# PilB from Streptococcus sanguinis is a bimodular type IV pilin with a direct role in adhesion

C. Raynaud, D. Sheppard, J.L. Berry, I. Gurung, V. Pelicic

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C. Raynaud, D. Sheppard, J.L. Berry, I. Gurung, V. Pelicic. PilB from Streptococcus sanguinis is a bimodular type IV pilin with a direct role in adhesion. Proceedings of the National Academy of Sciences of the United States of America, 2021, 118 (22), pp.e2102092118. 10.1073/pnas.2102092118. hal-03512351

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1 PilB from *Streptococcus sanguinis* is a bimodular type IV pilin with a direct role

2 in adhesion

3

4 Claire Raynaud<sup>a,1</sup>, Devon Sheppard<sup>a,1</sup>, Jamie-Lee Berry<sup>a</sup>, Ishwori Gurung<sup>a</sup>, Vladimir

5 Pelicic<sup>a,b,2</sup>

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7 <sup>a</sup>MRC Centre for Molecular Bacteriology and Infection, Imperial College London, SW7

8 2AZ London, United Kingdom

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10 <sup>b</sup>Laboratoire de Chimie Bactérienne, Aix-Marseille Université-CNRS (UMR 7283),

11 Institut de Microbiologie de la Méditerranée, 13009 Marseille, France

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13 <sup>1</sup>C.R. and D.S. contributed equally to this work.

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15 <sup>2</sup>To whom correspondence may be addressed. Email: vladimir.pelicic@inserm.fr
16 Abstract

17 Type IV pili (T4P) are functionally versatile filamentous nanomachines, nearly
18 ubiquitous in prokaryotes. They are predominantly polymers of one major pilin, but also
19 contain minor pilins whose functions are often poorly defined, and likely to be diverse.

20 Here, we show that the minor pilin PilB from the T4P of S. sanguinis displays an
21 unusual bimodular 3D structure, with a bulky von Willebrand factor A-like (vWA)
22 module "grafted" onto a small pilin module via a short loop. Structural modelling
23 suggests that PilB is only compatible with a localisation at the tip of T4P. By performing
24 a detailed functional analysis, we found that (i) the vWA module contains a canonical
25 metal ion-dependent adhesion site (MIDAS), preferentially binding Mg²⁺ and Mn²⁺, (ii)
26 abolishing metal-binding has no impact on the structure of PilB or piliation, (iii) metal-
27 binding is important for S. sanguinis T4P-mediated twitching motility and adhesion to
28 eukaryotic cells, and (iv) the vWA module shows an intrinsic binding ability to several
29 host proteins. These findings reveal an elegant, yet simple, evolutionary tinkering
30 strategy to increase T4P functional versatility, by grafting a functional module onto a
31 pilin for presentation by the filaments. This strategy appears to have been extensively
32 used by bacteria, in which modular pilins are widespread and exhibit an astonishing
33 variety of architectures.
34 Significance

35 Type IV pili (T4P) are functionally versatile filaments widespread in prokaryotes, 36 composed of type IV pilins and assembled by conserved multi-protein machineries. It 37 remains unclear how such rather simple filaments can be so versatile. Our 38 structure/function analysis of PilB, a minor pilin of S. sanguinis T4P, offers an elegant 39 explanation for this paradox. We show that PilB is a modular pilin with a bulky module 40 "grafted" onto a small pilin module, which directly mediates adhesion of S. sanguinis 41 to host cells/proteins. This evolutionary tinkering strategy appears to be prevalent in 42 bacteria, since a global analysis reveals that modular pilins are widespread and exhibit 43 an astonishing variety of architectures.
## 44 Introduction

Type IV pili (T4P) are functionally versatile filaments widespread in prokaryotes, implicated in a variety of functions such as adhesion, twitching motility, DNA uptake etc (1). T4P are helical polymers consisting of type IV pilins, usually one major pilin and several minor (low abundance) ones, assembled by conserved multi-protein machineries. These defining features are shared by a superfamily of filamentous nanomachines known as type IV filaments (T4F) (1), ubiquitous in prokaryotes (2).

T4P have been intensively studied for decades in diderm bacteria because they play a central role in pathogenesis in important human pathogens (3). The following global picture of T4P biology has emerged from these studies. The pilus subunits, type IV pilins, are characterised by a short N-terminal sequence motif known as class III signal peptide, which consists of a hydrophilic leader peptide ending with a small residue (Gly or Ala), followed by a tract of 21 predominantly hydrophobic residues (4). This tract constitutes the N-terminal segment (α1N) of an α-helix (α1) of ~50 residues, which is the universally conserved structural feature in type IV pilins. Usually, the α1N helix protrudes from a globular head most often consisting of a β-sheet composed of several antiparallel β-strands, which gives pilins their characteristic "lollipop" shape (4). The hydrophilic leader peptide is then processed by a dedicated prepilin peptidase (5) after pilin translocation across the cytoplasmic membrane (CM) by the general secretory pathway (6, 7). Processed pilins remain embedded in the CM via their α1N, generating a pool of subunits ready for polymerisation. Filament assembly, which occurs from tip to base, is mediated at the CM by a complex multi-protein machinery (10-20 components) (1), centred on an integral membrane platform protein and a cytoplasmic extension ATPase (8). Recent cryo-EM structures have revealed that T4P are right-handed helical polymers where pilins are held together by extensive interactions between their α1N helices, which are partially melted and run approximately parallel to each other within the filament core (9, 10). One of the properties of T4P key for their functional versatility is their ability to retract, which has
been best characterised for T4aP (where "a" denotes the subtype). In T4aP, retraction
results from rapid filament depolymerisation powered by the cytoplasmic retraction
ATPase PilT (11), which generates important tensile forces (12, 13).

Studying T4P in monoderm bacteria represents a promising alternative research
avenue (14). *Streptococcus sanguinis*, a commensal of the oral cavity that commonly
causes life-threatening infective endocarditis (IE), has emerged as a monoderm model
for deciphering T4P biology (15). Our comprehensive functional analysis of S.
*sanguinis* T4P (16) revealed that they are canonical T4aP. Indeed, filaments are (i)
assembled by a multi-protein machinery similar to diderm T4aP species, but simpler
with only ten components, (ii) retracted by a PilT ATPase, generating tensile forces
similar to diderm species, and (iii) powering intense twitching motility, leading to
spreading zones around bacteria growing on plates, visible by the naked eye.
Subsequently, we performed a global biochemical and structural analysis of S.
*sanguinis* T4P (17), showing that (i) they are hetero-polymers composed of two major
pilins, PilE1 and PilE2, rather than one as usually seen, (ii) the major pilins display
classical type IV pilin 3D structure, and (iii) the filaments contain a low abundance of
three minor pilins (PilA, PilB, and PilC), which are required for piliation.

The present study was prompted by a perplexing observation, i.e., the minor pilin
PilB harbours a protein domain that has been extensively studied in eukaryotic proteins
where it mediates adhesion to a variety of protein ligands (18). This suggested that
PilB might be an adhesin, promoting T4P-mediated adhesion of S. *sanguinis* to host
cells and proteins. Therefore, since both the molecular mechanisms of T4P-mediated
adhesion and the exact role of minor pilins in T4P biology remain incompletely
understood (1), we performed a structure/function analysis of PilB, which is reported
here. This uncovered a widespread strategy for minor pilins to enhance the functional
properties of T4P.
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## Results

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### PilB displays a modular pilin architecture

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PilB, one of the three minor pilins in *S. sanguinis* T4P (17), exhibits a canonical N-terminal class III signal peptide, the defining feature of type IV pilins (4). This sequence motif consists of a seven-residue leader peptide composed predominantly of hydrophilic and neutral amino acids (aa), ending with a Gly (Fig. 1A). This leader peptide, which is processed by the prepilin peptidase PilD (17), is followed by a stretch of 21 predominantly hydrophobic aa, except for a negatively charged Glu in position 5 (Fig. 1A). Processed PilB is unusually large for a pilin, with a predicted molecular mass of 50.5 kDa (Fig. 1B). For comparison, the two major pilins of *S. sanguinis* T4P, PilE1 and PilE2 (16), have typical pilin sizes of 14.7 and 14.1 kDa, respectively (Fig. 1B). The larger size of PilB is due to the presence of a C-terminal domain (Fig. 1B) readily detectable by bioinformatics, which belongs to the von Willebrand factor A-like domain superfamily (InterPro entry IPR036465). We will refer to this domain as vWA. The prototypical vWA domain is found in the von Willebrand factor (vWF), a human blood protein required for haemostasis (19), the physiological process that prevents/stops bleeding. vWA domains, which are found in more than 300,000 proteins in the three domains of life, have been extensively studied in eukaryotic proteins where they mediate adhesion to a variety of protein ligands (18). They have been much less studied in bacteria. Of note, the vWA domain in PilB is predicted to contain a metal coordination site known as MIDAS, for *metal ion-dependent adhesion site* (20) (Fig. 1A), which is important for ligand-binding in several eukaryotic vWA-containing proteins (21).

