Régine Dazzoni

Yuanyuan Li

Aracelys López-Castilla

Sébastien Brier

Ariel Mechaly

Florence Cordier

Ahmed Haouz

Michael Nilges

Olivera Francetic

Benjamin Bardiaux
email: benjamin.bardiaux@pasteur.fr

Nadia Izadi-Pruneyre

Structure and dynamic association of an assembly platform subcomplex of the bacterial type II secretion system

Keywords: Type II Secretion System, Assembly platform, Type IV pili, protein-protein interaction, Ferredoxin-like fold

Type II secretion systems (T2SS) allow diderm bacteria to secrete hydrolytic enzymes, adhesins or toxins important for growth and virulence. To promote secretion of folded proteins, T2SSs assemble periplasmic filaments called pseudopili or endopili at an inner membrane subcomplex or the assembly platform (AP). Here, we combined biophysical approaches, NMR and X-ray crystallography to study the Klebsiella AP components PulL and PulM. We determined the structure and associations of their periplasmic domains and describe the first structure of the heterodimer formed by their ferredoxin-like domains. We show how structural complementarity and plasticity favor their association during the secretion process. Cysteine scanning and cross-linking data provided additional constraints to build a structural model of the PulL-PulM assembly in the cellular context. Our structural and functional insights, together with the relative cellular abundance of its components, support the role of AP as a dynamic hub that orchestrates pilus polymerization.

Introduction

Gram-negative bacteria have developed multiple protein secretion systems that are important for their survival and pathogenesis [START_REF] Maffei | Tracking Proteins Secreted by Bacteria: What's in the Toolbox[END_REF]. Among these, the type II secretion system (T2SS), discovered in the late 1980s (d'Enfert et al., 1987), is one of the most widespread and at the same time most relevant, from biomedical and environmental standpoints. It allows the bacterium to secrete fully folded proteins with a wide range of functions -toxins, adhesins, cytochromes and hydrolytic enzymes [START_REF] Cianciotto | Expanding Role of Type II Secretion in Bacterial Pathogenesis and Beyond[END_REF].

The T2SS is a transmembrane nano-machine composed of 12 to 14 proteins designated here by the Gsp (General secretory pathway) nomenclature [START_REF] Pugsley | The complete general secretory pathway in gram-negative bacteria[END_REF]. It is organized into four sub-complexes (reviewed in [START_REF] Naskar | The structure and mechanism of the bacterial type II secretion system[END_REF]): (1) an outer membrane secretin channel (GspD); (2) a periplasmic filament called endopilus (previously called pseudopilus) composed of a non-covalent polymer of major pilin GspG and minor pilins GspH, I, J, K;

(3) an assembly platform (AP) composed of GspL, M, C and F [START_REF] Py | An inner membrane platform in the type II secretion machinery of Gram-negative bacteria[END_REF] ; and (4) a cytosolic ATPase GspE forming a hexamer. The T2SS has a common evolutionary origin [START_REF] Denise | Diversification of the type IV filament superfamily into machines for adhesion, protein secretion, DNA uptake, and motility[END_REF][START_REF] Hobbs | Common components in the assembly of type 4 fimbriae, DNA transfer systems, filamentous phage and protein-secretion apparatus: a general system for the formation of surface-associated protein complexes[END_REF][START_REF] Peabody | Type II protein secretion and its relationship to bacterial type IV pili and archaeal flagella[END_REF] with nanomachines involved in assembly of type IV pili (T4P) and archaeal flagella and pili and shares a similar architecture. Together they form the superfamily of type IV filament assembly systems [START_REF] Berry | Exceptionally widespread nanomachines composed of type IV pilins: the prokaryotic Swiss Army knives[END_REF]. Since the T2SS pili are not extracellular, they have been called pseudopilus. However, we prefer to call them endopili.

Although the general architecture of the T2SS has been extensively investigated, the biogenesis of this nanomachine and the secretion mechanism are still largely unknown. Current models propose that the substrate secretion is coupled with the polymerization of the endopilus in the periplasm, which is driven by the assembly platform (AP) complex by an unknown mechanism [START_REF] Naskar | The structure and mechanism of the bacterial type II secretion system[END_REF]. Information on the interaction mode of AP components and their structure is therefore essential to understand this key step of the secretion process.

GspL and GspM are the peripheral AP components that share a similar domain organization, suggesting their common evolutionary origin. Both are predicted to insert into the inner membrane via a single hydrophobic helix that is followed by a periplasmic a-helical region and a C-terminal globular domain. X-ray crystallography has provided structural information for the periplasmic domains of GspM [START_REF] Abendroth | The X-ray structure of the type II secretion system complex formed by the N-terminal domain of EpsE and the cytoplasmic domain of EpsL of Vibrio cholerae[END_REF] and GspL [START_REF] Abendroth | The dimer formed by the periplasmic domain of EpsL from the Type 2 Secretion System of Vibrio parahaemolyticus[END_REF][START_REF] Fulara | Structure and oligomerization of the periplasmic domain of GspL from the type II secretion system of Pseudomonas aeruginosa[END_REF]. Additionally, GspL has an N-terminal cytoplasmic domain, which forms a stable complex with the GspE ATPase motor, as demonstrated in the Vibrio cholerae T2SS [START_REF] Abendroth | The X-ray structure of the type II secretion system complex formed by the N-terminal domain of EpsE and the cytoplasmic domain of EpsL of Vibrio cholerae[END_REF]. This interaction anchors the GspE hexamer to the cytoplasmic base of the AP complex, and couples the energy of the ATPase motor to the endopilus polymerization. In vivo, GspM forms a complex with GspL, protecting it from degradation, as shown in V. cholerae [START_REF] Sandkvist | Direct interaction of the EpsL and EpsM proteins of the general secretion apparatus in Vibrio cholerae[END_REF] and studies in Dickeya dadantii highlight a dynamic nature of this complex [START_REF] Lallemand | Dynamic interplay between the periplasmic and transmembrane domains of GspL and GspM in the type II secretion system[END_REF]. An electron-microscopy study of a purified T2SS subcomplex from Klebsiella pneumoniae suggested a C6 symmetry of the GspE-GspL-GspM complex [START_REF] Chernyatina | Core architecture of a bacterial type II secretion system[END_REF]. However, neither that study, nor the in situ analysis of the Legionella pneumophila T2SS by cryo-tomography [START_REF] Ghosal | In vivo structure of the Legionella type II secretion system by electron cryotomography[END_REF] were able to provide a clear view of the L and M complex architecture required to understand their molecular function.

Here we focused on two essential AP components GspL and GspM from Klebsiella oxytoca T2SS called respectively PulL and PulM, with reference to the lipoprotein pullulanase (PulA), the only identified exoprotein secreted by this system. This enzyme degrades branched maltotriose polymers, allowing them to be taken up and used as nutrient (d'Enfert et al., 1987).

To understand the assembly of PulL and PulM, we first analyzed their periplasmic globular Cterminal domains (CTDs). We solved their structures by NMR and X-ray crystallography, and combined these approaches to determine the first structure of their heterodimer complex available to date. Mutational and functional studies based on this structure showed the key role of the heterodimer interface in the secretion process. To reach a comprehensive view of PulL-PulM assembly in the cellular and membrane context, we used bacterial two-hybrid (BACTH) and cysteine crosslinking. These data and the information on the relative abundance of PulL and PulM in bacterial cells, allowed us to propose a model of the dynamic association of AP proteins and of the way this drives the endopilus formation during protein secretion.

Results

Previous studies provide evidence for a direct interaction between the AP components PulL and PulM in K. oxytoca [START_REF] Nivaskumar | Pseudopilin residue E5 is essential for recruitment by the type 2 secretion system assembly platform[END_REF][START_REF] Possot | Multiple interactions between pullulanase secreton components involved in stabilization and cytoplasmic membrane association of PulE[END_REF]. In the Dickeya dadantii T2SS, pulldown, bacterial two-hybrid and cysteine crosslinking assays suggest that GspL and GspM interact via their periplasmic C-terminal domains [START_REF] Lallemand | Dynamic interplay between the periplasmic and transmembrane domains of GspL and GspM in the type II secretion system[END_REF].

However, their assembly mode is still not understood at the molecular and atomic levels. Here, to study their structure and interaction, we produced and purified the predicted globular C-terminal domains of PulL (PulLCTD, residues 312-398) and PulM (PulMCTD, residues 79-161) (See Figure S1 for details on the PulL and PulM domain organization).

PulLCTD structure

First, by using NMR and analytical ultracentrifugation (AUC), we determined the oligomerization state of PulLCTD at concentrations ranging from 10 to 300 µM (Figure S1 and Supplementary data). PulLCTD exists in a monomer-dimer equilibrium in solution depending on the protein concentration. At the concentration used for NMR experiments (300-400 µM), it is mainly found in dimeric form. Its 1 H-15 N HSQC spectrum was used for an initial structural analysis. In this spectrum, each signal arises from the backbone amide group of one residue.

The number of observed signals in the 1 H-15 N HSQC spectrum of PulLCTD ( 82) is consistent with the number of its backbone amide groups (83), and thus with a symmetrical homodimer (Figure 1A). To solve the structure of the PulLCTD dimeric form, we first determined the structure of its monomer by NMR. An ensemble of 15 monomeric structures was calculated based on chemical shift assignments obtained previously [START_REF] Dazzoni | 1 H, 15 N and 13 C resonance assignments of the C-terminal domain of PulL, a component of the Klebsiella oxytoca type II secretion system[END_REF] and NOESY experiments by using the program ARIA and CNS [START_REF] Allain | ARIAweb: a server for automated NMR structure calculation[END_REF][START_REF] Brünger | Crystallography & NMR system: A new software suite for macromolecular structure determination[END_REF] as detailed in Materials and Methods. The ensemble of PulLCTD monomer structures (Figure 1B) presents a backbone RMSD of 0.6 Å for its ordered parts (Table 1). PulLCTD displays a ferredoxin-like fold with 2 helices, a1 (S319-D332) and a2 (S359-R372), and an anti-parallel b-sheet formed by 4 b-strands, b1 (I336-D344), b2 (N348-A356), b3 (F373-Q376) and b4

(I387-G395) (Figure 1B).

