predicted to be at the pilus tip (6). We therefore wondered if PilA, B, C could co-exist
334 in one pilus, which we assessed by modelling using AlphaFold. The PilABC model
335 suggests that the two modular pilins PilB and PilC can be accommodated in the
336 same complex despite their bulky extra domains (Fig. 9B). The three subunits form a
337 quasihelical complex in which PilA interacts with PilC, which interacts with PilB.
338 Some level of movement for the extra modules in PilB and PilC is expected because
339 the short loops that connect them to their respective pilin moieties are likely to be
340 flexible. Although PilA is predicted to be added first, the extra modules in PilB and
341 PilC are capping the complex and look like "open wings" (Fig. 9B). Due to this
342 peculiar architecture, the PilABC complex could only be accommodated at the tip of
343 the T4P. It is possible that a portion of α1N needs to be melted, as in major pilins in
344 many T4F (11), to allow proper fitting. Tip localisation is likely to optimise the
345 presentation of the adhesive modules in PilB and PilC, promoting S. sanguinis
346 adhesion to host surfaces.
347 **Discussion**

348 T4F are a superfamily of filamentous nanomachines ubiquitous in bacteria and
349 archaea (1, 2), mediating a wide variety of functions ranging from adhesion to protein
350 secretion. Although T4F have been studied for 40 years – notably T4P (46) – many
351 aspects of their complex biology remain poorly understood. This holds especially true
352 for the filament – the "business end" of T4F nanomachines – where the roles of all
353 pilin subunits are yet to be understood. Therefore, in this report we used the inherent
354 simplicity of T4P in monoderm bacteria – which recently opened new research
355 avenues (30) – to complete a structure/function analysis of all the pilins in S.
356 *sanguinis* (31) T4P. This led to the notable findings discussed below, shining new
357 light on T4F.

358 Our first important finding – with widespread implications in T4F biology – is the
359 unexpected discovery that the minor pilin PilA from S. *sanguinis* is a structural
360 homolog of the I minor pilin of the widely conserved HIJK complex. The broader than
361 predicted conservation of that minor pilin is a testament to the central role it plays in
362 T4F biology. There are multiple analogies between the two systems, suggesting that
363 PilABC is perhaps a rudimentary/ancestral version of HIJK. The H,I,J,K pilins – which
364 are found in different T4F and are functionally interchangeable (24) – are encoded by
365 an operon, and K lacks the usually conserved Glu₅ (4). PilA,B,C pilins are also
366 encoded by an operon, and PilA lacks Glu₅ (35). Structural studies showed that HIJK
367 interact to form a quasihelical complex (25, 26), capped by the K subunit because no
368 additional subunits could fit above it without significant steric hindrance (25) and the
369 lack of Glu₅ in K would not allow neutralisation of the positively charged N-terminus
370 of a pilin above them. Importantly, this localisation was recently confirmed by cryo-ET
371 (27). Similarly, we found that PilA,B,C are likely to form a complex at the tip of S.
372 *sanguinis* T4P. Although PilA is added first, because it lacks a Glu₅, the bulky extra
373 modules in PilB and PilC cap the PilABC complex like open wings. Due to this
374 peculiar architecture – resembling the Winged Victory of Samothrace, a masterpiece
375 of Greek sculpture – PilABC could only be accommodated at the tip of the T4P.
376 Critically, both HIJK (21-23) and PilABC (34) pilins are essential for filament
377 assembly, which is compatible with PilA being a nucleator initiating filament
378 assembly, a role attributed to the I subunit in HIJK (28, 29). For the HIJK complex, it
379 was proposed that (i) I interacts with J in the membrane to form a staggered
380 complex, (ii) the IJ complex interacts with K via I, initiating filament assembly by
381 forming a quasihelical complex (25), (iii) the IJK complex interacts with H via J (26),
382 and (iv) the HIJK complex interacts with the major pilin via H (26). The series of
383 events is similar for PilABC, albeit simpler. (i) PilA interacts with PilC and stabilises it
384 (the I subunit in HIJK might play a similar role according to crystallisation studies
385 (47)), (ii) PilC interacts with PilB, and (iii) the PilABC complex most likely interacts
386 with the major pilins via PilB subunit. In both systems, the I/PilA subunit initiates
387 filament assembly and facilitates the presentation of "effectors" at the tip of filaments.
388 In S. sanguinis T4P, the effectors are the two modular pilins PilB and PilC with
389 adhesive properties. In T4P in diderm model species, the effector is the non-pilin
390 adhesin PilC/PilY1, an extremely large protein that is absent in monoderms.
391 Interestingly, cryo-ET analysis of M. xanthus T4P revealed that PilC/PilY1 forms a
392 kinked tip structure (27), which looks like one of the open wings in PilABC.