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The above type IV pilin architecture is unusual for two reasons. First, in contrast to classical pilins that consist only of a pilin module (4), defined by a short N-terminal IPR012902 motif (Fig. 1B), PilB apparently has an additional module. Second, the extra C-terminal module in PilB corresponds to a well-defined functional domain not
126 specific to T4P biology, which has not been previously seen in pilins. Specifically, the
127 second module – vWA – is often associated with adhesion to protein ligands (18, 21).
128 This is what we call a modular architecture, and why we refer to PilB as a modular
129 pilin.

130 Taken together, these findings suggest that PilB is a modular pilin in which a
131 functional module has been grafted during evolution onto a pilin moiety, in order to
132 promote T4P-mediated adhesion of S. sanguinis to protein ligands.

134 **Crystal structure of PilB reveals a bimodular pilin in which a small type IV pilin**
135 **module is linked to a bulky vWA module via a short loop**

136 High-resolution structural information was required to confirm that PilB is composed of
137 two modules, but also to understand how modular pilins are polymerised in the
138 filaments and how they modulate T4P functionality. We therefore endeavoured to solve
139 the 3D structure of PilB by X-ray crystallography. To facilitate protein purification, we
140 used a synthetic gene codon-optimised for expression in *Escherichia coli*, and
141 produced a recombinant PilB protein in which the N-terminal 35 aa (encompassing the
142 leader peptide and hydrophobic α1N) (Fig. 1A) were replaced by a hexahistidine tag
143 (6His) (17). This is a commonly used approach in the field to promote protein solubility
144 since the truncation of α1N has minimal structural impact on the rest of the protein
145 (22). The resulting 48.4 kDa 6His-PilB protein was soluble and could be purified using
146 a combination of affinity and gel-filtration chromatography. The protein readily
147 crystallised in multiple conditions, and after optimising the best diffracting crystals, we
148 collected a complete dataset on crystals forming in the space group *P*6₁ (Table 1).
149 After phase determination, done using crystals produced in the presence of seleno-
150 methionine (SeMet), we solved a high-resolution structure (2.26 Å) of native 6His-PilB.
151 As can be seen in Fig. 2A, this structure reveals a clear bimodular architecture with a
152 small pilin moiety (highlighted in blue) linked, via a short nine-residue loop (grey), to a
153 bulky vWA moiety (in red).
While bioinformatics could only predict that the extreme N-terminus of PilB corresponds to a class III signal peptide motif, our structure reveals that the first 180 residues of processed PilB clearly display a type IV pilin fold (4) and thus indeed correspond to a pilin module (Fig. 2B). The pilin module exhibits a long N-terminal α-helix packed against, not one β-sheet as usual, but two consecutive β-sheets consisting of six and three β-strands respectively, which together form the globular head of the pilin. The 432918 topology of the first β-sheet, i.e., the order of the β-strands (Fig. 2B), is unusual since the β-strands are not contiguous along the protein sequence. Moreover, the last portion of this β-sheet forms a Ψ-loop (23) in which two antiparallel strands (β8 and β9) are linked via β1 in between, connected to both of them by hydrogen bonds. This motif occurs rarely in proteins (23).

As for the vWA module (Fig. 2C), the structure strengthens the bioinformatic predictions. The vWA moiety of PilB adopts a canonical vWA fold (20, 24), with a central β-sheet (composed of five parallel and one antiparallel β-strands) surrounded on both sides by a series of α-helices (Fig. 2C). Consequently, the vWA module of PilB shows high structural similarity to many vWA-containing proteins with which it shares little sequence identity. For example, the vWA module of PilB is very similar to the third vWA domain of human vWF (25) (Fig. S1), with a root mean square deviation (RMSD) of 1.72 Å when the Cα atoms of the two structures are superposed. As in eukaryotic vWA-containing proteins (20, 24), PilB exhibits a MIDAS located on top of the central β-sheet (Fig. 2C). However, in contrast to these proteins, the MIDAS in PilB is flanked by two protruding "arms", which is reminiscent of the RrgA adhesin from *Streptococcus pneumoniae* (26). While the first arm is perhaps unremarkable, the second folds into a four-stranded β-sheet (Fig. 2C). The MIDAS motif in PilB, which is formed by residues conserved in vWA-containing proteins, non-contiguous in the sequence (Fig. 1A) but in close proximity in the 3D structure (Fig. 2C), is functional since it coordinates a metal ion in the crystal. We have modelled the metal as Mg⁺² because of its abundance in the growth medium and the high affinity of PilB for it (see below). The Ser<sub>206</sub>, Ser<sub>208</sub>,
Thr<sub>291</sub> and Asp<sub>319</sub> residues in the MIDAS motif (20) of PilB form direct hydrogen bonds with the metal through oxygen atoms (Fig. 2C), while two additional coordination sites are provided by water molecules.

An important biological implication of the PilB structure is that modular pilins, despite their large size, are likely to be polymerised into T4P in the same way as classical pilins (4), i.e., via their N-terminal pilin module. We therefore tested by structural modelling whether PilB could pack into filaments. First, we produced a full-length 3D structural model of PilB including the missing α1N (Fig. S2), which was absent in the recombinant protein that we purified. Since a portion of α1N in major pilins is melted during filament assembly, as observed in several T4aP cryo-EM structures (9, 10), the α1N of PilB was modelled with a melted segment. This is consistent with the presence of the helix-breaking Gly residue in position 21 of α1N (Fig. 1A). Then, we fitted this full-length PilB into a previously generated model of S. sanguinis T4P, a right-handed helical heteropolymer where major pilins PilE1/PilE2 are held together by interactions between their α1N helices (Fig. 3A), which was based on the cryo-EM structure of Neisseria meningitidis T4P (9). Despite its unusual modular structure, PilB can be readily modelled into T4P, its pilin module establishing extensive hydrophobic interactions via its α1N with the α1N of neighbouring major pilins (Fig. 3A). This suggests that PilB will assemble into filaments in the same way as classical pilins (9, 10). However, PilB can only be accommodated at the tip of the filaments because the bulky vWA module sits on top of the pilin module in the PilB structure, and essentially prevents other pilin subunits from being modelled above it (Fig. 3B). Accordingly, when PilB is modelled in the body of the filament (Fig. S3A), it exhibits important steric clashes with neighbouring major pilins (Fig. S3B).

Together, these structural findings show that PilB is a bimodular protein composed of two clearly distinct structural modules. The pilin module adopts a canonical type IV pilin fold (4), explaining how modular pilins are polymerised into T4P, most probably at their tip. The second module, which is linked to the end of the pilin
module via a short loop, adopts a vWA fold (20, 24) with a clearly defined MIDAS that coordinates a metal. Since the vWA motif in many eukaryotic proteins is involved in adhesion to protein ligands (18, 21), our structure strengthens our working hypothesis that PilB might be an adhesin.

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## Functional analysis of the MIDAS in PilB reveals that metal binding, although structurally dispensable, is important for T4P functionality

Our PilB structure revealed that Mg²⁺, despite not being added during crystallisation, is bound by the MIDAS. In eukaryotic proteins, the MIDAS sometimes coordinates Mn²⁺ as well (20, 24). We therefore tested the metal binding specificity of the MIDAS in PilB using ThermoFluor. This fluorescent-based method, which measures changes in thermal denaturation temperature, is a commonly used approach for quantifying protein-ligand interactions (27). We determined the affinity of purified PilB for Mg²⁺, Mn²⁺ and Ca²⁺ (Fig. 4A). While no binding was detected to Ca²⁺, we found that PilB binds Mg²⁺ and Mn²⁺ efficiently, in the micromolar range, with estimated Kd of 70 and 54 µM, respectively. To confirm that metal binding involves the MIDAS motif, we produced PilB<sub>D319A</sub> in which the key MIDAS residue Asp<sub>319</sub> (Fig. 2C) was changed into an Ala by site-directed mutagenesis. Binding assays performed with PilB<sub>D319A</sub> showed that changing this single residue abolishes the metal-binding ability of PilB for both Mg²⁺ and Mn²⁺ (Fig. 4B). These findings show that the MIDAS in PilB is functional and preferentially binds Mg²⁺ and Mn²⁺.