To determine the structure of the dimeric form, 13 C/ 15 N edited-filtered NOESY experiments were performed to collect intermolecular distance restraints between two protomers of PulLCTD. Unfortunately, no intermolecular cross-peaks could be observed in these experiments, although various conditions (temperature, magnetic field and pulse sequence)

were tested. The absence of signal is most likely due to conformational exchange at the µs-ms NMR time scale. Such dynamic behavior has been already suspected for several residues displaying very low NMR signal intensity [START_REF] Dazzoni | 1 H, 15 N and 13 C resonance assignments of the C-terminal domain of PulL, a component of the Klebsiella oxytoca type II secretion system[END_REF]. When mapping these residues on the PulLCTD monomer structure (Figure 1C), it appears that they are mostly located on the same exposed face of the molecule (a1, b1 and b2) suggesting that they are at the dimer interface and affected by a conformational exchange between monomeric and dimeric forms. We then used X-ray crystallography and solved the structure of PulLCTD at 1.8 Å resolution. The summary of data collection and refinement statistics is shown in Table 2. The structure of PulLCTD monomer exhibits a nearly identical ferredoxin-like fold (a1-b1-b2-a2-b3-b4) as observed in the NMR structure (Figure 1D). However, the b3 strand (residues F373-Q376) is not fully formed in the X-ray structure. As shown in Figure 1E, higher B-factor values are observed in this region compared to the rest of the protein, reflecting a disordered b-strand.

Consistently, higher flexibility on the ps-ns time scale in this strand in solution is evidenced by the low 1 H-15 N heteronuclear NOE values (Figure 1F).

Only one possible dimer interface with C2 symmetry exists in the crystal, across two asymmetric units in the crystal lattice and with a buried surface area of 526 Å 2 . This dimeric interface occurs between the a1 helix and the b1 strand oriented in an anti-parallel fashion (Figure 1D). Although the interaction surface is quite large relative to the size of the domain, only 6 hydrogen bonds are observed at the dimerization interface between residues of b1 and b1' belonging the other protomer: one between A345 and R339 and two between F343 and L341 (see also Figure 4G below). There are also a few interactions involving a1 helices, notably hydrophobic contacts between the I320 side-chains of each protomer. This small number of interprotein contacts explains that the assemblies are short-lived [START_REF] Janin | Macromolecular recognition in the Protein Data Bank[END_REF] and hence the monomer/dimer exchange of PulLCTD in solution observed by NMR and AUC.

PulMCTD structure

PulMCTD behaves as a stable dimer in solution at low and high concentrations (Figure S1 and Supplementary data). To determine its structure, we performed NMR experiments and crystallization trials in parallel. We obtained crystals in two days, with an asymmetric unit containing seven virtually identical PulMCTD molecules (average RMSD 0.28 Å for Ca atoms).

The final structure was refined at 1.5 Å of resolution (Figure 2A,B). The summary of data collection and refinement statistics is shown in Table 2. The structure of the PulMCTD protomer exhibits a ferredoxin-like fold (a1-b1-b2-a2-b3-b4) (Figure 2). The first helix a1 extends from P82 to H93, followed by strands b1 (V98-Q103), b2 (R106-V111), a2 helix (F116-A129), b3

(A134-A140) and b4 (V148-E156).

Among the molecular associations in the crystal, two possible dimer topologies of PulMCTD were found, named form A and form B. Form A was represented by four virtually superimposable anti-parallel dimers (average RMSD= 0.7 Å), with an interface involving the a2-b3 regions (Figure 2A). Form B was represented by a single homodimer with an a1-b1

interface and protomers oriented in a parallel fashion (Figure 2B).

These alternative dimeric forms A and B have, respectively, a buried surface area of 635 Å 2 and 453 Å 2 across the dimerization interface, as calculated by using the PISA server [START_REF] Krissinel | Inference of macromolecular assemblies from crystalline state[END_REF]. The A form is thus more favorable from a structural aspect, considering its larger buried interface and the fact that it is also the most abundant form in the crystal (80%). Since crystallization conditions and crystal packing might bias these results, we used NMR spectroscopy to determine which dimeric form of PulMCTD exists in solution. A total of 1623 intramolecular and 25 intermolecular restraints were used to calculate the structure of PulMCTD dimer with a backbone RMSD of 0.69 Å for the 15 lowest energy structures (Figure 2D and Table 1). Each PulMCTD protomer exhibits a ferredoxin-like fold composed of 2 a helices and one b sheet as follows: a1 (T84-H93); b1 (V98-Q101); b2 (L107-V111), a2 (F116-A129), b3 (M131-A140) and b4 (V148-R157). The dimer interface occurs between the a2 helix and the b3 strand of the two protomers, oriented in an antiparallel fashion (Figure 2D). This dimer in solution is therefore the form A obtained by X-ray crystallography (RMSD of backbone atoms 1.6 Å between the NMR dimer structure and the crystallographic form A, Figure S2).

Structure of the PulLCTD-PulMCTD complex

An analysis of the interactions between PulLCTD and PulMCTD gave us insight into their assembly and mode of association. Titration experiments of each protein with its unlabelled partner up to three times the molar ratio along with the rotational correlation time estimate highly suggested a stoichiometry of 1 PulLCTD for 1 PulMCTD (Figure S1A andB). For a more accurate characterization of the oligomeric state, we employed native mass spectrometry. Both PulLCTD (Figure 3A) and PulMCTD (Figure 3B) alone are detected as homodimers in accordance To further understand the PulLCTD-PulMCTD interaction mode, we solved the crystal structure of the PulLCTD-PulMCTD complex. A summary of the crystallographic parameters and data, as well as refinement statistics, is shown in Table 2. The asymmetric unit is organized in an arc-like arrangement where we found one PulLCTD molecule flanked on each side by one PulMCTD molecule with different orientations (Figure 3D). They form together a continuous anti-parallel b-sheet. This arrangement results in two different interfaces, either PulL !"# a$b$ -PulM !"# a%b& or PulL !"# a%b& -PulM !"# a%b& . Surface interface analysis (Table S1) indicates that PulL !"# a$b$ -PulM !"# a%b& is more likely to represent a biologically relevant arrangement. In addition to the complex form both A and B forms of the PulMCTD dimers, as present in the PulMCTD crystal, are observed (Figure S3A).

Functional validation of the PulLCTD-PulMCTD interface

To evaluate the role of the PulLCTD-PulMCTD association in protein secretion, we mutated the residues involved in the interface. We introduced single alanine substitutions in positions L319, L323, L341 and Q327 of PulL (Figure 5A, side chains shown as orange sticks)

and tested the ability of these variants to restore pullulanase (PulA) secretion in Escherichia coli strain PAP7460 carrying the pul gene cluster with a non-polar pulL gene deletion in plasmid pCHAP8251. In this strain, wherein the pul genes are moderately overexpressed, complementation with pulL WT allele in pCHAP8258 (WT) restored PulA secretion to native levels (> 90%). The single Leu to Ala substitutions in PulL did not affect the function, replacing polar residue Q327 with alanine in PulL Q327A led to a small but significant defect in PulA secretion (Figure 5B), which was further exacerbated in the presence of L319A or L341A substitutions (Figure 5B). On the PulM side, single Ala substitutions of F116, Q117, M120 and D124 of the a2 helix, or L136 of the b3 strand, did not affect the function (Figure 5C). However, the double substituted variant PulM F116A-L136A showed a significant secretion defect, suggesting that the interface was weakened (Figure 5C). Replacing the surface exposed M120 by charged residues in PulM M120D and PulM M120K variants also significantly reduced secretion (Figure 5C).

In comparison, charge inversions on the surface of the PulMCTD a1 helix, such as R88E and R92D did not show any effect, consistent with their position distal to the interface with PulLCTD.

Although we cannot exclude that these mutations affect interaction with other IM components of T2SS, their localization at the interface and the correlation between their effects with the more or less conservative amino acid changes argue for their direct effect on PulLCTD-PulMCTD C.

W T v e c t o r F 1 1 6 A Q 1 1 7 A M 1 2 0 A M 1 2 0 D M 1 2 0 K D 1 2 4 A L 1 3 6 A F 1 1 6 A L 1 3 6 A D 1 2 4 A L 1 3 6 A M 1 2 0 A L 1 3 6 A R 8 8 E R 9 2 D N 3 A 0 20 40 60 80 100 % PulA secreted ✱✱✱✱ ✱✱ ✱✱ ✱✱✱ W T V e c t o r L 3 1 9 A L 3 2 3 A Q 3 2 7 A L 3 4 1 A L 3 1 9 A L 3 4 1 A L 3 2 3 A L 3 4 1 A Q 3 2 7 A L 3 4 1 A Q 3 2 7 A L 3 1 9 A D 2 7

Nter Nter Cter

Cter encoding indicated PulM variants (Table S2). Data representation and analysis were performed as in (B).

Together, these data show that the intact PulLCTD-PulMCTD interface, and thus the PulL-PulM assembly, play an important role in the secretion. Possibly, other PulL and PulM regions might also contribute to this interaction in vivo. One indication is the PulM N3A variant mapping in the cytoplasmic N-terminus of the protein, which dramatically affected the stability of both proteins (Figure S4) and was defective in secretion (Figure 5C).

Interactions of full-length membrane-anchored PulL and PulM

To gain further insights into the interaction between full-length PulL and PulM in the cellular context and in the presence of the membrane, we combined bacterial adenylate cyclase two-hybrid system (BACTH) and cysteine cross-linking approaches. The BACTH approach allows us to analyze interactions between membrane proteins in their native environment [START_REF] Karimova | A bacterial two-hybrid system based on a reconstituted signal transduction pathway[END_REF]. To study how the full-length PulL and PulM interact, we fused them to the C-terminal ends of T18 and T25, fragments of the Bordetella pertussis adenylyl cyclase (CyaA) catalytic domain. The resulting plasmids (listed in Table S2) were introduced in the E. coli strain DHT1 [START_REF] Dautin | Sensitive genetic screen for protease activity based on a cyclic AMP signaling cascade in Escherichia coli[END_REF] carrying a deletion of the endogenous cya gene. The CyaA T18 and T25 fragments do not interact and do not restore adenylyl cyclase activity as indicated by white colonies on indicator plates (Figure 6A) and low expression levels of the chromosomal lacZ gene (Figure 6B). Bacteria containing T18-PulL and T25-PulL hybrids also showed white colonies and low activity, comparable to the negative controls, indicating that the full-length membrane-embedded PulL does not form homodimers. Bacteria carrying the T18-PulM and T25-PulM chimera were pale blue on X-gal indicator and showed somewhat higher activity, indicating a tendency of PulM to homodimerize (Figure 6). Finally, bacteria coproducing PulL and PulM chimera showed strong and highly significant interaction signals.