393 Drawing a picture of the roles of all the pilin subunits in a model T4F system is
394 the second major achievement in this study. S. sanguinis T4P are composed of five
395 pilins: two major (PilE1, PilE2) and three minor (PilA, PilB, PilC) ones. PilE1 and
396 PilE2 – 85 % identical in sequence (34) – follow universally shared principles for
397 processing by the PPase and display lollipop structures (35). This suggests that S.
398 sanguinis T4P are canonical T4F, i.e., helical polymers where pilins pack within the
399 core of the filament and are held together by interactions between their α1-helices
400 (11). Although unusual – T4F are most often homopolymers of only one major pilin –
401 the heteropolymeric composition of S. sanguinis T4P with two major pilins in nearly
equal amounts (35) is not unique. Recently, the structure of a heteropolymeric
archaeal T4F used for swimming by *Methanocaldococcus villosus* has been
elucidated by cryo-EM (16), showing an architecture with two alternating major pilin
subunits. Whether a similar alternating arrangement of PilE1 and PilE2 exists within
the *S. sanguinis* T4P remains to be tested. As for the three minor pilins in *S.
sanguinis* T4P they are divided into non-modular (PilA) and modular (PilB, PilC). We
have now defined their respective roles. PilA, which displays a canonical lollipop
structure, interacts strongly and exclusively with PilC. Pull-down experiments suggest
that PilA and PilC interact via their pilin moieties, which was confirmed by NMR. The
interaction interfaces determined by NMR and predicted by modelling are in strikingly
good agreement. As a functional consequence, PilA dramatically stabilises the PilC
pilin moiety, which is highly unstable on its own. Modelling suggests that this is done
by β-strand complementation. PilA thus acts as an intra-pilus chaperone, protecting
PilC from proteolysis by perhaps participating in its folding. We previously showed
that PilB displays a modular architecture with a bulky functional module grafted onto
a pilin moiety (6). The vWA module in PilB makes it a bona fide adhesin, playing a
key role in *S. sanguinis* adhesion to host cells by binding protein ligands such as
fibronectin and fibrinogen (6). Since *S. sanguinis* T4P are critical for disease
progression (48), these findings are consistent with PilB-mediated adhesion being
involved in IE. Here, we show that PilC is also a modular pilin with two extra
modules, an Ig-like fold followed by a ConA-like lectin/glucanase module
(SSF49899). While the function of the Ig-like module remains unclear, we showed
that the ConA-like module is a bona fide lectin. PilC binds specifically to two major
types of glycans – (i) sialylated glycans often terminated with α2-3-sialyl linkage (3'-
SL or 3'-SNL), and (ii) heparin oligosaccharide probes representative of sulphated
GAGs – both prevalent in the human glycome (45). The measured affinities for 3'-SL
and 3'-SLN are consistent with those previously determined for other SSF49899-
containing proteins (49). Affinities could be much higher for larger glycan
determinants, or when there is multivalent presentation (abundance) of a glycan
ligand at the cell surface (45, 49). The physiological ligands for PilC are yet to be
defined – identifying ligands for lectins is often difficult (45, 50) – but it is likely that
this modular pilin is involved in S. sanguinis commensalism or IE. Indeed, 3'-SL or 3'-
SLN terminated glycans are abundant in the oral cavity (32), especially on heavily
glycosylated salivary mucins (45). The full picture of S. sanguinis T4P that has now
emerged is the following. S. sanguinis T4P are heteropolymeric filaments composed
of PilE1 and PilE2, with helical symmetry parameters that are yet to be determined,
capped by a PilABC complex mediating bacterial adhesion to different eukaryotic
receptors. The adhesive modules in PilB and PilC are ideally placed to maximise
adhesion. However, although this is less likely, we cannot exclude the possibility that
S. sanguinis expresses two distinct pili, capped either by PilB or PilAC.

In conclusion, by providing perhaps the most detailed structure/function
characterisation of the role of all pilins in a model T4P, this study sheds light on
important aspects of T4F biology. The finding that PilABC might be a rudimentary
version of the widely conserved HIJK strengthens the notion that complexes of minor
pilins capping T4F facilitate the presentation of different effectors at the tip of
filaments, thus directly promoting T4F exceptional functional versatility (1). Our
findings have general implications for T4F and pave the way for further investigations
that will improve our understanding of these fascinating filaments.
450 **Material and methods**

451 Detailed Material and Methods are described in the Supporting information.

452

453 **Strains and plasmids**

454 The *E. coli* strains and plasmids used in this study are listed in Table S2. DH5α was
455 used for cloning, BL21(DE3) was used for routine protein expression and purification,
456 and B834(DE3) was used for purification of SeMet-labelled proteins.

457 DNA manipulations were performed using standard molecular biology
458 techniques (51). The primers used in this study are listed in Table S3. We amplified
459 *pilA* directly from the *S. sanguinis* 2908 genome, while *pilB* and *pilC* were synthetic
460 genes codon-optimised for expression in *E. coli* (GeneArt).

461

462 **Protein purification**

463 The recombinant proteins were purified in two steps: affinity chromatography and
464 SEC. 6His-tagged proteins were affinity-purified using Gravity Flow Chromatography
465 columns (Bio-Rad) loaded with Ni-NTA agarose resin (Qiagen). Strep-tagged
466 proteins were affinity-purified on an ÄKTA Purifier using StrepTrap HP columns (GE
467 Healthcare). Both the 6His- and Strep-tagged proteins were then further purified by
468 SEC on an ÄKTA Purifier using a Superdex 200 16/600 GL column (GE Healthcare),
469 and simultaneously buffer-exchanged into 50 mM HEPES pH 7.5, 150 mM NaCl.