Next, to determine whether metal presence/absence might impact the 3D structure of PilB, we solved the structure of PilB<sub>D319A</sub> by X-ray crystallography. The PilB<sub>D319A</sub> protein readily crystallised in the same condition as the wild-type (WT) protein. We collected a complete dataset on crystals diffracting to a resolution of 3 Å (Table 1) and solved the structure of PilB<sub>D319A</sub> (Fig. 5A) by molecular replacement. The structure of PilB<sub>D319A</sub> (Fig. 5A), solved by molecular replacement, clearly shows that no metal is occupying the mutated MIDAS pocket on top of the central β-sheet (Fig. 5B), which is

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consistent with metal-binding assays. Although PilB<sub>D319A</sub> resolution is significantly lower than WT, it nevertheless allows for meaningful structural comparison. When the vWA modules were compared, we found that they are essentially identical, the C<sub>α</sub> atoms superposing onto each other (Fig. 5C) with an RMSD of merely 0.45 Å, including the two arms flanking the MIDAS pocket. This shows that metal-binding by MIDAS has no detectable structural impact on PilB.

Next, we explored whether MIDAS-mediated metal-binding by PilB is important for piliation and/or T4P-powered twitching motility, both of which were previously shown to be abolished in a Δ*pilB* mutant (16). We therefore constructed an unmarked S. sanguinis mutant in which the endogenous *pilB* gene was altered by site-directed mutagenesis to produce PilB<sub>D319A</sub>. We first tested whether the *pilB*D319A mutant retains the ability to assemble T4P using filament purification (16). As can be seen in Fig. 6A, in which purified T4P were separated by SDS-PAGE and stained with Coomassie blue, the *pilB*D319A mutant is piliated. This is evidenced by the presence of the two bands corresponding to major pilins PilE1 and PilE2, which are absent in a non-piliated Δ*pilD* control (Fig. 6A). Moreover, the amount of T4P that can be purified from the *pilB*D319A mutant and WT strain appear comparable. We then tested whether the pili in the *pilB*D319A mutant are able to mediate twitching motility (16). For the WT strain, twitching motility is evidenced by spreading zones around bacteria grown on agar (Fig. 6B). Spreading zones were absent for the *pilB*D319A mutant (Fig. 6B). This shows that the MIDAS-mediated metal-binding ability of PilB, while dispensable for piliation, is important for T4P-mediated twitching motility.

Together, these findings show that the MIDAS in PilB is a functional metal-binding site, dispensable for piliation and protein folding, but essential for T4P functionality.

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T4P-mediated adhesion to eukaryotic cells requires PilB, which specifically
binds several human proteins

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Since vWA is involved in adhesion in many eukaryotic proteins (18, 21), our original hypothesis was that PilB might mediate S. sanguinis adhesion to host cells and/or proteins, which we aimed to test next. First, we determined whether T4P might be involved in its well-known ability of S. sanguinis to adhere to host cells (28). After testing a few eukaryotic cell lines, we opted for CHO cells because the WT strain adheres very efficiently to them. When CHO cells were infected by S. sanguinis at a multiplicity of infection (MOI) of 10, 31.6 ± 9.1 % of the bacterial inoculum adhered to the cells (Fig. 6C). In contrast, a non-piliated Δ*pilD* mutant showed a significantly reduced adhesion, with an 18-fold decrease relative to the WT (Fig. 6C). Next, we tested our original assumption that PilB might be an adhesin, by quantifying the adhesion of the *pilB*<sub>D319A</sub> mutant. As can be seen in Fig. 6C, although the *pilB*<sub>D319A</sub> mutant is piliated, its adhesion to CHO cells is dramatically impaired, with a 33-fold decrease when compared to the WT. These findings show that S. sanguinis T4P are multi-functional filaments important for adhesion to eukaryotic cells, and that PilB plays an important role.

Since the vWA domain in multiple eukaryotic proteins has been shown to mediate cell-extracellular matrix (ECM) interactions (21), we reasoned that PilB might recognise similar ligands because it exhibits a canonical vWA module (Fig. S1). We tested this hypothesis by performing binding assays with purified PilB using enzyme-linked immunosorbent assay (ELISA). In brief, we coated 96-well plates with selected putative ligands, added serial dilutions of purified 6His-PilB, and detected binding using an anti-6His antibody. We tested binding to fibrinogen and the ECM proteins fibronectin, elastin and laminin. While PilB exhibits no binding to BSA that was used as a negative control (Fig. 7A), we observed dose-dependent binding to fibronectin and fibrinogen, but not to the other ECM proteins that were tested (elastin and laminin).

Specific binding to fibronectin and fibrinogen was in the high nanomolar range, with calculated Kd of 865 and 494 nM, respectively (Fig. 7A). Under these *in vitro* experimental conditions, metal-coordination by the MIDAS is dispensable for binding
294 to fibronectin and fibrinogen since PilB<sub>D319A</sub> binds these ligands as well as PilB (Fig. 295 S4). Finally, to confirm the prediction that binding of PilB to the above ligands is 296 mediated by its vWA module, we produced and purified PilB<sub>vWA</sub> corresponding only to 297 the vWA module (see Fig. 1A). We found that PilB<sub>vWA</sub> binds to fibronectin and 298 fibrinogen (Fig. 7B), with calculated K<sub>d</sub> of 337 and 997 nM, respectively, which were 299 comparable to PilB. These findings confirm that the adhesive ability of PilB is due to 300 its vWA module.

301 Taken together, these findings show that *S. sanguinis* T4P are multi-functional 302 filaments mediating adhesion to eukaryotic cells, and that PilB is a *bona fide* adhesin 303 using its vWA module to bind several human protein ligands it shares with eukaryotic 304 vWA-containing proteins.

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**Pilins with modular architectures are widespread in bacteria**

307 PilB orthologs are ubiquitous in S. sanguinis, which also produces a second modular 308 pilin PilC (17), where the extra module belongs to the concanavalin A-like 309 lectin/glucanase domain superfamily (IPR013320) (Fig. 1B). We wondered how 310 widespread and diverse modular pilins might be. We therefore searched the InterPro 311 database (29) for all the proteins with an N-terminal IPR012902 domain, which also 312 contain an extra domain not specific to T4P biology. This showed that modular pilins 313 are (i) widespread with more than 1,200 proteins displaying such architecture 314 (Supplementary dataset 1), (ii) present both in monoderm and diderm species, and (iii) 315 highly diverse, with as many as 264 different architectures detected. Although a 316 bimodular architecture is the most prevalent, there are modular pilins with multiple 317 additional domains, the most extreme case being an 860-residue protein from 318 Candidatus Falkowbacteria, with 12 copies of the IPR013211 motif of unknown 319 function (Supplementary dataset 1). A closer inspection of the 15 most frequent 320 modular pilin architectures offers a glimpse of their diversity (Fig. 8). While in many of 321 these proteins the extra domain has no clear function (IPR007001, IPR011871,
322 IPR026906, PF05345, IPR006860, IPR003961, IPR021556), for others a function can
323 be predicted. These functions include (i) binding to carbohydrates via PF13385 (that
324 overlaps with the IPR013320 lectin domain superfamily), PF13620 (carbohydrate-
325 binding-like fold), or IPR011658 (PA14 carbohydrate-binding domain), (ii) binding to
326 proteins via IPR002035 (that overlaps with the IPR036465 vWA domain superfamily),
327 or even (iii) peptidase activity via IPR030392. These findings suggest that the rather
328 simple modular design strategy – where a functional module is grafted during evolution
329 onto a pilin – appears to have been used often during evolution both by monoderm
330 and diderm bacteria and is expected to increase the functional versatility of T4P.
331 **Discussion**

332 T4F are an important research topic because of their virtual ubiquity in prokaryotes
333 and their ability to mediate several key biological processes (1). Furthermore, the
334 molecular mechanisms of T4F-mediated functions and the exact role of minor pilins
335 remain incompletely understood. Therefore, in this report we focused on T4aP – the
336 prototypical T4F (1) – in the recently established monoderm model S. *sanguinis* (15),
337 and performed a structure/function analysis of the unusual minor pilin PilB, which we
338 predicted might play a role in T4P-mediated adhesion. This led to several notable
339 findings discussed below, and confirmed predictions that the study of T4P in
340 monoderms has the potential to shine new light on these filaments (14, 15).