These results show that full-length PulL and PulM interact strongly with each other and preferentially form heterodimers in vivo. as a starting construct to generate a series of PulM variants with single Cys substitution at positions 18 through 24. In a similar manner, we substituted Cys264, localized in the periplasmic part of PulL (PulL C264L ) and used it to generate single Cys substitutions at positions 249 to 256 of the TM segment. Bacteria producing all the combinations of single Cys variants of PulL and PulM single Cys variants were treated with CuCl2 as an oxidant and their total extracts were analyzed by Western blot with anti-PulL or anti-PulM antibodies (Figure 7). The PulL C264L variant did not produce any significant level of crosslinked species and a similar pattern was observed in the presence of single Cys residues at positions 249, 252, 253, 254, 255 and 256 (Figure 7). However, PulL C264L with Cys at positions 250 and 251 produced some homodimers and also heterodimers, notably with PulM with Cys at positions 17 (PulM WT ) and 18 (PulM C17L-L18C ). The latter variant gave the most intense heterodimer bands, which were also detected with anti-PulM antibodies (Figure 7). The PulM variants with Cys at positions 17, 18 and 19 also produced homodimer bands. These results show that PulL and PulM can interact via their transmembrane segments and that in this region they also use the same interface for homo-and heterodimerization. The levels of PulM homodimers were not affected by the presence of PulL I250C and PulL V251C , possibly because PulM was more abundant than PulL in these strains. Consistent with a higher fraction of PulL crosslinked with PulM, the L-M heterodimers were more readily detectable in anti-PulL Western blots compared to those with anti-PulM. Confirming the stabilizing role of PulM, the levels of all PulL variants were significantly reduced in the absence of PulM (Ø). In contrast, the levels of PulM did not change in the absence of PulL (Figure 7, last panel, Ø). The above experiments were performed in bacteria producing PulL and PulM in the absence of other T2SS components and at levels which may differ from those in the functional system. In the pulC-O operon encoding the Klebsiella T2SS, the pulL and pulM genes are adjacent and transcribed from the same promoter. To determine their relative abundance, we quantified the PulL and PulM amounts in bacteria producing the functional T2SS from a single, moderate copy-number plasmid pCHAP8185 (Table S2). We used Western blot analysis with antibodies raised against PulLCTD and PulMCTD to quantify the level of these proteins in bacterial extracts expressing the T2SS genes and compared these signals with those known amounts of purified PulLCTD and PulMCTD (Figure S5). These measurements indicated that PulM is present in large excess over PulL, with a molar ratio of about 20:1.

Combining the structural data on the PulLCTD-PulMCTD heterodimer with cysteine crosslinking experiments allowed us to build a 3D model of the membrane-anchored and periplasmic region of the PulL-PulM complex (Figure 8A,B). Sequence analysis revealed the presence of long periplasmic α-helices in both PulL and PulM, encompassing the transmembrane segments and upstream of the CTDs. Additionally, two regions involved in the formation of coiled-coil in both proteins are predicted, the first one overlapping with the transmembrane segments (Figure S6A-C). Consequently, periplasmic helices of PulL and PulM were modeled as a coiled-coil, in which knobs-into-holes packing was confirmed by SOCKET2 analysis [START_REF] Kumar | Socket2: A Program for Locating, Visualising, and Analysing Coiled-coil Interfaces in Protein Structures[END_REF]) (Figure S6D). In the model, the relative alignment of the predicted transmembrane segments is compatible with an orthogonal orientation of the connecting periplasmic helices with respect to the membrane. The coiled-coil formed by the periplasmic helices of PulL and PulM is followed by short disordered linkers connecting the a1 helices of the CTDs heterodimer. In the model of the complex, a potential salt-bridge is found between D276 of PulL and R42 of PulM (Figure 8B). Interestingly, the PulM D276K variant displayed significantly reduced secretion (Figure 5B) which could indicate that the coiled-coil also contributes to the overall interaction surface in the PulL-PulM complex.

Discussion

Here we report the first high-resolution structure of the PulL-PulM AP subcomplex from a bacterial T2SS and investigate its assembly in solution and in vivo. The structural data obtained here could be relevant for the PulL and PulM orthologues PilN and PilO in T4P assembly systems, for which no complex structures are not yet available. Due to the dynamics of PulL, PulM and their association, their structural study was only possible by an integrative approach. By combining native mass spectrometry, NMR and X-ray crystallography we show that while PulLCTD and PulMCTD each form a homodimer, they assemble as a heterodimer. The complementarity between NMR and X-ray crystallography was crucial for the structural study of such dynamic assemblies. While multiple molecules in the crystal asymmetric unit displayed several protein-protein interfaces, NMR gave us unambiguous insights on the dimerization interfaces in solution for both the isolated proteins and their assembly. Functional assays confirm that in vivo the dimerization interface and thus the PulLCTD-PulMCTD association play an important role in the secretion process.

Intriguingly, the same interfaces and residues are involved in both homo-and heterodimerization, however resulting in different orientations of the subunits. These different parallel and antiparallel topologies were also observed in homodimers of other GspL, GspM and their orthologues, indicating the high plasticity of their interfaces and their need for stabilization in dimeric form (Figure S7, S8). For instance, in T4P assembly systems, the CTD of the PulM orthologue PilO forms homodimers, however with β-sheets facing the opposite sides in the two protomers compared to PulM [START_REF] Leighton | Conserved, unstructured regions in Pseudomonas aeruginosa PilO are important for type IVa pilus function[END_REF][START_REF] Sampaleanu | Periplasmic domains of Pseudomonas aeruginosa PilN and PilO form a stable heterodimeric complex[END_REF].

The distribution of conserved residues in PulL and PulM homologs is remarkably consistent with the respective dimerization interfaces of their C-terminal domains (Figure S9). The ferredoxin-like fold of PulLCTD and PulMCTD and their orthologues explains well their structural complementarity and their dynamic assembly mode, both as homodimer and as heterodimer.

The ferredoxin-like fold is highly favorable for molecular association. Its symmetrical repetition has been proposed by Eck and Dayhoff (known as Dayhoff's theory) as the result of the repetition of simple peptides to form a protein and would represent the protein ancestor [START_REF] Alva | From ancestral peptides to designed proteins[END_REF][START_REF] Eck | Evolution of the structure of ferredoxin based on living relics of primitive amino Acid sequences[END_REF]. PulLCTD and PulMCTD indeed exhibit an internal pseudo-symmetry, where the two faces of the ferredoxin-like fold (α1-β1-β2 and α2-β3-β4) are structurally superimposable and present high level of sequence similarity (64% for PulMCTD and 66% for PulLCTD). Interestingly, the dimerization interfaces of PulLCTD and PulMCTD are also highly similar, at the sequence and structural levels, with overlapping residue positions involved in the interactions (Figure S10).

The PulLCTD and PulMCTD homodimers associate in an antiparallel fashion, whereas the PulLCTD-PulMCTD complex is parallel and thus more compatible with the membrane insertion of the proteins where both N-termini are found on the same side of the dimer (Figure 8). This interaction mode is consistent with the BACTH data in which full-length membrane-inserted proteins form heterodimers in parallel arrangement, and with previous data showing that the formation of GspL-GspM heterodimers in D. dadantii is favored over their homodimerization. In addition to the PulLCTD-PulMCTD interface, native full-length forms of PulL and PulM also interact via their transmembrane segments, as shown by Cys scanning experiments. Like for the CTDs, we found an overlap between homo-and heterodimer interfaces involving the transmembrane segments (Figure 7). This is consistent with the previously observed competition between L-M homo-and heterodimerization [START_REF] Lallemand | Dynamic interplay between the periplasmic and transmembrane domains of GspL and GspM in the type II secretion system[END_REF]. The relative abundance of these proteins in vivo is likely to determine whether homo-and heterodimers coexist and exchange. In most of the T2SS models, GspL and GspM are shown with a 1:1 stoichiometry, which finds confirmation in the purified T2SS subcomplex [START_REF] Chernyatina | Core architecture of a bacterial type II secretion system[END_REF]. However, in this study we showed that in the cellular context, when the whole system is expressed from endogenous promoters, the amount of PulM per cell is about 20 times higher than that of PulL (Figure S5). Higher cellular levels of PulM compared to PulL have also been observed by fluorescence microscopy using the GFP (green fluorescent protein) chimera fused to their N-terminal ends [START_REF] Buddelmeijer | Green fluorescent chimeras indicate nonpolar localization of pullulanase secreton components PulL and PulM[END_REF]. Considering the static role of PulL, which serves as an anchor for the PulE ATPase, the higher abundance of PulM would ensure its dynamic role in PulG targeting [START_REF] Nivaskumar | Pseudopilin residue E5 is essential for recruitment by the type 2 secretion system assembly platform[END_REF][START_REF] Santos-Moreno | Polar N-terminal Residues Conserved in Type 2 Secretion Pseudopilins Determine Subunit Targeting and Membrane Extraction Steps during Fibre Assembly[END_REF], which requires rapid exchange of its binding partners. The structural flexibility and complementarity of PulLCTD and PulMCTD is likely to facilitate this exchange during the highly dynamic endopilus assembly and turnover. The homodimerization would be more rapid than the heterodimerization, which is more stable, similarly to Jun-Fos proteins where the homodimers are formed with a favorable kinetics, while the heterodimer is more stable, both forms are found in vivo and in vitro [START_REF] O'shea | Mechanism of specificity in the Fos-Jun oncoprotein heterodimer[END_REF].