470 SeMet-labelled PilA and PilC for phasing were purified as above, but the
471 bacteria were grown in chemically defined medium containing SeMet. ¹⁵N and/or ¹³C-
472 labelled PilA for NMR analysis was purified as above, but the bacteria were grown in
473 M9 medium supplemented with ¹⁵NH₄Cl and/or ¹³C-labelled glucose.

474

475 **Protein crystallisation and structure determination**

476 Protein crystals for SeMet-labelled PilA were produced using 0.4 µl sitting drops with
477 a 1:1 ratio protein to mother liquor (0.1 M Tris pH 8, 23 % PEG 2K (w/v), 0.3 M
478 Mg(NO₃)₂·6H₂O). Protein crystals for SeMet-labelled PilC were produced using 0.2 µl
479 sitting drops with of 1:1 ratio protein to mother liquor (0.1 M Pipes pH 7 15 % w/v
480 PEG broad smear, 0.2 M (NH₄)₂SO₄, 0.0 1M cadmium chloride hemi (pentahydrate)).

481 Data was collected at the Diamond beamlines i04-1 and i03, respectively. Data was
482 processed using Xia2 (52), and phased using autoSHARP (53). Initial structure was
483 produced using CRANK2 (54) and autobuild, with some manual building in Coot (55).

484 Refinement was performed using Coot and phenix.refine (56). Structure validation
485 was performed using MolProbity (57). Protein crystals for PilC<sup>SK36</sup> were produced in
486 0.2 µl sitting drop with a 1:1 protein to mother liquor (0.2 M MgCl<sub>2</sub>·6H<sub>2</sub>O, 20 % w/v
487 PEG 3350). Data was collected at the Diamond beamline i04 and structure was
488 produced using the PilC structure from strain 2908 for molecular replacement.

489 All the 3D structures determined during this study have been deposited in the
490 PDB and are available under accession codes 7O5Y (PilA), 7OA7 (PilC), and 7OA8
491 (PilC<sup>SK36</sup>). The PilABC structural model is available in ModelArchive at
492 https://modelarchive.org/doi/10.5452/ma-zz3r2.

493

494 **Pull-down assays**

495 Pull-down assays were carried out using Dynabeads His-tag Isolation and Pulldown
496 (Invitrogen). The 6His-tagged soluble pilin domains were used as bait, while Strep-
497 tagged soluble pilins were used as prey. The pull-down assays were performed three
498 times for each combination. For each pull-down reaction, input, flow-through, and
499 elution samples were analysed by immunoblotting.

500

501 **SDS-PAGE and immunoblotting**

502 SDS-PAGE was carried out using standard molecular biology techniques (51). Gels
503 were either stained with Bio-Safe Coomassie (Bio-Rad) or transferred to a membrane
504 and analysed by immunoblotting. We used specific antibodies generated in rabbits
505 against PilE1, PilE2, PilA, PilB, PilC, which were previously described (34, 35). The
secondary was an anti-rabbit antibody conjugated to horseradish peroxidase (GE Healthcare).

508

## 509 SEC-MALS

SEC-MALS was performed using an ÄKTA Prime system with a S200 10/300 GL column (GE Healthcare) and a MALS detector (Wyatt). The UV, light scattering and refractive index signals were analysed with the ASTRA chromatography software.

513

## 514 ITC

ITC was used to study the interaction between the minor pilins PilA and PilC, as well as the binding of PilC<sub>Δpilin</sub> to sugar ligands. The experiments were performed on a MicroCal ITC200 Malvern machine. Each experiment was repeated three times. Data analysis was performed on the Origin ITC200 software. Statistical analyses were performed with Prism (GraphPad Software). Comparisons were done by one-way ANOVA, followed by Dunnett's multiple comparison tests with 99 % confidence interval. An adjusted *P* value <0.01 was considered statistically significant (** *P*<0.01, *** *P*<0.001, **** *P*<0.0001).

523

## 524 NMR assignment and chemical shift perturbations

We performed TROSY-based assignment experiments: HNCA, HNCOCA, HNCO, HNCACO, HNCACB, and CBCACONH. All data were collected at 25°C on a Bruker Avance III HD 800MHz triple resonance spectrometer with a cryoprobe.

Chemical shift perturbation experiments were performed with samples in NMR buffer containing <sup>15</sup>N labelled PilA with or without unlabelled PilC. Chemical shift perturbations were determined using the equation:

531

532

$$ \Delta\delta = \sqrt{(\Delta\delta_H)^2 + \left(\frac{\gamma_N}{\gamma_H}\Delta\gamma_N\right)^2} $$
533 Protein stability assays

534 To perform protein stability tests, PilA and PilC were purified and mixed at equal
535 concentrations. In long-term assays, the proteins were kept at 4°C for four weeks.
536 Samples were taken once a week and analysed by SDS-PAGE/Coomassie staining.
537 In trypsin sensitivity assays, the proteins were incubated with trypsin (Sigma) for 60
538 min on ice. Samples – taken at 1, 5, 10, 20, 40 and 60 min – were analysed by SDS-
539 PAGE/Coomassie staining.