341 The first important finding in this study is that modular type IV pilins – T4F
342 subunits in which an N-terminal pilin module is fused via a short linker to a functional
343 module with a direct role in a T4F-mediated function – are widespread and extremely
344 diverse. Modular pilins are likely to be tip-exposed in the filaments because of their
345 peculiar architecture. However, a location in the body of the filament cannot be
346 excluded provided that the linker is flexible enough. While previous 3D structures of a
347 few large minor pilins suggest that they are modular pilins, their second modules do
348 not correspond to protein domains readily identifiable by available bioinformatic tools.
349 For example, in CofB from enterotoxigenic *E. coli* (ETEC) T4bP, there are two
350 additional structural domains linked to the C-terminus of the pilin module by a flexible
351 linker, a β-repeat domain followed by a β-sandwich domain (30). CofB, which forms a
352 trimer predicted to be exposed at the tip of ETEC T4bP (31), appears to be an adapter
353 for a secreted protein CofJ (32) that has a direct role in adhesion. TcpB, from *Vibrio
354 cholerae* T4bP toxin-coregulated pilus, is a tip-located minor pilin forming trimers with
355 a structure very similar to CofB (33), which probably has a similar function. Incidentally,
356 CofB is also the receptor for the bacteriophage CTXφ (33). In ComZ from *Thermus
357 thermophilus* T4aP, the additional structural domain is a large β-solenoid inserted not
358 at the end of the pilin module but into the β-sheet (34), which is thought to mediate
binding of extracellular DNA during transformation (34). This modular architecture is not restricted to T4P as it is also observed for a minor pilin from another T4F, GspK from type II secretion systems (T2SS) (35). In GspK, the additional structural domain is an α-domain of unclear function inserted into the β-sheet of the pilin module. GspK has been proposed to be at the tip of T2SS pseudopili, together with two other non-modular minor pilins (Gspl and GspJ) with which it interacts to form a heterotrimer (35). These examples suggest that we have probably underestimated the global distribution of modular pilins, which are likely to be much more widespread because in many of them the additional modules are not yet defined by protein signatures in the databases. However, what is clear from our global analysis is that the functions associated with these modular pilins are potentially extremely diverse. Although a "common theme" appears to be the interaction of T4F with a variety of ligands – including proteins (via vWA in PilB, and the β-repeat/β-sandwich module in CofB), carbohydrates (via a variety of lectin domains including the concanavalin A-like lectin/glucanase domain in PilC), or DNA (the role of the β-solenoid module in ComZ) – other previously unreported T4F functions are possible. This is suggested by the modular architectures IPR012902-IPR030392 or IPR012902-IPR011493 in which the second module is a predicted peptidase belonging to S74 and M26 families, respectively.

The functional characterisation of the vWA module in PilB including its MIDAS, showing that it is a bona fide adhesin, is another significant achievement of this study. First, the vWA domain, which is ubiquitous in the three domains of life and has been extensively studied in eukaryotes (18, 21), has been much less studied in bacteria. Second, T4P-mediated adhesion remains among the least understood T4P functions (1). Our functional analysis of the vWA module in PilB significantly extends what was known for prokaryotic vWA-containing proteins and highlights important similarities and differences with eukaryotic vWA-containing proteins. Our 3D structure shows that the vWA module in PilB exhibits striking similarity to the vWA domain in eukaryotic proteins (20, 24), with a canonical MIDAS coordinating a metal. The main difference is
that the MIDAS in PilB is flanked by two protruding arms, similarly to what has been described for RrgA from S. pneumoniae (26). Interestingly, RrgA is a subunit with intrinsic adhesive properties (36) of sortase-assembled pili in monoderms (37), which are unrelated to T4P. The parallel between RrgA and PilB denotes a case of convergent evolution in which two unrelated types of pili have evolved a similar strategy to mediate adhesion. Testing metal binding by the MIDAS in PilB, which was previously done only for eukaryotic vWA-containing proteins (38), highlights important similarities. MIDAS shows no significant binding to Ca²⁺, and a slight preference for Mn²⁺ over Mg²⁺, although the difference in affinity is much smaller than in eukaryotic proteins (38). Metal binding can be abolished by altering the MIDAS motif, which has no impact on PilB structure (38, 39). Abolishing metal binding has no detectable effect on piliation, which is analogous to what has been reported for vWA-containing adhesins of sortase-assembled pili (40, 41), but it impairs T4P-mediated twitching motility. It is unclear at this stage whether the lack of motility of the *pilB*<sub>D319A</sub> mutant is due to reduced T4P-mediated adhesion to the agar, which would be consistent with PilB role in adhesion, or to impaired filament retraction (11). We also provide evidence that the vWA module of PilB binds several human protein ligands that it shares with eukaryotic vWA-containing proteins such as integrins and/or vWF (19, 21). However, unlike in these proteins where binding is often impaired when the MIDAS is inactivated (20), binding to fibronectin and fibrinogen is unaffected in a *PilB*<sub>D319A</sub> mutant. This either suggests that the MIDAS is not implicated in binding these specific ligands, which has been described for vWF binding to collagen (25), or that our *in vitro* binding assay is not sensitive enough to detect subtle but significant differences in binding.

The finding that PilB plays a key role in adhesion of S. sanguinis to host cells and structures, via its vWA module, has implications for the pathogenesis of this species in particular, and for our understanding of T4P-mediated adhesion in general. Our findings are consistent with the possibility that PilB-mediated adhesion to host proteins might play a role in IE (42), a life-threatening infection often caused by S.
sanguinis. Indeed, during IE, bacteria that have gained access to the bloodstream adhere to pre-existing sites of valvular damage where ECM proteins are exposed, and a blood clot is present containing large amounts of platelets, fibrinogen/fibrin and fibronectin (43). Our finding that PilB adheres directly to two of these proteins, but additional ligands cannot be excluded, suggests that PilB might be important at this early stage in IE, which could be tested in future studies. Our findings, which arguably make PilB the best characterised T4P adhesin alongside PilC/PilY1 found in diderm T4aP (44-48), have general implications for our understanding of T4P-mediated adhesion. The vWA module in PilB, which is most likely exposed at the pilus tip, is ideally placed to maximise bacterial adhesion to host protein receptors. T4P spring-like properties – gonococcal T4P can be stretched 3 times their length (49) – are expected to help bacteria that are bound via a tip-located adhesin to withstand adverse forces in their particular environment, e.g., blood flow in a heart valve. This is likely to apply to other modular pilins as well, which harbour different modules predicted to function in adhesion. The parallel with the best characterised T4P adhesin PilC/PilY1 is obvious. This protein, which is not a pilin, is an adhesin that has been proposed to be presented at the T4P tip (45) via its interaction with a tip-located complex of four widely conserved minor pilins (50). All PilC/PilY1 have in common a C-terminal IPR008707 β-propeller domain while their N-termini are different (51). Since this is analogous to the situation with modular pilins, we wondered whether it could be an indication of a modular design for PilC/PilY1. This indeed seems to be the case since a search of the InterPro database (29) for all the proteins with an IPR008707 domain shows that 68 different PilC/PilY1 modular architectures are detected (Supplementary dataset 2). Strikingly, many of the N-terminal modules in PilC/PilY1 are shared with modular pilins, including vWA that was identified in PilY1 from *Pseudomonas aeruginosa* (52). These observations suggest that the same tinkering strategy has been used both by pilins and PilC/PilY1 to increase the functional versatility of T4P. In both instances, a "carrier" module for presentation at the tip of the filaments (either a
pilin, or an IPR008707 domain that interacts with a tip-located complex of minor pilins)
443

has been fused to variety of "effector" modules, directly involved in a variety of
444

functions.
445

In conclusion, by performing a detailed structure/function of the minor pilin PilB
446
447 from S. sanguinis, we have shed light on several aspects of T4P biology. Our findings
448
449 are not only of relevance for S. sanguinis, most notably for colonisation of its human
520
450 host, they have general implications for T4F by uncovering a prevalent strategy used
451
452 by these widespread filamentous nanomachines to promote their well-known
453 way for further investigations, which will further improve our understanding of these
fascinating filaments.
453
454 **Materials and methods**

455

456 **Strains and growth conditions**

457 Strains and plasmids used in this study are listed in Table S1. For cloning, we used *E. coli* DH5α. For protein purification we used *E. coli* BL21(DE3) or *E. coli* BL21 B834(DE3) (Supplementary appendix). Chemically competent *E. coli* cells were prepared as described (53). DNA manipulations were done using standard molecular biology techniques (54). PCR were done using high-fidelity DNA polymerases (Agilent). Primers used in this study are listed in Table S2. The pET-28b (Novagen) derivative, pET28-*pilB*<sub>36-461</sub> for expressing 6His-*PilB*<sub>36-461</sub> was described previously (17). In this plasmid, the portion of a synthetic *pilB* gene codon-optimised for expression in *E. coli*, encoding the soluble portion of *PilB*, was fused to a non-cleavable N-terminal 6His tag. Similarly, we constructed pET28-*pilB*<sub>192-461</sub> for expressing *PilB*<sub>vWA</sub>. To construct pET28-*pilB*<sub>D319A</sub> for expressing 6His-*PilB*<sub>D319A</sub>, we introduced a missense mutation in pET28-*pilB*<sub>36-461</sub> using QuickChange site-directed mutagenesis (Agilent).