We have previously shown that PulG proteins that are assembled to form the endopilus [START_REF] Lopez-Castilla | Structure of the calciumdependent type 2 secretion pseudopilus[END_REF] interact with PulM via their transmembrane regions [START_REF] Nivaskumar | Pseudopilin residue E5 is essential for recruitment by the type 2 secretion system assembly platform[END_REF][START_REF] Santos-Moreno | Polar N-terminal Residues Conserved in Type 2 Secretion Pseudopilins Determine Subunit Targeting and Membrane Extraction Steps during Fibre Assembly[END_REF]. By binding to PulL, PulM might therefore channel PulG to the assembly site defined by the PulE-PulL complex (Figure 8B). The higher abundance of PulM is consistent with its role in targeting of the even more abundant PulG subunits [START_REF] Santos-Moreno | Polar N-terminal Residues Conserved in Type 2 Secretion Pseudopilins Determine Subunit Targeting and Membrane Extraction Steps during Fibre Assembly[END_REF] to ensure their insertion into the growing endopilus. While PulG does not directly interact with PulL [START_REF] Nivaskumar | Pseudopilin residue E5 is essential for recruitment by the type 2 secretion system assembly platform[END_REF], crosslinking studies in V. cholerae showed their physical proximity [START_REF] Gray | In vivo cross-linking of EpsG to EpsL suggests a role for EpsL as an ATPase-pseudopilin coupling protein in the Type II secretion system of Vibrio cholerae[END_REF] supporting the existence of either a ternary complex or their rapid exchange of partners. Rapid influx of pilins would ensure efficient incorporation into helical filaments, thought to be the driving force for protein secretion. In addition to their role in endopilus assembly, the PulL and PulM homologs have been implicated in interactions with the secreted substrate in the P. aeruginosa T2SS [START_REF] Michel-Souzy | Direct interactions between the secreted effector and the T2SS components GspL and GspM reveal a new effector-sensing step during type 2 secretion[END_REF]. Together, these data suggest that the PulL-PulM complex play a major role in the dynamic coupling of endopilus assembly and protein transport. Future studies are needed to address the influence of the pilin and the secreted substrate on the AP assembly and to define the sequence of events during pilin and substrate targeting to the secretion complex.

STAR Methods

Key resources table

REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies

Anti-MalE-PulL rabbit polyclonal antibodies [START_REF] Possot | Multiple interactions between pullulanase secreton components involved in stabilization and cytoplasmic membrane association of PulE[END_REF] N/A Anti-MalE-PulM rabbit polyclonal antibodies [START_REF] Possot | Multiple interactions between pullulanase secreton components involved in stabilization and cytoplasmic membrane association of PulE[END_REF] N/A Anti-PulA rabbit polyclonal antibodies (d'Enfert et al., 1987) N/A Goat anti-Rabbit antibody coupled to HRP Amersham Bacterial Strains

Escherichia coli DH5a F'lacI Q Laboratory collection N/A Escherichia coli BL21(DE3) Star
Novagen Cat# 69450 Escherichia coli PAP7460 [START_REF] Possot | Multiple interactions between pullulanase secreton components involved in stabilization and cytoplasmic membrane association of PulE[END_REF] N/A Escherichia coli DHT1 [START_REF] Dautin | Sensitive genetic screen for protease activity based on a cyclic AMP signaling cascade in Escherichia coli[END_REF] N/A Biological Samples Escherichia coli strain PAP7460 carrying plasmids pCHAP8251 and pCHAP8258 or derivatives listed in Table S2 This study N/A Escherichia coli strain PAP7460 carrying plasmids pCHAP8496 and pCHAP1353 or derivatives listed in Table S2 This study N/A Escherichia coli strain DHT1 carrying plasmids pKT25 and pUT18C or derivatives listed in Table S2 This

Plasmid constructions

Escherichia coli K-12 strain DH5a F'lacI Q was used as a host for cloning purposes. The plasmids used in this study are listed in Table S2. To construct plasmids pMS1222 and pMS1229, the pulL gene was PCR-amplified from plasmid pCHAP8258 as template using primers PulL Kpn 5 and PulL Eco 3 with the high-fidelity Q5 DNA polymerase (New England Biolabs). The PCR products were purified on a Qiaquick spin column, digested with EcoRI and KpnI restriction enzymes (NEB) and ligated with the EcoRI -KpnI digested plasmids pUT18C and pKT25. Site-directed mutagenesis to generate derivatives of pCHAP8258 and pCHAP1353

was performed using a two-step amplification with perfectly overlapping mutagenic primers.

Two separate reactions with single mutagenic primers were performed for 6 cycles using the Q5 DNA polymerase under conditions recommended by the manufacturer. The reactions were mixed and additional 15 cycles of amplification were performed. The PCR reactions were treated with DpnI and transformed into DH5a F'lacI Q ultracompetent cells. The purified plasmids were verified by DNA sequencing (Eurofins). The list of oligonucleotides (Eurofins) is shown in Table S3.

Proteins PulLCTD and PulMCTD were produced in E. coli BL21 (DE3) under control of the T7 promoter. Bacteria were grown at indicated temperatures in LB medium or in minimal M9 medium [START_REF] Miller | Experiments in Molecular Genetics[END_REF]. Antibiotics were added as required at following concentrations: ampicillin (Ap) 100 µg.mL -1 , chloramphenicol (Cm), 25 µg.mL -1 and kanamycin (Km) at 25 µg.mL -1 . When required, the genes under placZ control were induced with 1 mM isopropyl b-D-1-thiogalactopyranoside (IPTG).

Production of unlabeled and isotope-labeled proteins

Derived pMalP2 vectors were used to express either the CTD of PulM (PulMCTD: from residues 79 to 161) or the CTD of PulL (PulLCTD: from residues 312 to 398) in E. coli BL21(DE3) cells (Table S2). Each protein was expressed in the periplasm fused to maltose binding protein (MBP) followed by a His6-tag and a TEV protease cleavage site that is used to remove the MBP and the His6-tag. Only an additional N-terminal serine remains after the cleavage.

Uniformly 15 N and 15 N/ 13 C labeled PulMCTD, PulLCTD, were produced in M9 minimal medium using 1 g/L of 15 NH4Cl and 4 g/L 13 C glucose, as the sole nitrogen and carbon sources, respectively. Gene expression was induced with 1 mM IPTG (isopropyl b-D-1thiogalactopyranoside) overnight at 18 o C in E. coli BL21 (DE3) cells. Unlabeled protein samples were prepared from E. coli BL21 (DE3) cell cultures in LB medium and expression was induced with 1 mM IPTG for 4 hours at 30 o C.

Protein purification

Proteins were purified from the supernatant after the sonication of bacterial cells and centrifugation at 16000 g, during 1 hour at 4 °C. The supernatant of the cell lysis was filtered on a 0.22 µm filter, then loaded onto a HisTrap HP column (Cytiva) equilibrated with 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 10 mM imidazole. Bound proteins were eluted with a linear imidazole gradient going from 10 to 500 mM. The eluted fractions were then incubated with the TEV-His6 protease overnight at 10 o C. The mixture was loaded on a HisTrap HP column to remove TEV-His6 and MBP-His6. Pul proteins from the unbound fractions were collected, concentrated on a 3 kDa cutoff centricon device (Cytiva) and applied on a Sephacryl S-100 column (Cytiva) equilibrated with 50 mM HEPES, pH 7.0, 50 mM NaCl. The fractions containing the purified Pul proteins were pooled and concentrated using centricon devices (Cytiva). Protease inhibitor cocktail, EDTA-free (Roche) was added to all buffers used during the purification. SDS-PAGE was used for analyzing the purity and protein contents of the fractions at each step of the purification.

PulMCTD protein concentration was determined spectrophotometrically using the absorbance at 280 nm and a calculated extinction coefficient of 11000 M -1 .cm -1 . PulLCTD concentration was obtained by BCA (BiCinchoninic acid Assay) method [START_REF] Simpson | Quantifying protein by bicinchoninic Acid[END_REF], since this protein lacks tryptophan.

NMR experiments for assignment

NMR spectra were acquired with a range of 0.3 to 0.4 mM 15 N/ 13 C labeled proteins in 50 mM HEPES, pH 7.0 (PulMCTD) or pH 6.5 (PulLCTD), 50 mM NaCl at 25°C on a 600 MHz Avance III HD and a 800 MHz Avance NEO spectrometers (Bruker Biospin) both equipped with a cryogenically cooled triple resonance 1 H [ 13 C / 15 N] probe (Bruker Biospin). The pulse sequences were used as implemented in the TOPSPIN 3.6.1 (Bruker Biospin) and IBS libraries [START_REF] Favier | NMRlib: user-friendly pulse sequence tools for Bruker NMR spectrometers[END_REF]. TOPSPIN 3.6.1 (Bruker Biospin) was used for NMR data acquisition and processing. The 1 H, 15 N, and 13 C backbone and side chain resonance assignments were carried out as previously described [START_REF] Dazzoni | 1 H, 15 N and 13 C resonance assignments of the C-terminal domain of PulL, a component of the Klebsiella oxytoca type II secretion system[END_REF]. Briefly standard experiments [START_REF] Cavanagh | Protein NMR Spectroscopy: Principles and Practice[END_REF] were used: 2D 15 N-HSQC, 13 C-HSQC, and 3D

HNCA/HN(CO)CA, HNCACB/ HN(CO)CACB pair, HNCO/HN(CA)CO pair, HCCH-TOCSY, C(CO)NH-TOCSY and H(CCO)NH-TOCSY. For the assignment of CdHd and CeHe of phenylalanines and tyrosines, the 2D 13 C-1 H HBCBCGCDHD and HBCBCGCDCEHE spectra were used [START_REF] Yamazaki | Complete assignments of magnetic resonances of ribonuclease H from Escherichia coli by double-and triple-resonance 2D and 3D NMR spectroscopies[END_REF]. Side chain resonance assignments were completed by using 3D 13 C and 15 N NOESY-HSQC each with a mixing time of 120 ms and optimized for the detection of either aliphatic or aromatic carbon [START_REF] Iwahara | Improved NMR spectra of a protein-DNA complex through rational mutagenesis and the application of a sensitivity optimized isotopefiltered NOESY experiment[END_REF], together with 2D 1 H-1 H NOESY (120 ms mixing time) and TOCSY in D2O.

2,2-Dimethyl-2-silapentane-5-sulfonate (DSS) signal was taken as 0 ppm for referencing proton chemical shifts and 15 N and 13 C chemical shifts were indirectly referenced to DSS [START_REF] Wishart | 1H, 13C and 15N chemical shift referencing in biomolecular NMR[END_REF]. CcpNmr Analysis [START_REF] Vranken | The CCPN data model for NMR spectroscopy: development of a software pipeline[END_REF] was used for NMR data analysis. Secondary structure analysis was performed by using HN, Ha, Ca, Cb, CO, and N chemical shifts with the TALOS-N prediction server [START_REF] Shen | Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks[END_REF].