540

541 Glycan microarrays

542 The binding specificities of 6His-PilC, 6His-PilC<sub>Δpilin</sub> and 6His-PilC<sup>SK36</sup> were analysed
543 using a NGL-based microarray system (42). A previously described broad-spectrum
544 microarray of 672 sequence-defined lipid-linked glycan probes was used (58). The
545 list of glycan probes is given in the Dataset S1. Details of the preparation of the
546 glycan probes and the generation of the microarrays are listed in Table S4 in
547 accordance with the MIRAGE guidelines (59). The microarray analyses were
548 performed essentially as described (60). Binding was detected with Alexa Fluor-647-
549 labelled streptavidin (Molecular Probes). Imaging and data analysis are described in
550 Table S4.

551

552 Bioinformatics and modelling

553 This was done as previously described (6). Modelling was done using AlphaFold
554 2.2.0 (38) and AlphaFold-Multimer (61). Five predictions were generated in total, with
555 a final relaxation step. The predictions were ranked according IPTM+PTM and the
556 top predictions were chosen.
557 Acknowledgements

558 This work was funded by the Medical Research Council (MR/P022197/1). The glycan
559 microarray studies were performed in the Carbohydrate Microarray Facility at the
560 Glycosciences Laboratory, which is supported by Wellcome Trust biomedical
561 resource grants (099197/Z/12/Z, 108430/Z/15/Z, and 218304/Z/19/Z) and partially by
562 the March of Dimes Prematurity research centre grant (22-FY18-82). The sequence-
563 defined glycan microarrays contain many saccharides provided by collaborators
564 whom we thank. We acknowledge the use of the crystallisation facility at Imperial
565 College London, which is supported by the Biotechnology and Biological Sciences
566 Research Council (BB/D524840/1) and Wellcome Trust (202926/Z/16/Z). We thank
567 Angelika Gründling (Imperial College London), Sophie Helaine (Harvard Medical
568 School) and Romé Voulhoux (CNRS Marseille) for critical reading of the manuscript.
569 **References**

570 1. J. L. Berry, V. Pelicic, Exceptionally widespread nano-machines composed of type
571 IV pilins: the prokaryotic Swiss Army knives. *FEMS Microbiol. Rev.* **39**, 134-154
572 (2015).

573 2. R. Denise, S. S. Abby, E. P. C. Rocha, Diversification of the type IV filament
574 superfamily into machines for adhesion, protein secretion, DNA uptake, and motility.
575 *PLoS Biol.* **17**, e3000390 (2019).

576 3. Y. W. Chang et al., Architecture of the type IVa pilus machine. *Science* **351**,
577 aad2001 (2016).

578 4. C. L. Giltner, Y. Nguyen, L. L. Burrows, Type IV pilin proteins: versatile molecular
579 modules. *Microbiol. Mol. Biol. Rev.* **76**, 740-772 (2012).

580 5. Z. Szabó et al., Identification of diverse archaeal proteins with class III signal
581 peptides cleaved by distinct archaeal prepilin peptidases. *J. Bacteriol.* **189**, 772-778
582 (2007).

583 6. C. Raynaud, D. Sheppard, J. L. Berry, I. Gurung, V. Pelicic, PilB from
584 *Streptococcus sanguinis* is a bimodular type IV pilin with a direct role in adhesion.
585 *Proc. Natl. Acad. Sci. USA* **118**, e2102092118 (2021).

586 7. M. R. Kaufman, J. M. Seyer, R. K. Taylor, Processing of TCP pilin by TcpJ typifies
587 a common step intrinsic to a newly recognized pathway of extracellular protein
588 secretion by Gram-negative bacteria. *Genes Dev.* **5**, 1834-1846 (1991).

589 8. D. N. Nunn, S. Lory, Product of the *Pseudomonas aeruginosa* gene *pilD* is a
590 prepilin leader peptidase. *Proc. Natl. Acad. Sci. USA* **88**, 3281-3285 (1991).

591 9. C. F. LaPointe, R. K. Taylor, The type 4 prepilin peptidases comprise a novel
592 family of aspartic acid proteases. *J. Biol. Chem.* **275**, 1502-1510 (2000).

593 10. M. McCallum, S. Tammam, A. Khan, L. L. Burrows, P. L. Howell, The molecular
594 mechanism of the type IVa pilus motors. *Nat. Commun.* **8**, 15091 (2017).

595 11. E. H. Egelman, Cryo-EM of bacterial pili and archaeal flagellar filaments. *Curr.
596 Opin. Struct. Biol.* **46**, 31-37 (2017).
12. S. Kolappan et al., Structure of the *Neisseria meningitidis* type IV pilus. *Nat. Commun.* **7**, 13015 (2016).

13. F. Wang et al., Cryoelectron microscopy reconstructions of the *Pseudomonas aeruginosa* and *Neisseria gonorrhoeae* type IV pili at sub-nanometer resolution. *Structure* **25**, 1423-1435 (2017).

14. A. Lopez-Castilla et al., Structure of the calcium-dependent type 2 secretion pseudopilus. *Nat. Microbiol.* **2**, 1686-1695 (2017).

15. A. Luna Rico, W. Zheng, N. Petiot, E. H. Egelman, O. Francetic, Functional reconstitution of the type IVa pilus assembly system from enterohaemorrhagic *Escherichia coli*. *Mol. Microbiol.* **111**, 732-749 (2019).