469 The WT S. *sanguinis* 2908 strain and deletion mutants (Δ*pilD*, Δ*pilB*) were described previously (16). S. *sanguinis* genomic DNA was prepared from overnight (O/N) liquid cultures using the XIT Genomic DNA from Gram-Positive Bacteria kit (G-Biosciences). Strain 2908, which is naturally competent, was transformed as described (16, 55). The unmarked S. *sanguinis* *pilB*<sub>D319A</sub> mutant was constructed using a previously described two-step gene editing strategy (55) (Supplementary appendix).

475

476 **Protein purification**

477 To purify native *PilB*, *PilB*<sub>D319A</sub> and *PilB*<sub>vWA</sub> proteins, the corresponding pET-28b derivatives were transformed in *E. coli* BL21(DE3). Expression and purification are detailed in the Supplementary appendix. To purify SeMet-labelled *PilB* for phasing, the corresponding pET-28b derivative was transformed in *E. coli* BL21 B834(DE3). Expression and purification are detailed in the Supplementary appendix.

478

479

480

481
482

483 **Crystallisation and structure determination**

484 Purified proteins in 50 mM HEPES pH 7.4, 200 mM NaCl were concentrated to 50
485 mg/ml and tested for crystallisation using sitting-drop vapor diffusion, with 100 nl drops
486 of protein solution and mother liquor. We tested a range of commercially available kits
487 (Molecular Dimensions, Hampton Research and Rigaku Reagents), which yielded a
488 number of hits, mainly in high salt conditions. Crystallisation conditions were optimised
489 to yield larger and better diffracting crystals. All data was collected and processed
490 using the Diamond Light Source beamline i03, and integrated in P6₁ using the 3dii
491 pipeline in xia2 (56). Initial molecular replacement was performed with Phaser (57) on
492 the 2.26 Å resolution PilB dataset using a low-resolution partial model produced from
493 the SeMet data using autoSHARP (58). Manual building in Coot (59) was performed
494 on the high-resolution dataset, and the full model was then used for molecular
495 replacement in the low-resolution datasets. All structures were produced using Coot
496 and phenix.refine (60) and validated using MolProbity (61).

497

498 **Assaying metal-binding by purified PilB**

499 The metal binding specificity of PilB was tested using ThermoFluor, a fluorescent-
500 based method measuring changes in thermal denaturation temperature (27). Assays
501 were done in a 96-well plate (Applied Biosystems) format. In the wells, we added to a
502 final volume of 40 µl (i) 0-1 mM range of concentrations of MgCl₂, MnCl₂ and CaCl₂,
503 (ii) 20 µM purified PilB or PilB<sub>D319A</sub>, and (iii) 1/5,000 dilution of SYBR Orange (Thermo
504 Fisher Scientific). Plates were then analysed using a temperature gradient, from 25 to
505 99°C, on a StepOnePlus real-time qPCR machine (Applied Biosystems). The data
506 were exported MATLAB and analysed in GraphPad. Analyses were performed with
507 Prism (GraphPad Software). Kd were calculated using non-linear regression fits,
508 applying saturation binding equation (One site - Total and non-specific binding) using
509 Ca²⁺ as non-specific binding control.
510

511 Assaying protein ligand-binding by purified PilB

512 Binding of PilB, PilB<sub>VWA</sub>, PilB<sub>D319A</sub> to a variety of eukaryotic proteins was tested by
513 ELISA as follows. Putative ligand proteins (elastin from human skin, fibrinogen from
514 human plasma, laminin from human placenta, fibronectin from human plasma) (all from
515 Sigma) were resuspended in carbonate-bicarbonate buffer (Sigma) at 5 µg/ml. Fifty µl
516 was dispatched into the wells of MaxiSorp plates, and adsorbed O/N at 4°C. Wells
517 were washed three times with PBS (Gibco) and blocked during 1 h with 3% BSA
518 (Probumin) or 1% gelatin (Sigma) in PBS. After washing with PBST (PBS containing
519 0.05% tween 20), serial twofold dilutions of PilB (from 40 to 0.625 µg/ml) were added
520 to the wells and incubated for 2 h at 37°C. After five washes with PBST, we added 50
521 µl anti-6His RTM antibody (Abcam) at 1/500 dilution in PBS, and incubated for 1 h at
522 RT. After five washes with PBST, we added 50 µl Amersham ECL anti-rabbit IgG HRP-
523 linked whole antibody (GE Healthcare) at 1/500 dilution in PBS, and incubated for 1 h
524 at RT. After five washes with PBST, we added 100 µl/well of TMB solution (Thermo
525 Scientific) and incubated the plates during 20 min at RT in the dark. Finally, we stopped
526 the reaction by adding 100 µl/well of 0.18 M sulfuric acid, before reading the plates at
527 450 nm using a plate reader. Analyses were performed with Prism (GraphPad
528 Software). Kd were calculated using non-linear regression fits, applying saturation
529 binding equation (One site - Total and non-specific binding) using BSA or gelatin as
530 non-specific binding control.

531

532 Assaying piliation of S. sanguinis

533 S. sanguinis T4P were purified as described (16, 17) (Supplementary appendix). Pili
534 were resuspended in pilus buffer, separated by SDS-PAGE, before gels were stained
535 with Bio-Safe Coomassie (Bio-Rad).

536

537 Assaying twitching motility of S. sanguinis
538 Twitching motility was assessed on agar plates as described (16) (Supplementary
539 appendix). Plates were photographed using an Epson Perfection V700 photo scanner.

540

541 Assaying adhesion of S. sanguinis to eukaryotic cells

542 We tested adhesion of S. sanguinis to CHO cells (Public Health England) as follows.
543 Cells were replicated in flasks in DMEM medium (Gibco) containing 1 x MEM non-
544 essential amino acid mix (Gibco) and 5 % fetal bovine serum (Gibco) and seeded at
545 100,000 cells/cm² in 24-well plates, which were incubated O/N at 37°C in the presence
546 of 5 % CO₂. The next day, cell monolayers were gently rinsed with PBS, and infected
547 at an MOI of 10 with bacteria grown in TH. In brief, bacteria were grown for a few hours
548 to OD₆₀₀ 0.5 units, adjusted at the same OD, pelleted by centrifugation at 1,100 g
549 during 10 min, and resuspended in PBS. Bacteria in the inoculum were quantified by
550 performing CFU counts on TH plates. After 1h of infection at 37°C, cell monolayers
551 were gently rinsed four times with PBS, before cells with adherent bacteria were
552 scraped in distilled water. Adherent bacteria were then quantified by performing CFU
counts. Statistical analyses were performed with Prism. Comparisons were done by
553 one-way ANOVA, followed by Dunnett's multiple comparison tests. An adjusted *P*
554 value < 0.05 was considered significant (* *P*<0.05, ** *P*<0.01, *** *P*<0.001, ****
555 *P*<0.0001).

557

558 Bioinformatics

559 Protein sequences were routinely analysed using DNA Strider (62). Prediction of
560 protein domains, their global distribution and associated architectures was done by
561 using InterProScan (29) to interrogate the InterPro database. This database was also
562 used to download all the protein entries discussed in this paper. Molecular visualisation
563 of protein 3D structures was done using PyMOL (Schrödinger). The PDBsum Generate
564 (63) server was used to provide at-a-glance overviews – secondary structure, topology
565 diagram, protein motifs, schematic diagram of metal-protein interactions – of the 3D
566 structures determined during this work. The DALI (64) server was used for comparing
567 protein structures in 3D. Protein 3D structures were downloaded from the RCSB PDB
568 server. The 3d-SS (65) server was used to superpose 3D protein structures with the
569 STAMP algorithm (66).

570 The cryo-EM structure of *N. meningitidis* T4P (PDB 5KUA) (9) was used to
571 model, using SWISS-MODEL (67), the N-terminal helices of PilE1, PilE2 and PilB
572 within the filaments. Coot and PyMOL were then used to place the full-length structures
573 within the T4P model.
574 **Data availability**

575 3D structures have been deposited in the PDB, under accession codes 7B7P (PilB),

576 and 7BA2 (PilB<sub>D319A</sub>). All the datasets generated during this study are included in this

577 paper and its Supplementary appendix.
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729 64. L. Holm, L. M. Laakso, Dali server update. *Nucleic Acids Res.* **44**, W351-355 (2016).