The assignment of PulMCTD 13 C/ 15 N/ 1 H resonances was deposited to the BMRB under accession number 34719. The assignment of PulLCTD 13 C/ 15 N/ 1 H resonances was obtained in our previous work [START_REF] Dazzoni | 1 H, 15 N and 13 C resonance assignments of the C-terminal domain of PulL, a component of the Klebsiella oxytoca type II secretion system[END_REF].

PulMCTD and PulLCTD NMR structure calculation

The structure of PulMCTD and PulLCTD in their dimeric and monomeric form respectively, were determined by performing several cycles of calculation with ARIA 2.3 software [START_REF] Rieping | ARIA2: automated NOE assignment and data integration in NMR structure calculation[END_REF] coupled to CNS 1.2 software and ARIAweb [START_REF] Allain | ARIAweb: a server for automated NMR structure calculation[END_REF][START_REF] Brünger | Crystallography & NMR system: A new software suite for macromolecular structure determination[END_REF], making use of the standard torsion angle simulating annealing protocol.

Each cycle consisted of automatic 3D 15 N-NOESY-HSQC and 3D 13 C-NOESY-HSQC spectra assignment and structure calculations with 9 or 8 iterations with default parameters. In the last iteration 200 or 50 structures were calculated and further refined in an explicit water box [START_REF] Linge | Refinement of protein structures in explicit solvent[END_REF]. Some corrections to the NOE assignment were made manually. The 15 lowest energy structures exhibiting no NOE restraint violations > 0.5 Å and no dihedral angle violations > 5 ° were selected as the final ensemble.

For PulLCTD, an ensemble of 15 monomer structures was calculated. During the calculation process, most of the NOESs restraints from 3D 15 N-NOESY-HSQC and 3D 13 C-NOESY-HSQC spectra were automatically assigned by ARIA based on the chemical shifts previously obtained [START_REF] Dazzoni | 1 H, 15 N and 13 C resonance assignments of the C-terminal domain of PulL, a component of the Klebsiella oxytoca type II secretion system[END_REF].

PulMCTD dimer structure was calculated in two steps, first by using intramolecular distance restraints derived from 3D 13 C-and 15 N-NOESY-HSQC spectra and the PulMCTD 13 C/ 15 N/ 1 H resonances assignment to determine the monomer structure. A 3D 13 C/ 15 N filtered NOESY-HSQC experiment (120 ms mixing time) was performed on a 1:1 doublelabeled:unlabeled PulMCTD mixture to obtain intermolecular distance restraints to calculate the dimer structure with ARIA. Chemical shift tolerances were set to 0.045 ppm for protons and 0.4 ppm for the bound heteroatoms. Phi and psi dihedral angles were predicted with TALOS-N [START_REF] Shen | Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks[END_REF], and predictions classified as "strong" or "good" were incorporated as dihedral angle restraints. The structure ensemble was visualized and inspected with PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC), their quality was evaluated with PROCHECK-NMR [START_REF] Laskowski | AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR[END_REF] and the PSVS server [START_REF] Bhattacharya | Evaluating protein structures determined by structural genomics consortia[END_REF]. The atomic coordinates of PulMCTD dimer and restraints used in the calculation were deposited in the Protein Data Bank (PDB ID: 7ZE0).

Protein-protein interaction analysis by NMR

The protein-protein interactions were monitored by comparison of the 1 H-15 N HSQC spectra of one labeled protein alone and in the presence of its unlabeled partner at 25 °C. between the two experimental conditions. CSP higher than 1.5, 2 or 3 standard deviations (s) from the mean were considered for the analysis.

Native mass spectrometry

Prior to performing native mass analysis, the quality of each protein was assessed by intact mass measurement under denaturing conditions. For native mass spectrometry, protein samples were buffer exchanged against 250 mM ammonium acetate (pH 7.0) using Zeba spin desalting columns with a 7 kDa cutoff (Thermo Fisher Scientific, Waltham, MA, USA). The PulLCTD-PulMCTD complex was formed before or after buffer exchange by mixing PulMCTD with an equimolar or a 2-fold molar excess PulLCTD.

Samples (at a final concentration of 5 to 10 µM) were analyzed on a SynaptG2-Si HDMS mass spectrometer (Waters) equipped with a nano-electrospray source. The instrument was calibrated in sensitivity mode using a 2 mg/ml cesium iodide solution prepared in 50% isopropanol (v/v), 0.1% formic acid (v/v) in the 50 to 5000 m/z range, and the quadrupole profile was adjusted to ensure the best transmission in the selected mass range. To preserve the integrity of non-covalent complexes in the gas phase, the instrument settings were carefully adjusted to collection and model refinement statistics are summarized in Table 2. All structure figures were generated with Chimera (version 1.13rc) [START_REF] Pettersen | UCSF Chimera--a visualization system for exploratory research and analysis[END_REF] or with PyMOL (version 2.5.2

The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC).

Protein secretion assays

Strain PAP7460 ∆(lac-argF)U169 araD139 relA1 rpsL150 ∆malE444 malG501 [F' (lacI Q ∆lacZM15 pro+ Tn10)] (Tc R )) was used as a pul gene expression host [START_REF] Possot | Multiple interactions between pullulanase secreton components involved in stabilization and cytoplasmic membrane association of PulE[END_REF]. PAP7460 bacteria harboring plasmids pCHAP8251 or pCHAP8496 (Table S2) were transformed with compatible plasmids encoding PulL and PulM variants (Table S2). Bacteria were cultured overnight in LB containing Ap and Cm, 0.4% D-maltose (w/v) and 0.1 vol of M63 salts [START_REF] Miller | Experiments in Molecular Genetics[END_REF] at 30°C. The next day, 6 mL of the same fresh medium was inoculated with 300 µL of precultures and grown for 5 hours to OD600nm > 1.8. Cultures were normalized to OD600nm of 1 in a total volume of 1 mL and fractionated as follows: 0.1 mL of cultures were centrifuged for 10 min at 16000 x g in an Eppendorf centrifuge, the supernatant was aspirated off and the bacterial pellets were resuspended in 0.1 mL of SDS sample buffer to give the cell fraction. The remaining 0.9 mL was centrifuged for 5 min at 16000 x g. The supernatant was transferred to a fresh tube and centrifuged for another 10 min at 16000 x g. A fraction (0.1 mL)

of the supernatant was mixed with 0.1 mL of 2x SDS sample buffer to give the supernatant fraction. The bacterial pellets of the 0.9 mL of normalized cultures were resuspended in 90 µL of SDS sample buffer for analysis of PulL and PulM levels (Figure S4).

Bacterial two-hybrid assays

Bacterial two-hybrid assays [START_REF] Karimova | A bacterial two-hybrid system based on a reconstituted signal transduction pathway[END_REF] were performed in strain DHT1 [START_REF] Dautin | Sensitive genetic screen for protease activity based on a cyclic AMP signaling cascade in Escherichia coli[END_REF]. Plasmids encoding compatible pUT18C and pKT25 vectors and their derivatives were co-transformed in calcium-competent DHT1 bacteria and selected on LB plates containing Ap and Km. After 47-72 hours of growth at 30 °C, single colonies were picked at random and inoculated in LB containing the same antibiotics. Bacteria were grown overnight at 30°C and precultures were used to inoculate the same medium supplemented with 1 mM IPTG. After 4 hours, cultures were placed on ice and beta-galactosidase assays were performed as described previously [START_REF] Miller | Experiments in Molecular Genetics[END_REF]. Beta-galactosidase activity from at least 8 independent cultures (indicated by the number of dots on the plot) was plotted and the data was analysed using GraphPad Prism 9 software. For the qualitative plate tests, 10 µl of bacterial cultures was spotted on LB plates containing Ap, Km, X-gal (0.2 mg. mL -1 ) and 100 µM IPTG. The plates were cultured for 24-36 hours at 30 °C and images were recorded with a digital camera.

Cysteine crosslinking assays

Plasmids encoding different PulL and PulM variants or the empty vectors were cotransformed into strain PAP7460 and selected on LB Ap Cm plates at 30 °C. Single colonies were inoculated into 5 mL of LB containing Ap and Cm and grown overnight at 30 °C with shaking at 200 rpm. The next day, 0.2 mL of the precultures was diluted into 5 mL of fresh LB Ap Cm medium and bacteria were incubated for 5 hours at 30 °C with vigorous shaking.

Cultures were normalized to OD600nm of 1 and 1 mL of bacteria was centrifuged for 3 min at 16000 x g in a table-top Eppendorf centrifuge. Bacterial pellets were washed once with phosphate buffer saline (PBS) and resuspended in 1 mL of buffer containing 50 mM MOPS pH

Full-length PulL-PulM heterocomplex modeling

To model the structure of the membrane embedded and periplasmic regions of the PulL/PulM complex, we used the structure of the PilN/PilO complex from the piliated state of the type IV pilus machine from Myxococcus xanthus (PDB ID: 3JC8) [START_REF] Chang | Architecture of the type IVa pilus machine[END_REF].

First, the structure of PulLCTD-PulMCTD heterodimer was superimposed onto the C-terminal domains (ferredoxin like-domains) of PilN and PilO. In both proteins, DeepCoil [START_REF] Ludwiczak | DeepCoil-a fast and accurate prediction of coiled-coil domains in protein sequences[END_REF] predicts two regions potentially involved in coiled-coil formation. Thus, the Nterminal helical region of PilN/PilO was used as template for comparative modeling of the homologous PulL/PulM region as a coiled-coil. The sequences of PulL and PulM encompassing the transmembrane segments (TMS) and the long helices between the TMS and the C-terminal domains, as predicted by PSIPRED [START_REF] Jones | Protein secondary structure prediction based on position-specific scoring matrices[END_REF], were aligned on the corresponding regions of PilN and PilO, respectively. In addition to sequence similarity, we consider the following to guide the alignment: i) the positions of the hydrophobic TMS, ii) the length of the predicted helices and iii) the expected spatial proximity of PulL residues 250/251 and PulM residues 17/18 from the cysteine cross-linking experiments. Using the resulting alignments (Figure S7C)

and the PulLCTD-PulMCTD heterodimer structure, a full atom model of PulL 239-398 -PulM 1-161 was built by using Modeller [START_REF] Sali | Comparative protein modelling by satisfaction of spatial restraints[END_REF]. SOCKET2 [START_REF] Kumar | Socket2: A Program for Locating, Visualising, and Analysing Coiled-coil Interfaces in Protein Structures[END_REF] identified a coiled-coil interface involving helical segments from PulL (L254-R300) and PulM Statistics for the highest-resolution shell are shown in parentheses.