16. L. Gambelli et al., An archaellum filament composed of two alternating subunits. *Nat. Commun.* **13**, 710 (2022).

17. A. Cehovin et al., Specific DNA recognition mediated by a type IV pilin. *Proc. Natl. Acad. Sci. USA* **110**, 3065-3070 (2013).

18. J. L. Berry et al., A comparative structure/function analysis of two type IV pilin DNA receptors defines a novel mode of DNA binding. *Structure* **24**, 926-934 (2016).

19. J. P. Barnier et al., The minor pilin PilV provides a conserved adhesion site throughout the antigenically variable meningococcal type IV pilus. *Proc. Natl. Acad. Sci. USA* **118** (2021).

20. S. Helaine, D. H. Dyer, X. Nassif, V. Pelicic, K. T. Forest, 3D structure/function analysis of PilX reveals how minor pilins can modulate the virulence properties of type IV pili. *Proc. Natl. Acad. Sci. USA* **104**, 15888-15893 (2007).

21. H. C. Winther-Larsen et al., *Neisseria gonorrhoeae* PilV, a type IV pilus-associated protein essential to human epithelial cell adherence. *Proc. Natl. Acad. Sci. USA* **98**, 15276-15281 (2001).

22. E. Carbonnelle, S. Helaine, L. Prouvensier, X. Nassif, V. Pelicic, Type IV pilus biogenesis in *Neisseria meningitidis*: PilW is involved in a step occuring after pilus assembly, essential for fiber stability and function. *Mol. Microbiol.* **55**, 54-64 (2005).
23. C. L. Giltner, M. Habash, L. L. Burrows, *Pseudomonas aeruginosa* minor pilins are incorporated into type IV pili. *J. Mol. Biol.* **398**, 444-461 (2010).

24. D. A. Cisneros, G. Pehau-Arnaudet, O. Francetic, Heterologous assembly of type IV pili by a type II secretion system reveals the role of minor pilins in assembly initiation. *Mol. Microbiol.* **86**, 805-818 (2012).

25. K. V. Korotkov, W. G. Hol, Structure of the GspK-Gspl-GspJ complex from the enterotoxigenic *Escherichia coli* type 2 secretion system. *Nat. Struct. Mol. Biol.* **15**, 462-468 (2008).

26. C. A. Escobar et al., Structural interactions define assembly adapter function of a type II secretion system pseudopilin. *Structure* **29**, 1116-1127 (2021).

27. A. Treuner-Lange et al., PilY1 and minor pilins form a complex priming the type IVa pilus in *Myxococcus xanthus*. *Nat. Commun.* **11**, 5054 (2020).

28. D. A. Cisneros, P. J. Bond, A. P. Pugsley, M. Campos, O. Francetic, Minor pseudopilin self-assembly primes type II secretion pseudopilus elongation. *EMBO J.* **31**, 1041-1053 (2012).

29. B. Douzi et al., The XcpV/Gspl pseudopilin has a central role in the assembly of a quaternary complex within the T2SS pseudopilus. *J. Biol. Chem.* **284**, 34580-34589 (2009).

30. S. Melville, L. Craig, Type IV pili in Gram-positive bacteria. *Microbiol. Mol. Biol. Rev.* **77**, 323-341 (2013).

31. V. Pelicic, Monoderm bacteria: the new frontier for type IV pilus biology. *Mol. Microbiol.* **112**, 1674-1683 (2019).

32. J. Kreth, R. A. Giacaman, R. Raghavan, J. Merritt, The road less traveled – defining molecular commensalism with *Streptococcus sanguinis*. *Mol. Oral. Microbiol.* **32**, 181-196 (2017).

33. T. J. Cahill, B. D. Prendergast, Infective endocarditis. *Lancet* **387**, 882-893 (2016).
34. I. Gurung et al., Functional analysis of an unusual type IV pilus in the Gram-positive *Streptococcus sanguinis*. *Mol. Microbiol.* **99**, 380-392 (2016).

35. J. L. Berry et al., Global biochemical and structural analysis of the type IV pilus from the Gram-positive bacterium *Streptococcus sanguinis*. *J. Biol. Chem.* **294**, 6796-6808 (2019).

36. S. Imam, Z. Chen, D. S. Roos, M. Pohlschröder, Identification of surprisingly diverse type IV pili, across a broad range of Gram-positive bacteria. *PLoS One* **6**, e28919 (2011).

37. L. Craig et al., Type IV pilin structure and assembly. X-ray and EM analyses of *Vibrio cholerae* toxin-coregulated pilus and *Pseudomonas aeruginosa* PAK pilin. *Mol. Cell* **11**, 1139-1150 (2003).

38. J. Jumper et al., Highly accurate protein structure prediction with AlphaFold. *Nature* **596**, 583-589 (2021).

39. P. Bork, L. Holm, C. Sander, The immunoglobulin fold. Structural classification, sequence patterns and common core. *J. Mol. Biol.* **242**, 309-320 (1994).

40. E. Wong et al., The Vibrio cholerae colonization factor GbpA possesses a modular structure that governs binding to different host surfaces. *PLoS Pathog.* **8**, e1002373 (2012).