731 65. K. Sumathi, P. Ananthalakshmi, M. N. Roshan, K. Sekar, 3dSS: 3D structural superposition. *Nucleic Acids Res.* **34**, W128-W132 (2006).

733 66. R. B. Russell, G. J. Barton, Multiple protein sequence alignment from tertiary structure comparison: assignment of global and residue confidence levels. *Proteins* **14**, 309-323 (1992).

736 67. A. Waterhouse et al., SWISS-MODEL: homology modelling of protein structures and complexes. *Nucleic Acids Res.* **46**, W296-W303 (2018).
738 Acknowledgements

739 This work was funded by the Medical Research Council (MR/P022197/1). We
740 acknowledge the use of the crystallisation facility at Imperial College London, which is
741 supported by the Biotechnology and Biological Sciences Research Council
742 (BB/D524840/1) and Wellcome Trust (202926/Z/16/Z). We thank Claire Poyart (Institut
743 Cochin) for advice on adhesion experiments. We thank Angelika Gründling (Imperial
744 College London), Sophie Helaine (Harvard Medical School), Christoph Tang
745 (University of Oxford), and Romé Voulhoux (Laboratoire de Chimie Bactérienne) for
746 critical reading of the manuscript.
747 **Figure legends**

748

749 **Fig. 1. Bioinformatic analysis of PilB.** A) Relevant features of PilB from *S. sanguinis* 2908. The N-terminal class III signal peptide is boxed. The 7-aa long leader peptide contains mostly hydrophilic (shaded in orange) and neutral (no shading) residues, and it ends with a conserved Gly. This leader peptide is processed by the prepilin peptidase PilD, which is indicated by the vertical arrow, generating a protein of 454 residues (50.5 kDa). The processed protein starts with a tract of 21 predominantly hydrophobic residues (shaded in blue), which invariably form an extended α-helix that is the main assembly interface within filaments. The C-terminal vWA module (IPR036465) is boxed, with the conserved residues forming the MIDAS highlighted in yellow. Arrowheads indicate the proteins that were produced and purified in this study, consisting of either two modules (black arrowhead) or just the vWA module (red arrowhead). B) Modular architectures of PilB and PilC minor pilins compared to the major pilins PilE1/PilE2. The proteins, from *S. sanguinis* 2908, have been drawn to scale. The black rounded rectangles correspond to the IPR012902 motif that is part of the class III signal peptide. The C-terminal domains in PilB and PilC are highlighted by coloured rounded rectangles, vWA domain (red) and lectin domain (yellow).

765

766 **Fig. 2. Crystal structure of PilB.** A) Orthogonal cartoon views of the PilB structure in 767 which the two distinct modules have been highlighted in blue (pilin module) and red 768 (vWA module), while the short loop connecting them is in grey. The orange sphere 769 represents a magnesium ion. B) Left, close-up cartoon view of the pilin module 770 coloured in rainbow spectrum from blue (N-terminus) to red (C-terminus). Right, 771 topology diagram of the pilin module structure. C) Left, close-up cartoon view of the 772 vWA module in which the β-strands composing the central β-sheet are highlighted in 773 red, while the surrounding α-helices are highlighted in yellow. The connecting loops
774 are in grey, except for the two "arms" on top of the structure (coloured in orange and blue), which surround the MIDAS. Right, diagram of the magnesium coordination by the conserved MIDAS residues. Coordinating oxygen atoms are shown, with dashed lines corresponding to hydrogen bonds.
775
776
777

778
779 **Fig. 3.** 3D model of PilB in S. sanguinis T4P. **A)** Packing of PilB (red) into S. sanguinis T4P, which is a right-handed helical heteropolymer of two major pilins PilE1 (blue) and PilE2 (grey). **B)** View of the T4P tip capped by PilB, or not.
780
781
782

783
784
785
786
787
**Fig. 4.** Metal binding by PilB. Purified PilB was incubated with increasing concentrations of divalent ions ($Ca^{2+}$, $Mg^{2+}$, $Mn^{2+}$) and binding was quantified by ThermoFluor. **A)** Metal binding by PilB. **B)** Metal binding by $PilB_{D319A}$, with an inactive MIDAS. Results are the average ± standard deviations from 3 independent experiments.
788
789
790
791
792
793
794

795
796
797
798
799
**Fig. 5.** 3D crystal structure of $PilB_{D319A}$. **A)** Close-up cartoon view of the vWA module in $PilB_{D319A}$. **B)** Comparison of electron density maps in the MIDAS pocket for the $PilB_{D319A}$ (upper panel) and PilB (lower panel) structures. **C)** Superposition of the vWA modules of PilB with bound $Mg^{2+}$ (grey) and $PilB_{D319A}$ (orange). The two structures superpose with a RMSD of 0.45 Å.
800

795
796
797
798
799
800
**Fig. 6.** Phenotypic characterisation of a S. sanguinis mutant expressing $PilB_{D319A}$ with an inactive MIDAS. **A)** Piliation was quantified by purifying T4P from cultures adjusted to the same $OD_{600}$. Purified T4P (identical volumes were loaded in each lane) were separated by SDS-PAGE and stained with Coomassie blue. A molecular weight marker (MW) was run in the first lane. Molecular weights are indicated in kDa. **B)** Twitching motility was assessed by a macroscopic motility assay. Bacteria were
streaked on plates, which were incubated several days at 37°C in a humid atmosphere and then photographed. Twitching motility is characterised by spreading zones around colonies. C) Adhesion of *S. sanguinis* to eukaryotic cells was quantified by incubating bacteria (MOI 10) with CHO cells for 1 h. After removing non-adherent bacteria by several washes, bacteria adhering to cells were enumerated by performing CFU counts. Results are expressed as adhesion relative to WT (set to 1) and are the average ± standard deviations from five independent experiments. For statistical analysis, one-way ANOVA followed by Dunnett's multiple comparison tests were performed (****P < 0.0001).

**Fig. 7. Dose-dependent binding of PilB to various protein ligands.** Increasing concentrations of purified PilB was added to constant concentrations of immobilised putative ligands, and binding was quantified by ELISA. BSA served as negative control. Results are the average ± standard deviations from at least three independent experiments. A) Binding of PilB to fibrinogen, fibronectin, elastin and laminin. B) Binding of PilB<sub>vWA</sub>, consisting only of the vWA module, to fibrinogen and fibronectin.

**Fig. 8. Global distribution of modular pilins.** Fifteen most widespread modular pilin architectures in the InterPro database are presented. The numbers in parenthesis represent the number of proteins displaying that architecture. The representative proteins depicted, drawn to scale, are from the following species. 1, *Candidatus Magasanikbacteria* (UniProtKB/TrEMBL protein A0A0G0IU57). 2, *Photorhabdus luminescens* (A0A022PI42). 3, *Desulfuribacillus stibiiarsenatis* (A0A1E5L295). 4, *Candidatus Falkowbacteria* (A0A1J4TDE2). 5, *Candidatus Wolfebacteria* (A0A0G1WFE5). 6, *Clostridiales bacterium* (A0A101H8M7). 7, *Corynebacterium glutamicum* (A0A1Q6BQB1). 8, *Thermosulfidibacter takaii* (A0A0S3QUH2). 9, *Candidatus Gracilibacteria* (A0A1J5F7A7). 10, *Candidatus Saccharibacteria* (A0A1Q3NLQ9). 11, *Planctomycetes bacterium* (A0A1G2ZHU9). 12, *Actinoplanes*
829 *awajinensis* subsp. *mycoplanecinus* (A0A101J7V4). 13, *Actinoplanes derwentensis*

830 (A0A1H2D7E9). 14, Desulfobacterales bacterium (A0A1V1WSE4). 15, Parcubacteria