Figure 1 .

 1 Figure 1. PulLCTD homodimer structure. A. 1 H-15 N HSQC NMR spectrum of PulLCTD (400 µM in 50 mM HEPES buffer pH 6.5, 50 mM NaCl). Backbone resonance assignments are indicated in one-letter amino acid code and side chain NH 2 peaks of Asn (N) and Gln (Q) are connected by horizontal lines. B. NMR structure ensemble of PulLCTD exhibiting a ferredoxin-like fold (a1-b1-b2-a2-b3-b4; a-helix in red, b-sheet in yellow, loops and turns in green). C. Cartoon representation of the lowest-energy NMR structure of the PulLCTD monomer. The intensity of the 1 H-15 N HSQC signals of PulLCTD is reported with a specific colour code on the structure from blue to red for decaying peak intensity, outlining the likely homodimeric interface. D. Superposition of the PulLCTD NMR structure (coloured as in C) and the dimer structure of PulLCTD obtained by X-ray crystallography in grey (PDB ID: 8A9W). E. Representation of the B-factor per residue of PulLCTD in the X-ray crystallography dimer structure, from blue to red, with increasing B-factor. F. 1 H-15 N heteronuclear NOE values along the PulLCTD sequence.

Figure 2 .

 2 Figure 2. PulMCTD homodimer structure. A. Main topology (form A) found in the PulMCTD crystal. The four homodimers observed in the asymmetric unit are superimposed. B. Minor topology (form B) found in the PulMCTD crystal, represented by a single homodimer. The vertical lines indicate the twofold symmetry axis. C. 1 H-15 N HSQC NMR spectrum of PulMCTD in 50 mM HEPES pH 7.0, 50 mM NaCl. The backbone resonance assignments are indicated in one-letter amino acid code. Side chain NH2 signals of Asn (N) and Gln (Q) are connected by horizontal lines. D. NMR structure ensemble of the PulMCTD homodimer. Helices are coloured in red, b-strands in yellow and turns/loops in green.

Figure 3 .

 3 Figure 3. Oligomeric state and interactions of the PulLCTD-PulMCTD complex. Native mass spectra of PulLCTD (A) and PulMCTD (B) alone and in an equimolar mixture of PulLCTD and PulMCTD (C). From mass spectra we observe m/z corresponding to the presence of monomeric, homodimeric and heterodimeric species (annotated in grey for PulLCTD and in red for PulMCTD). Charge states are indicated for all species. D. PulLCTD-PulMCTD complexes in the X-ray crystallography asymmetric unit. E. Overlay of 1 H-15 N HSQC spectra of PulLCTD in the absence (grey) and presence (red) of unlabeled PulMCTD. F. Overlay of 1 H-15 N HSQC spectra of PulMCTD in the absence (grey) and presence (red) of unlabeled PulLCTD.. G. X-ray structure of the 𝑃𝑢𝑙𝐿 '() a$b$ -𝑃𝑢𝑙𝑀 '() a%b& complex found in solution. The residues at the heterodimer interface displaying the highest CSP (>3 s in black, >1.5 s in blue) are colored.

Figure 4 .

 4 Figure 4. Comparison of the interfaces in homodimers of PulLCTD or PulMCTD and thePulLCTD-PulMCTD heterodimer complex. A, B, C and D. Hydrophobic interactions (cyan) inPulMCTD (X-ray structure), PulMCTD (NMR structure), PulLCTD (X-ray structure), and the

  Figure 6. BACTH analysis of PulL and PulM interactions. A. Plate assay and B. Betagalactosidase activity of E. coli strains DHT1 producing T18, T25 or their chimera with yeast leucin Zipper (Z) (as positive control), PulL (L) or PulM (M). The b-galactosidase activities measured for independent bacterial cultures (n 8) are plotted as dots and horizontal red lines show median values. Statistical analysis was performed with One-way ANOVA and multiple comparisons test, and plotted with GraphPad Prism 9 software.

Figure 7 .

 7 Figure 7. Cysteine scanning and crosslinking of PulL and PulM transmembrane segments. Bacteria of strain PAP7460 producing the indicated PulL and PulM variants, or empty vector (Ø) were oxidized with CuCl2 and the total extract from 0.05 OD600nm of bacteria was analyzed by SDS-PAGE and Western blot. Molecular weight markers are shown on the left, and migration of PulL (L) and PulM (M) monomers, homodimers (L-L, M-M) and heterodimers (L-M) are indicated on the right. The same total fractions were analyzed with anti-PulL antibody (top) and anti-PulM antibody (bottom).

  is colored in grey, PulM in red, transmembrane segments in yellow. Residues close in space as revealed by crosslinking are annotated on the structural model. C. Model of full-length PulL and PulM in the context of the T2SS. The highly abundant PulG endopilus subunit interacts with PulM. By binding to PulL, PulM targets PulG to the PulL-PulE-PulF complex. PulG, the endopilus subunit, is released from PulM and added to the growing endopilus.

  1 H-15 N HSQC experiments were acquired on 30 µM of either 15 N-PulMCTD or 15 N-PulLCTD alone or in the presence of 60 µM, 120 µM and 250 µM of unlabeled PulLCTD or PulMCTD, respectively. To avoid dilution, we lyophilized the unlabeled protein in the same buffer and added it to the labeled protein sample in solution. Chemical shift perturbations (CSP) of backbone amide cross-peaks were quantified by using the equation CSP = [ΔδH 2 + (ΔδN*0.159) 2 ] 1/2 , where ΔδH and ΔδN are the observed 1 H and 15 N chemical shift changes

  The cell-and supernatant fractions corresponding to the 0.05 OD600nm of bacteria were analyzed on 10% Trisglycine SDS-PAGE and transferred to the nitrocellulose membranes. The membranes were probed by Western blot with anti-PulA antibodies and revealed by fluorescence using ECL-2 and Typhoon SLA-9000. Concentrated cell pellets (0.1 OD600nm) were analyzed on 10% Tristricine SDS-PAGE and Western blot using anti-PulL and anti-PulM antibodies. Membranes were incubated with secondary anti-rabbit antibodies coupled to HRP (Cytiva) and revealed with ECL-2 (Thermo). The fluorescence signals were recorded using Typhoon SLA-9000. The signal intensity of PulA bands in the cell-and supernatant fractions was quantified using ImageJ to calculate the fraction of PulA in the supernatant. Data were analyzed with Prism GraphPad software, using Ordinary one-way ANOVA test with multiple comparisons.

7. 0

 0 , 5 mM MgCl2, 10% (v/v) glycerol. Bacterial suspensions were prewarmed at 23 °C, CuCl2 was added to 300 µM (final concentration) and incubated for 23 min at 23 °C in a Thermomixer shaken at 650 rpm. To stop the reaction, EDTA pH 8.0 was added to a final concentration of 22.5 mM. Reaction mixtures were pelleted by centrifugation and bacteria were resuspended in the SDS sample buffer at 10 OD600nm mL -1 . Total extracts were analyzed by sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) on 10% Tris-Tricine gels. Proteins were transferred to nitrocellulose membranes (Cytiva) using the fast blotting system PowerBlot (Invitrogen) and a 1-Step transfer buffer (Thermo). The membranes were blocked in 5% skim milk (w/v) in Tris buffer saline solution containing 0.05% Tween-20 (TBST) and probed with anti-PulL or anti-PulM antibodies. The signals were revealed with ECL chemiluminescence kit (Thermo) and recorded on Amersham 680 imager.Quantification of cellular PulL and PulM levelsTo estimate the ratio between the cellular levels of PulL and PulM, we used semiquantitative Western blot analysis. Different amounts of total bacterial extracts in parallel with the purified PulLCTD or PulMCTD were analyzed on 10% Tris-Tricine SDS-PAGE. Total bacterial extracts were prepared from cultures of strains PAP7460 harboring plasmid pCHAP8185 containing the pul operons. Bacteria were grown in LB supplemented with Ap, 0.4% D-maltose (w/v) and 0.1 vol of M63 salts. Colony forming units from these cultures were counted by plating bacterial serial dilutions in triplicate. The proteins were transferred on nitrocellulose membranes, probed with antibodies directed against PulLCTD and PulMCTD domains followed by secondary goat anti-rabbit antibodies coupled with HRP. The blots were developed using the ECL-2 kit and quantified on Typhoon FLA-9000 imager. The bands were quantified with ImageJ. The standard curves of signal intensities were plotted as a function of known PulLCTD or PulMCTD molar concentrations and linear regression analysis was used to determine the amounts of PulL and PulM in total bacterial extracts and their molar ratio, using the GraphPad Prism software.

5. Functional characterization of the PulLCTD-PulMCTD interface. A. Cartoon representation

binding. Figure of the complex of PulLCTD (grey) with PulMCTD (red). The substituted residues are highlighted as sticks and marked with a single letter code, those at the PulL-PulM interfaces are colored in orange (PulLCTD) or yellow (PulMCTD), and those far from the interface are colored in cyan. B. PulA secretion in strain PAP7460 carrying plasmid pCHAP8251 (DpulL) complemented with pCHAP8258 encoding PulL (WT) or its derivatives encoding mutant PulL variants (Table S2). Details of the secretion assays are provided in Materials and Methods. The bar graph heights represent the mean values and dots represent the percentage of secreted PulA from independent experiments (n 3). One-way ANOVA and multiple comparisons were done with GraphPad PRISM 9. Statistically significant differences relative to the wild-type PulL are indicated. C. PulA secretion in strain PAP7460 carrying plasmid pCHAP8496 (DpulM) complemented with pCHAP1353 encoding WT PulM or its mutant derivatives

Table 2 :