41. D. D. Leonidas et al., Structural basis for the recognition of carbohydrates by human galectin-7. *Biochemistry* **37**, 13930-13940 (1998).

42. Y. Liu et al., Neoglycolipid-based oligosaccharide microarray system: preparation of NGLs and their noncovalent immobilization on nitrocellulose-coated glass slides for microarray analyses. *Meth. Mol. Biol.* **808**, 117-136 (2012).

43. W. L. Marsh et al., Anti-Sdx: a "new" auto-agglutinin related to the Sda blood group. *Transfusion* **20**, 1-8 (1980).

44. J. A. Morton, M. M. Pickles, A. M. Terry, The Sda blood group antigen in tissues and body fluids. *Vox Sang.* **19**, 472-482 (1970).
679 45. R. D. Cummings, The repertoire of glycan determinants in the human glycome.
680 Mol. Biosyst. **5**, 1087-1104 (2009).

681 46. V. Pelicic, Type IV pili: *e pluribus unum*? *Mol. Microbiol. 68*, 827-837 (2008).

682 47. M. E. Yanez, K. V. Korotkov, J. Abendroth, W. G. Hol, The crystal structure of a
683 binary complex of two pseudopilins: EpsI and EpsJ from the type 2 secretion system
684 of *Vibrio vulnificus*. *J. Mol. Biol.* **375**, 471-486 (2008).

685 48. A. M. Martini, B. S. Moricz, L. J. Woods, B. D. Jones, Type IV pili of
686 *Streptococcus sanguinis* contribute to pathogenesis in experimental infective
687 endocarditis. *Microbiol. Spectr.* **9**, e0175221 (2021).

688 49. S. Böcker, L. Elling, Binding characteristics of galectin-3 fusion proteins.
689 *Glycobiology* **27**, 457-468 (2017).

690 50. P. R. Crocker, T. Feizi, Carbohydrate recognition systems: functional triads in
691 cell-cell interactions. *Curr. Opin. Struct. Biol.* **6**, 679-691 (1996).

692 51. J. Sambrook, D. W. Russell, *Molecular cloning. A laboratory manual* (Cold Spring
693 Harbor Laboratory Press, Cold Spring Harbor, New York, 2001).

694 52. G. Winter, C. M. Lobley, S. M. Prince, Decision making in *xia2*. *Acta Crystallogr.*
695 *Sect. D: Biol. Crystallogr.* **69**, 1260-1273 (2013).

696 53. C. Vonrhein, E. Blanc, P. Roversi, G. Bricogne, Automated structure solution with
697 autoSHARP. *Meth. Mol. Biol.* **364**, 215-230 (2007).

698 54. P. Skubak et al., A new MR-SAD algorithm for the automatic building of protein
699 models from low-resolution X-ray data and a poor starting model. *IUCRJ* **5**, 166-171
700 (2018).

701 55. P. Emsley, B. Lohkamp, W. G. Scott, K. Cowtan, Features and development of
702 Coot. *Acta Crystallogr. Sect. D: Biol. Crystallogr.* **66**, 486-501 (2010).

703 56. P. V. Afonine et al., Towards automated crystallographic structure refinement
704 with phenix.refine. *Acta Crystallogr. Sect. D: Biol. Crystallogr.* **68**, 352-367 (2012).

705 57. V. B. Chen et al., MolProbity: all-atom structure validation for macromolecular
706 crystallography. *Acta Crystallogr. Sect. D: Biol. Crystallogr.* **66**, 12-21 (2010).
707 58. N. McAllister et al., Chikungunya virus strains from each genetic clade bind
708 sulfated glycosaminoglycans as attachment factors. *J. Virol.* **94**, e01500-01520
709 (2020).

710 59. Y. Liu et al., The minimum information required for a glycomics experiment
711 (MIRAGE) project: improving the standards for reporting glycan microarray-based
712 data. *Glycobiology* **27**, 280-284 (2017).

713 60. U. Neu et al., A structure-guided mutation in the major capsid protein retargets
714 BK polyomavirus. *PLoS Pathog.* **9**, e1003688 (2013).

715 61. R. Evans et al., Protein complex prediction with AlphaFold-Multimer. *BioRxiv*
716 https://doi.org/10.1101/2021.10.04.463034 (2022).
717 **Legends to Figures**

718

719 **Fig. 1. PilA and PilC sequence characteristics.** A) Protein architecture of the five prepilins in the T4P-encoding *pil* locus in *S. sanguinis* 2908 (34). Each protein 720 harbours a type 4 pilin-defining class 3 SP (black rounded rectangle) at its N- 721 terminus. PilB and PilC are modular pilins, which contain additional domains at their 722 C-terminus (yellow and red rounded rectangles). PilB contains a von Willebrand 723 factor A-like domain (IPR036465) (6), while PilC contains a concanavalin A-like 724 lectin/glucanase structural domain (SSF49899). Proteins are drawn to scale. B) 725 Relevant sequence features of PilA. The class 3 SP is black boxed and contains an 726 8-aa long leader peptide with mostly hydrophilic (orange shading) and neutral (no 727 shading) residues, followed by a tract of 21 mainly hydrophobic residues (blue 728 shading). The processing of the leader peptide by the PPase, indicated by the 729 vertical arrow, generates a pilin of 17 kDa. The arrowhead indicates the soluble 730 portion of PilA that was produced and purified in 6His-PilA. C) Relevant sequence 731 features of PilC. The class 3 SP is black boxed. The processing of the leader peptide 732 by the PPase (black vertical arrow), generates a pilin of 52.8 kDa. The lectin module 733 (SSF49899) is red boxed. The Ig-like fold module revealed by our structure is green 734 boxed. Arrowheads indicate the soluble proteins that were produced and purified in 735 this study: black arrowhead is 6His-PilC, grey arrowhead is 6His-PilC<sub>Δpilin</sub> without the 736 pilin moiety. The site of spontaneous proteolysis in 6His-PilC is indicated by a star. 737