831 group bacterium (A0A2D6FLV5).
<figure><img src="image_2.png" /></figure>

GKATSIFILDDSKFKGSKQGLTREWSYIGLSADGKVKMNYVWNKQKQDWDVSELGTKSLYNMKLD

LEFKTEGAYQDNRLISYNLTGKYPDTNNKLGIDTAISALNTKQVFSKVAKGKKGIAIAYRTDIQ

GQMNAIVSFVFDTSGSMDWDLQGRNVKKTGNESRMIDLIRKKSVIMIKDLAEIGNSVNLVGFSTS

AKYIQQNFSNLDNGTNTIIATITKRENLNPDGVTNPGDGLRYGMISLQSQPAQLKYIVLLTGDIP

NAYLVDSRALYAGNRVDLSQGAGRVTFFNNPIYDLSPTLGYEYSRLGYDLYSRDSITRENSIAYAG

EVSKFGLGIGRKNVNVIGFSGVNHEIAYGQSLTDRIGEGGMETKYVSATNEEALQKTFSDIKKQIQ

QDLWFVSGP 461

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

IPR012902

IPR036465

IPR013320
<figure><img src="image_4.png" /><figcaption>B</figcaption></figure>

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

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

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

<figure><img src="image_8.png" /></figure>
<figure><img src="image_9.png" /><figcaption>B</figcaption></figure>

pilus tip without PilB

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

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

pilus tip with PilB
A

PilB

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

B

PilB<sub>D319A</sub>

<figure><img src="image_13.png" /></figure>
<figure><img src="image_14.png" /><figcaption>PiIB<sub>D319A</sub></figcaption></figure>

<figure><img src="image_15.png" /><figcaption>empty MIDAS (PiIB<sub>D319A</sub>)</figcaption></figure>

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

<figure><img src="image_17.png" /><figcaption>metal-bound MIDAS (PiIB)</figcaption></figure>
A

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

MW

WT

ΔpilD

pilB<sub>D319A</sub>

B

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

WT

ΔpilD

pilB<sub>D319A</sub>

C

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

PilB

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

B

PilB<sub>vWA</sub>

<figure><img src="image_22.png" /></figure>
<figure><img src="image_23.png" /></figure>
1 PilB from *Streptococcus sanguinis* is a bimodular type IV pilin with a direct

2 role in adhesion

3

4 Claire Raynaud<sup>a,1</sup>, Devon Sheppard<sup>a,1</sup>, Jamie-Lee Berry<sup>a</sup>, Ishwori Gurung<sup>a</sup>, Vladimir

5 Pelicic<sup>a,b,2</sup>

6

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

8 London SW7 2AZ, United Kingdom

9

10 <sup>b</sup>Laboratoire de Chimie Bactérienne, Aix-Marseille Université-CNRS (UMR7283),

11 Institut de Microbiologie de la Méditerranée, 13009 Marseille, France

12

13 <sup>1</sup>C.R. and D.S. contributed equally to this work

14

15 <sup>2</sup>To whom correspondence may be addressed. Email: vladimir.pelicic@inserm.fr
16 **Materials and methods**

17

18 **Strains and growth conditions**

19 *E. coli* was grown in liquid or solid Lysogenic Broth (LB) (Difco) containing, when required, 100 µg/ml spectinomycin or 50 µg/ml kanamycin (both from Sigma). For purification of protein labelled with seleno-methionine (SeMet), bacteria were grown in chemically defined medium (CDM) supplemented with 20 mg/ml SeMet (Sigma).

23 *S. sanguinis* was grown on plates containing Todd Hewitt (TH) broth (Difco) and 1 % agar (Difco), incubated at 37°C in anaerobic jars (Oxoid) under anaerobic conditions generated using Anaerogen sachets (Oxoid) (1). Liquid cultures were grown statically under aerobic conditions in THT, i.e., TH broth containing 0.05 % tween 80 (Merck) to limit bacterial clumping(1). When required, 500 µg/ml kanamycin was used for selection and 15 mM *p*-Cl-Phe (Sigma) for counterselection (2). To construct the unmarked *S. sanguinis* *pilB*<sub>D319A</sub> mutant, we replaced the gene in the WT by a promoterless *pheS*\* *aphA*-3 double cassette, which confers sensitivity to *p*-Cl-Phe and resistance to kanamycin (2). To do this, we fused by splicing PCR the upstream and downstream regions flanking *pilB* to *pheS*\* *aphA*-3, directly transformed the PCR product into the WT, and selected allelic exchange mutants on kanamycin plates. Allelic exchange was confirmed by PCR. In the second step, we replaced the *pheS*\* *aphA*-3 double cassette in this primary mutant by allelic exchange, with an unmarked *pilB*<sub>D319A</sub> mutation. To do this, we first constructed the missense mutation by site-directed mutagenesis, using as a template a pCR8/GW/TOPO (Invitrogen) derivative in which the WT gene was cloned. Then, the PCR product was directly transformed into the primary mutant, with plating on *p*-Cl-Phe-containing plates. Markerless allelic exchange mutants, which are the only one sensitive to kanamycin, were identified by re-streaking colonies on TH plates with and without antibiotic.

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42
43 Protein purification

44 To purify native PilB, PilB<sub>D319A</sub> and PilB<sub>VWA</sub> proteins, the corresponding pET-28b
45 derivatives were transformed in *E. coli* BL21(DE3). Transformants were grown O/N at
46 37°C in liquid LB with kanamycin. The next day, this culture was diluted (1/500) in 1 l
47 of the same medium and grown at 37°C to an OD<sub>600</sub> of 0.4-0.6. Proteins were purified
48 as described (3). The temperature was then set to 16°C, the culture allowed to cool
49 for 30 min, before protein expression was induced O/N by adding 0.5 mM IPTG
50 (Merck). The next day, cells were harvested by centrifugation at 8,000 g for 20 min
51 and subjected to one cycle of -80°C freeze/thaw in binding buffer (50 mM HEPES pH
52 7.4, 200 mM NaCl, 15 mM imidazole), to which we added SIGMAFAST EDTA-free
53 protease inhibitor cocktail (Sigma). Cells were disrupted by repeated cycles of
54 sonication, i.e., pulses of 5 sec on and 5 sec off during 3-5 min, until the cell
55 suspension was visibly less viscous. The cell lysate was then centrifuged for 30 min
56 at 17,000 g to remove cell debris. The clarified lysate was first affinity-purified on an
57 ÄKTA Purifier using His-Trap HP columns (GE Healthcare) and eluted with elution
58 buffer (50 mM HEPES pH 7.4, 200 mM NaCl, 300 mM imidazole). Affinity-purified
59 proteins were further purified, and simultaneously buffer-exchanged into (50 mM
60 HEPES pH 7.4, 200 mM NaCl), by gel-filtration chromatography on an ÄKTA Purifier
61 using a Superdex 75 10/300 GL column (GE Healthcare). Protein concentration was
62 quantified spectrophotometrically using a NanoDrop Lite (Thermo Fisher Scientific).

63 To purify SeMet-labelled PilB for phasing, the corresponding pET-28b
64 derivative was transformed in *E. coli* BL21 B834(DE3). Transformants were grown at
65 37°C in liquid LB with kanamycin, until OD<sub>600</sub> reached 0.6-0.7. Next, the cells were
66 pelleted at 8,000 g for 5 min, and washed twice with 2 ml of CDM, which contains no
67 Met. The pellet was then washed with 2 ml of CDM supplemented with 20 mg/ml L-
68 Met (Sigma) and used to inoculate, at 1/200 dilution, 20 ml of CDM supplemented
69 with 20 mg/ml Met. This culture was grown at 37°C for 16-18 h. Cells were pelleted
70 and washed three times with CDM. Then, the pellet was re-suspended in 20 ml of
71 CDM supplemented with 20 mg/ml SeMet, which was used to inoculate 1 l of CDM
72 supplemented with SeMet. Cells were grown at 37°C until OD₆₀₀ reached 0.5-0.7.
73 The temperature was then set to 16°C, the culture allowed to cool for 30 min, before
74 protein expression was induced O/N by adding 1 mM IPTG (Merck) and 4 ml of 36 %
75 glucose (w/v). Two and half hours later, we again added 4 ml of 36 % glucose to the
culture. The next day, SeMet-labelled PilB was purified as above.
76
77

78 **Crystallisation and structure determination**

79 The PilB crystals used for the high-resolution structure determination were obtained
80 when the purified protein was mixed 1:1 with crystallisation liquor (0.1 M Bis-tris
81 propane pH 7, 3 M NaCl). The lower resolution PilB<sub>D319A</sub> crystals were obtained with
82 crystallisation liquor (0.1 M Bis-tris pH 6.5, 3 M NaCl). Crystals were cryoprotected
83 with 30 % glycerol in crystallisation liquor, and flash-frozen in liquid nitrogen.
84

85 **Assaying piliation of S. sanguinis**

86 To purify S. sanguinis T4P (1, 3), bacteria grown in 100 ml THT until the OD₆₀₀
87 reached 1-1.5, at which point OD were normalised, bacteria pelleted and re-
88 suspended in pilus buffer (20 mM Tris, pH 7.5, 50 mM NaCl). This suspension was
89 vortexed for 2 min at full speed to shear T4P. After removing bacterial cells by two
90 centrifugation steps and filtration through a 0.22 µm pore size syringe filter
91 (Millipore), pili were pelleted by ultracentrifugation and resuspended in pilus buffer.
92

93 **Assaying twitching motility of S. sanguinis**

94 To assess twitching motility on agar plates (1), bacteria grown O/N were streaked as
straight lines on freshly poured TH plates containing 1 % Eiken agar (Eiken
95 Chemicals). Plates were grown for several days at 37°C in anaerobic condition under
96 high humidity, which is necessary for twitching.
97
98 **References**

99 1. I. Gurung *et al.*, Functional analysis of an unusual type IV pilus in the Gram-positive *Streptococcus sanguinis*. *Mol. Microbiol.* **99**, 380-392 (2016).