 2 Crystallography data collection and refinement statistics.

	Parameters	PulLCTD	PulLCTD-PulMCTD	PulMCTD
	Beamline	Proxima 2A	Proxima 1	Proxima 1
	Resolution range (Å)	40.15 -1.895 (1.963 -1.895)	58.95 -2.771 (2.87 -2.771)	67.765 -1.523 (1.549 -1.523)
	Space group	I 21 3	P 63 2 2	C 2 2 21
	Unit cell (Å, °)	80.3 80.3 80.3 90 90 90	117.899 117.899 110.002 90 90 120	80.85 135.529 109.408 90 90 90
	Total reflections	277247 (26424)	464974 (47811)	1238306 (62080)
	Unique reflections	7012 (687)	11980 (1165)	91712 (4530)
	Multiplicity	39.5 (38.5)	38.8 (41.0)	13.5 (13.7)
	Completeness (%)	99.97 (100.00)	99.92 (100.00)	100.0 (100.0)
	Mean I/sigma(I)	33.73 (3.03)	29.12 (2.73)	17.6 (2.2)
	Wilson B-factor	41.28	95.24	25.02
	R-merge	0.06935 (1.739)	0.09584 (1.471)	0.070 (0.951)
	R-meas	0.07029 (1.762)	0.09716 (1.49)	0.073 (0.987)
	R-pim	0.01132 (0.2832)	0.01573 (0.2311)	0.020 (0.264)
	CC1/2	1 (0.86)	1 (0.846)	0.999 (0.900)
	CC*	1 (0.962)	1 (0.958)	1 (0.975)
	Reflections used in refinement	7012 (687)	11980 (1165)	91712 (4530)
	Reflections used for R-free	339 (32)	581 (50)	4683 (494)
	R-work	0.2408 (0.3641)	0.2569 (0.3558)	0.2167 (0.2828)
	R-free	0.2765 (0.2755)	0.2980 (0.4413)	0.2305 (0.2933)
	CC(work)	0.946 (0.725)	0.907 (0.681)	0.954 (0.874)
	CC(free)	0.915 (0.713)	0.952 (0.552)	0.954 (0.820)
	Number of non-hydrogen atoms	606	2365	4680
	Macromolecules	579	2326	4124
	Ligands	10	0	0
	Solvent	17	39	556
	Protein residues	75	308	549
	RMS (bonds) (Å)	0.010	0.012	0.016
	RMS (angles) (°)	1.41	1.62	1.53
	Ramachandran plot
	favored (%)	94.37	90.00	98.88
	allowed (%)	2.82	8.00	1.12
	outliers (%)	2.82	2.00	0.00
	Rotamer outliers (%)	10.00	10.29	1.37
	Clashscore	11.83	9.52	2.04
	Average B-factor	64.98	122.89	32.90
	Macromolecules	65.09	123.41	32.01
	Ligands	79.76
	Solvent	52.56	92.02	39.48

Acknowledgements:

This work was supported by the French Agence Nationale de la Recherche (ANR Synergy-T2SS ANR-19-CE11-0020-01) and the Fondation pour la Recherche Médicale (Equipe FRM 2017M.DEQ20170839114). YL was funded by the Pasteur Paris University (PPU) international PhD program. We thank Ingrid Guilvout and Maylis Lejeune for their constant help. We acknowledge Iñaki Guijarro, Rémy Le Meur, Bertrand Raynal, Sébastien Brûlé and Christophe Thomas of C2RT for their help and assistance. The 800-MHz NMR spectrometer and the optima AUC of the Institut Pasteur were partially funded by the Région Ile de France (SESAME 2014 NMRCHR grant no 4014526) and DIM One Health, respectively. The authors are grateful to the staff of the Institut Pasteur Crystallography platform for robot-driven crystallization screening. We acknowledge the Synchrotron SOLEIL (St Aubin, France) staff for assistance and advice during data collection on PROXIMA-1 and PROXIMA-2A beamlines. This work used the computational and storage service (TARS cluster) provided by the IT Department at Institut Pasteur, Paris.

Software and Algorithms

PRISM version 9

GraphPad https://www.graphpa d.com/scientificsoftware/prism/ Image J [START_REF] Abramoff | Image Processing with ImageJ[END_REF] https://imagej.nih.go v/ij/ TOPSPIN 3.6.1 Bruker Biospin. N/A CcpNmr Analysis 2.4 (Vranken et al., 2005) https://www.ccpn.ac. uk/ ARIA 2.3 (Rieping et al., 2007) http://aria.pasteur.fr ARIAweb (Allain et al., 2020) http://ariaweb.pasteu r.fr CNS 1.2 (Brünger et al., 1998) http://cns-online.org TALOS-N (Shen and Bax, 2013) http://spin.niddk.nih. gov/bax/software/TA LOS-N Modeller 9.24 (Sali and Blundell, 1993) https://salilab.org/mo deller/ Pymol 2 Schrödinger, LLC https://pymol.org/ UCSF Chimera 1.13 (Pettersen et al., 2004) https://www.cgl.ucsf. edu/chimera/ PROCHECK-NMR (Laskowski et al., 1996) https://www.ebi.ac.u k/thorntonsrv/software/PROCH ECK/ PSVS 1.5 (Bhattacharya et al., 2007) https://montelionelab .chem.rpi.edu/PSVS/ PSVS/ MassLynx 4.1 Waters N/A Phaser (McCoy et al., 2007) https://www.phaser.c imr.cam.ac.uk/ Coot (Emsley and Cowtan, 2004) https://www2.mrclmb.cam.ac.uk/perso nal/pemsley/coot/ Buster (Bricogne et al., 2011) http://www.globalpha sing.com/buster/ DeepCoil (Ludwiczak et al., 2019) https://github.com/la bstructbioinf/DeepCo il PSIPRED (Jones, 1999) http://bioinf.cs.ucl.ac. uk/psipred/ SOCKET2 (Kumar and Woolfson, 2021) http://coiledcoils.chm .bris.ac.uk/socket2/h ome.html PISA server (Krissinel and Henrick, 2007) https://www.ebi.ac.u k/pdbe/pisa/

During the revision of this manuscript, we became aware of a recent work using molecular docking and proposing a similar arrangement of the GspL-GspM ferredoxin-like domains from E. coli O18:K1:H7, but as a tetramer [START_REF] Pacheco Gutierrez | Structural studies of the bacterial Type II secretion system[END_REF]. Although the antiparallel homodimerization of PulLCTD and PulMCTD would require a tangled arrangement with the respect to the membrane (Figure 8A), we cannot exclude that it might occur during the process.

The long coiled-coil regions and flexible linkers that connect them to the CTDs could provide additional plasticity in the periplasmic regions of the proteins allowing this tangled association.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Nadia Izadi-Pruneyre (nadia.izadi@pasteur.fr).

Materials availability

Plasmids and strains produced in this study are available from the authors and detailed in Table S2.

Data and code availability

Atomic coordinates and structure factors have been deposited in the RCSB Protein Data Bank under the accession codes 8A9W (PulLCTD), 7ZE0 (PulMCTD, NMR), 8A9X (PulMCTD, X-ray) and 8AB1 (PulLCTD-PulMCTD). PulM 13 C/ 15 N/ 1 H resonance assignments were deposited to the BMRB under accession number 34719.

Experimental model and subject details

Escherichia coli strains used in this study are listed in Key resources table. They were cultured in M9 or LB medium [START_REF] Miller | Experiments in Molecular Genetics[END_REF] at temperatures indicated in the corresponding Methods sections. The genes encoding the type II secretion system components, including PulL and PulM proteins studied here originate from the Klebsiella oxytoca strain UNF5023 (formerly classified and Klebsiella pneumoniae) described in (d'Enfert et al., 1987).

Method details

the following values: capillary voltage, 1.5-2.0 kV; sampling cone, 150 V; source offset, 150 V; trap gas flow: between 5 and 7 mL/min, trap collision energy, 4 V; cone gas, 20 L/h; source temperature, 30 °C. Spectra were acquired in positive mode for 5 to 10 minutes to obtain a good signal-to-noise ratio and processed with MassLynx 4.1 software (Waters) with minimal smoothing. Validation of the settings of the above-mentioned instrument was performed using equine skeletal muscle myoglobin (Sigma Aldrich) prepared in 250 mM ammonium acetate buffer, pH 7.0 and using the same experimental procedure as for PulLCTD and PulMCTD.

Crystallization and diffraction data collection

For PulLCTD-PulMCTD complex analysis, an equimolar mixture of two proteins was prepared at a final concentration of 1 mM and co-eluted on a Sephacryl S-100 column (Cytiva) equilibrated with 50 mM HEPES, pH 7.0, 50 mM NaCl. Initial screening of crystallization conditions was carried out using the vapor diffusion method with a MosquitoTM nanoliterdispensing system (TTP Labtech, Melbourn, United Kingdom) following the established protocols [START_REF] Weber | High-Throughput Crystallization Pipeline at the Crystallography Core Facility of the Institut Pasteur[END_REF]. Briefly, sitting drops were set up using 400 nl of a 1:1 mixture of each sample protein and crystallization solutions (672 different commercially available conditions) equilibrated against a 150-μl reservoir in multiwell plates (Greiner Bio-one, GmbH, Frichenhausen, Germany). The crystallization plates were stored at 18°C in an automated RockImager1000 ® (Formulatrix, Bedford, MA, United States) imager to monitor crystal growth. The best crystals were obtained in crystallization conditions containing 30% (w/v) PEG 4K, 0.1 M HEPES pH 7.5, 0.2 M CaCl2 for PulMCTD; 0.5 M LiSO4 and 15% (w/v) PEG 8K for PulLCTD; and 20% (w/v) PEG 3350, 0.2M KCl for the PulLCTD-PulMCTD complex. Crystals were then flash cooled in liquid nitrogen using the crystallization condition supplemented with 30% (V/V) of glycerol as cryoprotectant.

Diffraction data were collected at cryogenic temperatures (100 K) on the PROXIMA-1 and PROXIMA-2A beamlines at the SOLEIL synchrotron (St Aubin, France) and processed with autoPROC [START_REF] Vonrhein | Data processing and analysis with the autoPROC toolbox[END_REF].

X-ray structure determination and model refinement

The crystal structures of the PulLCTD, PulMCTD and the PulLCTD-PulMCTD complex were solved by the molecular replacement method with Phaser [START_REF] Mccoy | Phaser crystallographic software[END_REF], using NMR models as search probes. Final models were obtained through interactive cycles of manual model building with Coot [START_REF] Emsley | Coot: model-building tools for molecular graphics[END_REF] and reciprocal space refinement with Buster [START_REF] Bricogne | BUSTER version 2.11.1[END_REF] and REFMAC [START_REF] Murshudov | REFMAC5 for the refinement of macromolecular crystal structures[END_REF]. X-ray diffraction data (G21-I67). Regions with knobs-into-holes packing between PulL and PulM correspond to the ones predicted as coiled-coils (Figure S6D).