738

739 **Fig. 2. Crystal structure of PilA.** A) Cartoon view of the PilA structure rainbow- 740 coloured from blue (N-terminus) to red (C-terminus). B) Cartoon view of the head-to- 741 toe PilA dimers in the crystal packing. C) Structural similarity between PilA (blue) and 742 a widely conserved minor pilin in T4F (pink). The GspI protein from enterotoxigenic 743 *E. coli* T2SS (PDB 3CI0) is presented. Although these two proteins share only 9.4 % 744 sequence identity, they superpose with an RMSD of 1.52 Å.
745

746

**Fig. 3. PilA interacts with PilC.** A) Interactions between PilA and the four other pilins were tested by performing pull-down assays using purified proteins corresponding to the soluble portion of the pilins. The 6His-tagged bait protein was first incubated with Strep-tagged prey protein, and then pulled-down using cobalt-based magnetic beads. Proteins bound to the beads were eluted and identified by immunoblotting using specific anti-Pil antibodies. IN, input. NB, not bound. B, bound.

747

748

749

750

751

752

* Non-specific binding of Strep-PilB to the beads, which was also observed with the protein on its own. B) Characterisation of the interaction between PilA and PilC by SEC-MALS. Upper panel, chromatogram traces for the PilAC complex. dRI, differential refractive index, LS, MALS signal. Mw, weight average molecular weight.

753

754

755

756

Lower panel, SEC-MALS data for PilA, PilC and PilA-PilC. Mn, number average molecular weight. C) Quantification of the PilA-PilC interaction by ITC. A representative ITC titration curve (upper panel) and binding isotherm (lower panel) are presented. The calculated K_D is the average ± SD from three independent experiments.

757

758

759

760

761

**Fig. 4. Mapping the PilA-PilC binding interface.** A) Testing the interaction between PilA and PilC<sub>Δpilin</sub> by performing pull-down assays. PilC<sub>Δpilin</sub> corresponds to the protein missing the pilin moiety. Proteins bound to the beads were eluted and identified by immunoblotting using anti-6His and anti-PilA antibodies. IN, input. NB, not bound. B, bound. B) NMR analysis of PilAC complex formation. PilA residues for which NMR assignment could be performed are highlighted in blue on our crystal structure (180° view). Residues experiencing significant chemical shift perturbations in the presence of PilC are highlighted in yellow. C) ITC quantification of the binding of PilA mutant proteins to PilC. The results, presented as K_D ratios to the WT, are the average ± SD from 2-3 independent experiments. Statistical significance was calculated using one-way ANOVA followed by Dunnett's multiple comparison tests.

762

763

764

765

766

767

768

769

770

771

772
773

774

**Fig. 5. PilA stabilises PilC.** A) Long-term protein stability experiments. Purified PilA and PilC were mixed in equimolar amounts, and the PilAC complex was isolated by SEC. The complex, as well as PilA and PilC on their own, were kept at 4°C for a month. Samples were taken once a week and analysed by SDS-PAGE/Coomassie.

775

776

777

778

The degradation products (PilC<sub>Nter</sub> and PilC<sub>Cter</sub>) are identified on the right side. Three independent experiments were performed yielding same results, representative gels are shown. The molecular masses are in kDa.

779

780

781

B) Trypsin sensitivity assays. Purified PilA, PilC and PilAC were incubated with trypsin (1:1,000 dilution) for 60 min on ice. Samples were taken at various times and analysed by SDS-PAGE/Coomassie. The degradation products are indicated on the right side. Three independent experiments were performed yielding similar results, representative gels are shown.

782

783

784

785

786

**Fig. 6. Crystal structures of PilC.** A) Cartoon views at 180° rotation of the structure of PilC<sub>Δpilin</sub> from strain 2908 in which two distinct modules have been identified highlighted in green (Ig-like fold) and red (lectin). B) Left, Close-up view of the Ig-like fold module (PilC<sub>lg</sub>). The two β-sheets packed against each other have been highlighted in green. Right, superposition of PilC<sub>lg</sub> with the Ig-like fold domain in V.

787

788

789

790

791

cholerae colonisation factor GbpA (PDB 2XWX) (40). C) Left, orthogonal cartoon views of the lectin module (PilC<sub>lectin</sub>) in which the β-sandwich of two opposing antiparallel β-sheets is highlighted in red. The putative CRD is indicated by an arrow.