101 2. I. Gurung, J. L. Berry, A. M. J. Hall, V. Pelicic, Cloning-independent markerless gene editing in *Streptococcus sanguinis*: novel insights in type IV pilus biology. *Nucleic Acids Res.* **45**, e40 (2017).

104 3. 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).

107 4. R. A. Romijn *et al.*, Identification of the collagen-binding site of the von Willebrand factor A3-domain. *J. Biol. Chem.* **276**, 9985-9991 (2001).

109 5. S. Kolappan *et al.*, Structure of the *Neisseria meningitidis* type IV pilus. *Nat. Commun.* **7**, 13015 (2016).

111 6. P. Jones *et al.*, InterProScan 5: genome-scale protein function classification. *Bioinformatics* **30**, 1236-1240 (2014).
113
114

<table><caption>Table S1. Strains and plasmids used in this study.</caption><thead><tr><th>Name</th><th>Details</th><th>Source</th></tr></thead><tbody><tr><td colspan="3"><strong>E. coli</strong> strains</td></tr><tr><td>DH5α</td><td>used for cloning</td><td></td></tr><tr><td>BL21(DE3)</td><td>used for protein expression/purification</td><td></td></tr><tr><td>BL21 B834(DE3)</td><td>used for SeMet protein expression/purification</td><td></td></tr><tr><td colspan="3"><strong>S. sanguinis</strong> strains</td></tr><tr><td>2908</td><td>sequenced WT isolate</td><td>(1)</td></tr><tr><td>ΔpilB</td><td>ΔpilB::aphA-3 deletion mutant</td><td>(1)</td></tr><tr><td>ΔpilD</td><td>ΔpilD::aphA-3 deletion mutant</td><td>(1)</td></tr><tr><td>ΔpilB primary mutant</td><td>ΔpilB::pheS*aphA-3 deletion mutant</td><td>this study</td></tr><tr><td>pilB<sub>D319A</sub></td><td>pilB point mutant expressing PilB<sub>D319A</sub></td><td>this study</td></tr><tr><td colspan="3"><strong>Plasmids</strong></td></tr><tr><td>pCR8/GW/TOPO</td><td>TA cloning vector</td><td>Invitrogen</td></tr><tr><td>TOPO-pheS*aphA-3</td><td>pheS*aphA-3 double cassette in TOPO</td><td>(2)</td></tr><tr><td>TOPO-pilB</td><td>pilB in TOPO</td><td>this study</td></tr><tr><td>TOPO-pilB<sub>D319A</sub></td><td>pilB<sub>D319A</sub> in TOPO</td><td>this study</td></tr><tr><td>pET-28b</td><td>T7-based expression vector</td><td>Novagen</td></tr><tr><td>pMK-pilB</td><td>codon-optimised synthetic pilB in pMK</td><td>(3)</td></tr><tr><td>pET28-pilB</td><td>pET-28 derivative for expressing 6His-PilB<sub>36-461</sub></td><td>(3)</td></tr><tr><td>pET28-pilB<sub>D319A</sub></td><td>pET-28 derivative for expressing 6His-PilB<sub>D319A</sub></td><td>this study</td></tr><tr><td>pET28-pilB<sub>VWA</sub></td><td>pET-2b derivative for expressing 6His-PilB<sub>192-461</sub></td><td>this study</td></tr></tbody></table>

115
116

pilB, codon-optimised synthetic gene. pheS*, point mutant encoding PheS<sub>A316G</sub>.
<table><caption>Table S2. Primers used in this study.</caption><thead><tr><th>Name</th><th>Sequence</th></tr></thead><tbody><tr><td>pilB<sub>D319A</sub> #1</td><td>GAAATATATCGTTCTGCTGACCGcTGGTATTCCGAATGCATATCTGG</td></tr><tr><td>pilB<sub>D319A</sub> #2</td><td>CCAGATATGCATTCGGAATACCAgCGGTCAGCAGAACGATATATTTC</td></tr><tr><td>pilB<sub>vWA</sub>-pET-F</td><td>gggccatggatcatcatcatcatcatCAGGGCCAGATGAATATTGC</td></tr><tr><td>pilB<sub>vWA</sub>-pET-R</td><td>cccggatccTTACGGACCGCTAACAAACC</td></tr><tr><td>pilB-F</td><td>TACAACTGGACCGAAGCTGG</td></tr><tr><td>pilB-R</td><td>TTTGGCCTATCGTTCCCACT</td></tr><tr><td>pilB-F1</td><td>TACAACTGGACCGAAGCTGG</td></tr><tr><td>pilB-R1</td><td>gttcttcaatcgttttcgtcatcaTTCCTACCTATTTATTTTTACTTCTG</td></tr><tr><td>pilB-F2</td><td>ttttactggatgaattgttttagAGGATTTGTGGTTTGTATCAGGG</td></tr><tr><td>pilB-R2</td><td>TTTGGCCTATCGTTCCCACT</td></tr><tr><td>pheS*-F</td><td>ATGACGAAAACGATTGAAGAAC</td></tr><tr><td>aph-R</td><td>CTAAAACAATTCATCCAGTAAAA</td></tr><tr><td>pilB<sub>D319A</sub> #1</td><td>GCTTAAATATATAGTTCTATTGACAGcTGGCATACCTAATGCTTATTTAGTAG</td></tr><tr><td>pilB<sub>D319A</sub> #2</td><td>CTACTAAATAAGCATTAGGTATGCCAgCTGTCAATAGAACTATATATTTAAGC</td></tr></tbody></table>

119

120 *pilB*, codon-optimised synthetic gene. Overhangs are in lower case. Restriction sites

121 are in bold. Mismatches are in red.
<figure><img src="image_24.png" /><figcaption>**Fig. S1. Structural similarity of vWA domains in PilB and human vWF.** Left, A3 vWA domain in vWF (from PDB 1FE8) (grey) (4). Center, vWA module of PilB (purple). Right, superposition of the two structures. While the two sequences share only 15.6 % sequence identity, the structures superpose with an RMSD of 1.72 Å.</figcaption></figure>
<figure><img src="image_25.png" /></figure>

127

128

129

**Fig. S2.** 3D model of full-length PilB with a melted α1N segment. The cryo-EM structure of the *N. meningitidis* T4P (PDB 5KUA) (5) has been used as a template.
A

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

B

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

130

131

Fig. S3. Modelling of PilB in the body of a T4P leads to important steric clashes. A) Packing of PilB (red) into S. sanguinis T4P composed of PilE1 (blue) and PilE2 (grey). B) Close-up view of the important steric clashes between PilB and PilE subunit above in the filament.

132

133

134
<figure><img src="image_28.png" /></figure>

135

136

137

138

139

140

**Fig. S4. Binding of PilB<sub>D319A</sub> to fibrinogen and fibronectin.** Increasing concentrations of purified PilB were added to constant concentration of immobilised ligands, and binding was quantified by ELISA. Results are represented as Kd relative to WT, which is set to 1. Results are the average ± standard deviations from 3-4 independent experiments.
141 Supplementary datasets legends

142

143 Supplementary dataset 1. List of all the pilin architectures in the InterPro
144 database. This list was generated by searching the database (November 2020) for
145 entries displaying an IPR012902 pilin motif (6). Modular pilins display an N-terminal
146 pilin motif together with additional module(s) not specific to T4P biology. Proteins in
147 which the pilin motif is not N-terminal are listed as unclear.

148

149 Supplementary dataset 2. List of all the PiIC/PiIY1 architectures in the InterPro
150 database. This list was generated by searching the database (November 2020) for
151 entries displaying the IPR008707 PiIC/PiIY1 β-propeller domain (6). Modular
152 PiIC/PiIY1 display a C-terminal IPR008707 motif together with additional module(s)
153 not specific to T4P biology. Proteins in which the IPR008707 motif is not C-terminal
154 are listed as unclear.