792

793

794

Right, superposition of PilC<sub>lectin</sub> with human Gal-7 (PDB 2GAL) (41). The bound galactose in the Gal-7 structure is highlighted in red. D) Left, structure of PilC from S. sanguinis SK36. Right, superposition of PilC<sup>2908</sup> and PilC<sup>SK36</sup>. These two proteins, which share 57 % sequence identity (Fig. S4), superpose almost perfectly with an RMSD of only 0.79 Å.

795

796

797

798

799
Fig. 7. PilC specifically binds sialylated glycans and GAG. A) Glycan microarray screening analyses of 6His-PilC, as a protein-antibody pre-complex. Multiple experiments were performed yielding similar results. Results of one representative experiment are shown as the means of fluorescence intensities of duplicate spots of glycan probes, printed at 5 fmol/spot. The 672 lipid-linked probes are grouped according to sialyl linkages as annotated by the coloured panels. The full list of glycan probes, their sequences, and binding scores are given in Dataset S1. * Signals with large error bars due to artefacts on the array slides. B) ITC quantification of PilC<sub>Δpilin</sub> affinity for sialylated glycans, sialic acid and lactose. The calculated K<sub>D</sub> are the average ± SD from three independent experiments. For binding to 3'-SL, representative ITC titration curve (upper panel) and binding isotherm (lower panel) are presented.

812

Fig. 8. Probing the putative carbohydrate-recognition domain in PilC by mutagenesis. A) Cartoon view of PilC<sub>lectin</sub> where the residues in the putative CRD – targeted by SDM – are highlighted in orange. B) ITC quantification of binding to 3'-SL by PilC<sub>Δpilin</sub> mutant proteins. The results, presented as K<sub>D</sub> ratios to the WT, are the average ± SD from three independent experiments. Statistical significance was calculated using one-way ANOVA followed by Dunnett's multiple comparison tests.

819

Fig. 9. Modelling the complex of minor pilins in S. sanguinis T4P. A) Model of the PilAC complex. B) Model of the PilABC complex. Left, cartoon views at 180° rotation. Right, surface representation.

821

822
B

<figure><img src="image_2.png" /></figure>

C

<figure><img src="image_3.png" /></figure>

A

<figure><img src="image_4.png" /></figure>
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

PilA

GspI
A

PilA + PilB

PilA + PilC

PilA + PilE1

PilA + PilE2

Bait

<figure><img src="image_8.png" /></figure>

Prey

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

B

<figure><img src="image_12.png" /></figure>

<table><thead><tr><th>protein</th><th>Mw (kDa)</th><th>Mn (kDa)</th><th>polydispersity (Mw/Mn)</th></tr></thead><tbody><tr><td>PilA</td><td>31.7 ± 0.35</td><td>31.7 ± 0.03</td><td>1.000 ± 0.015</td></tr><tr><td>PilC</td><td>57.7 ± 0.63</td><td>57.8 ± 0.06</td><td>1.001 ± 0.016</td></tr><tr><td>PilA-PilC</td><td>70.8 ± 0.07</td><td>70.8 ± 0.07</td><td>1.000 ± 0.015</td></tr></tbody></table>

C

<figure><img src="image_13.png" /></figure>
A

Bait

<figure><img src="image_14.png" /></figure>

Prey

<figure><img src="image_15.png" /></figure>

B

<figure><img src="image_16.png" /></figure>

C

<figure><img src="image_17.png" /></figure>
A

0 1 2 3 4

PilA

<figure><img src="image_18.png" /></figure>

0 1 2 3 4

PilC

<figure><img src="image_19.png" /></figure>

0 1 2 3 4 Weeks

<figure><img src="image_20.png" /></figure>

B

0 1 5 10 20 40 60

PilA

<figure><img src="image_21.png" /></figure>

0 1 5 10 20 40 60

PilC

<figure><img src="image_22.png" /></figure>

0 1 5 10 20 40 60 min

<figure><img src="image_23.png" /></figure>

<PilC

<PilC<sub>Cter</sub>

<PilA

<PilC<sub>Nter</sub>
A

lectin module

<figure><img src="image_24.png" /></figure>

B

<figure><img src="image_25.png" /></figure>

C

<figure><img src="image_26.png" /></figure>

D

<figure><img src="image_27.png" /></figure>

C

<figure><img src="image_28.png" /></figure>

PilC<sub>SK36</sub>

PilC<sub>2908</sub>

CRD
A

<figure><img src="image_29.png" /></figure>

B

<table><thead><tr><th>glycan</th><th>ΔH<br>(kcal/mol)</th><th>K<sub>D</sub><br>(μM)</th></tr></thead><tbody><tr><td>3'-SL</td><td>-7.94 ± 0.42</td><td>79.1 ± 0.36</td></tr><tr><td>3'-SLN</td><td>-8.28 ± 0.39</td><td>143.73 ± 7.36</td></tr><tr><td>sialic acid</td><td>—</td><td>—</td></tr><tr><td>lactose</td><td>—</td><td>—</td></tr></tbody></table>

Time (min)

<figure><img src="image_30.png" /></figure>
A

<figure><img src="image_31.png" /></figure>

B

<figure><img src="image_32.png" /></figure>
<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>