Quantification and statistical analysis

For the PulA secretion assays, the fluorescence signals revealed by Western blot and ECL2 were quantified using Image J from 3 or more independent experiments (indicated by the number of dots on the graphs representing individual values). For each sample, the fraction of secreted PulA was calculated as the signal intensity in the supernatant fraction divided by the sum of signal intensities of PulA in the cell and supernatant fractions. Data were analyzed with Prism GraphPad software, using Ordinary one-way ANOVA test with multiple comparisons.

For the BACTH experiments, beta-galactosidase activity measured from at least 8 independent cultures as described in Methods [START_REF] Miller | Experiments in Molecular Genetics[END_REF]. The mean values were plotted and the data was analyzed using GraphPad prism 9 software using Ordinary one-way ANOVA test with multiple comparisons.

Quantification of cellular protein levels of PulL and PulM was performed by comparing

Western blot ECL signals from defined number of bacteria with the signals from define quantities of purified proteins using with ImageJ. The standard curves of signal intensities were plotted as a function of known PulLCTD or PulMCTD molar concentrations and linear regression analysis was used to determine the amounts of PulL and PulM in total bacterial extracts and their molar ratio, using the GraphPad Prism software. [START_REF] Laskowski | AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR[END_REF] c Calculated using PSVS ver. 1.5 [START_REF] Bhattacharya | Evaluating protein structures determined by structural genomics consortia[END_REF] d Average root mean square deviation (RMSD) over the ensemble atomic coordinates with respect to the average structure.

e Ordered residues [S(phi) + S(psi) > 1.8].

Author contributions:

RD

Conflict of interest:

The authors declare that they have no conflict of interest.


Leplat Mélody

Loheac Youenn
email: youenn.loheac@rennes-sb.com

Teillet Eric

Preferences & choices experiments with real products consumption: application with plant-based proteins

Keywords: substituts à la viande, évaluation sensorielle, expériences de choix, produits réels meat substitutes, sensory evaluation, choices experiment, real products

Dans un contexte de végétalisation des repas se développe une offre de substituts végétaux imitant les propriétés gustatives et nutritionnelles des produits carnés. Par ailleurs, les motivations à réduire la consommation de produits carnés ou à les remplacer par des substituts végétaux repose sur plusieurs arguments tels que la santé, l'environnement ou le bien-être animal. A travers deux études, nous explorons les préférences pour des substituts végétaux à la viande en associant les outils de l'évaluation sensorielles à ceux de l'économie expérimentale. Ainsi les sujets dégustent des produits réels et s'expriment à leur sujet, puis ils sont soumis à des choix relatifs à ces produits (maintient de la consommation ou retour à leur produit habituel). Nos résultats montre qu'un tiers des participants apprécient les substituts et qu'ils sont prêt à en renouveler la consommation.

Preferences & choices experiments with real products consumption: application with plant-based proteins. EXTENDED ABSTRACT

People say that they are interested by meat reduction or meat substitution for many arguments: health, environmental and animal welfare. On this basis, firms explore new models and new supply (better meat or meat substitutes) in order to take, to keep or to increase market shares. But what consumers really do when they face to the real product? In a study with German, French and English meat consumers, [START_REF] Michel | A multi-national comparison of meat eaters' attitudes and expectations for burgers containing beef, pea or algae protein[END_REF] compare expectations about taste, healthiness and environmental friendship of beef burger and two vegetable substitutes (pea and algae). These consumers expected that vegetable burgers are healthier and environmentally friendly but less tasty than beef burger.

A growing literature propose to study consumers preferences for meat substitutes that are close to meat attributes, as burgers, through choice experiments. [START_REF] Slade | If you build it, will they eat it? Consumer preferences for plant-based and cultured meat burgers[END_REF] conduct a discrete choice experiment (DCE) with five burgers (natural beef, plant-based proteins, cultured meat, each organic or not organic). 65% of their participants prefer the natural beef. The alternatives are preferred by vegetarians, younger, educated people. They underline that women prefer the plant-based proteins (PBP). [START_REF] Loo | Consumer preferences for farm-raised meat, labgrown meat, and plant-based meat alternatives: Does information or brand matter?[END_REF] conduct a DCE with 4 burgers: natural beef, pea alternative, yeast alternative and cultured meat. They consider four treatments depending on informational context: no information, information on brand, information on environmental impact of meat and alternatives, and information on technologies used for alternatives and cultured meat. 72% of participants prefer natural beef. Information has a side effect: consumers quite the market (no choice). Alternatives are preferred by vegetarians, males, younger, educated peoples. [START_REF] Carlsson | How much does it take? Willingness to switch to meat substitutes[END_REF] conduct a choice experiment with 4 burgers: natural beef, plant-based proteins, meat-like, cultured meat. They first endow people with a natural beef hamburger and propose them to change for an alternatives, and ask what they do if the price will change. The price matter: participants switch from natural beef to alternatives at 2/3 of price. 50% of participants do not want cultured burger even if free. Environmental and health consciousness support substitution. [START_REF] Apostolidis | To meat or not to meat? Comparing empowered meat consumers' and anti-consumers' preferences for sustainability labels[END_REF] conduct a DCE on 500g of products with various attributes: fat content, carbon footprint, method of production, type of mince, brand, region of origin, price. They consider 3 groups of consumers: meat eaters, meat reducers, vegetarians. They observe that meat reducers have higher health than environmental motivations. They underline the importance to know more on empowerment and motivation of consumers for better targeted policies (price, information, campaigns, education, labels, etc.). The results of these works are based on hypothetical choices and hypothetical products. If in panels some products really exist, it is not the case for others.

Another growing literature includes real tasting with sensory evaluation and some valuation of products and information (purchase preference -PP-, willingness to buy -WTB-, willingness to pay -WTP-). [START_REF] Grasso | Effect of information on consumers' sensory evaluation of beef, plant-based and hybrid beef burgers[END_REF] evaluate sensory dimensions of three burgers (100% beef, 100% plant-based, hybrid with 60% beef and 40% vegetables) and include WTB and WTP question (even if hypothetical). In a blind tasting condition, the hybrid product is largely preferred to the two others, and the score of linking increase a little with information about product. The plant-based burger is significantly less preferred in sensory evaluations, and in WTB and WTP evaluations. [START_REF] Martin | Importance of additional information, as a complement to information coming from packaging, to promote meat substitutes: A case study on a sausage based on vegetable proteins[END_REF] compare au pork-based sausage with a plantbased sausage in blind tasting and in tasting with packaging information. They introduce some health and environmental information too. They measure preferences, PP and WTP. The meat is preferred to the vegetable substitute, but information from the packaging and information of health or environmental issues decrease the gap between the two products. The WTP with all the information are very close. [START_REF] Bazoche | Does the provision of information increase the substitution of animal proteins with plant-based proteins? An experimental investigation into consumer choices[END_REF] study the substitution between a meat meal and a vegetarian alternative (with comparable products as couscous, chili or pasta) when introducing information about health and environment. At the first stage, before any information, between 15% and 21% of participants choose the vegetarian meal. In the third stage, after two waves of information, the shares of vegetarian choice are from 24% to 35%. Those who react the more are in the group receiving environmental information (a little less for the group with health information). The study identifies a share of population who are information sensitive but does not find a strong average effect of information on vegetarian choices. These results are strong arguments to include real products or real choices in studies in order to better understand the preferences for alternatives to meat consumption.

The studies we present include real products that consumers really eat. Our question is if consumers are ready to substitute their usual meat product by a plant-based proteins (PBP) product. The first study is based on a sensory evaluation and a choice experiment with the same products. The second study introduce the PBP product in a real context of consumption and a real choice between new product and usual product.

The two are conducted at home with real consumers for two reasons. First, we wish to get closer to the real context of consumption. Second, we adapt the design to the Covid-19 restrictions. All participants answer in an online questionnaire which guide them in the design. The participants are consumers of sliced products, take part of food purchases in their home. They could be omnivore or flexitarian (restricted-omnivore), but not vegetarians. The products tested are sliced products from meat or PBP. The two studies use methods from sensory evaluation and economic choices.

Study 1 is divided in two part. The first part is a sensory evaluation of various PBP products (experimental processed food) declined in two aromas and three forms (slices, dices, sticks). 6 samples are given to participants with a drive process. The tests of products are made at home following close laboratory instructions (blind tasting). Participants answered to three questions:

(1) General liking of the product, (2) Appearance / color liking of the product, and (3) Open question on improvment on appearance / color. The second part is a discrete choice experiment. The decisions are hypothetical but based on the products subjects just ate. Three products presented: 2 slices of ham (pork), 2 slices of chicken, and 2 slices of plant-based proteins. Products could have five prices: 0.95€ / 1.07€ / 1.19 € / 1.31€ / 1.43€. The full design contains 243 choices. We use a reduction of 8 questions of choice between three options without opt-out option (participants are product consumers and they just test the plant-based product). 101 subjects form the Dijon area participated to this study in June 2020. They are 49% women and the average age is 42. They are selected for their meat reduction behavior. They could conduct this with various and multiple ways (multiple answers): 73.3% do not eat meat at each meal, 24.7% reduice the size of portions, and 20.8% do a day without meat.

Study 2 included the same product but only in slices of two aromas (experimental processed food). The first week of experiment, participants received a number of slices of the PBP based on their week habit consumption (multiples of two) though a drive. Participants are invited to consume the product as they do for their usual product. They follow the online instructions for questions: Description of the meal (photo), number of people, general liking (+ appearance, smell, meal), and have a choice about the product for the second week. The day of consumption, we ask them the following non hypothetical question: "For the week 2, do you prefer to receive (at the drive) the same product (PBP) or do you prefer to receive your usual product?" (we have the information of the usual product, but for logistical reason we give them a gift card in supermarket). We add hypothetical question on what could change their choice. The second week, they receive the PBP product or their usual product (gift card). 166 subjects from Dijon area participated to this second study in December 2020. They are 55% women and the average age is 42 years. They are selected on their meat consumption (omnivore or restricted omnivore / flexitarian): 74.7% declare to have omnivore diet and 25.3% declare to have flexitarian diet.

We present the preliminary results of two studies on choices with plant-based meat-substitut which could be tasted by consumers (non-vegetarians). In study 1, after blind tasting of PBP, there is no significant reject of this kind of product when consumers have the choice. We observe a negative price effect, and a potential substitution between pork ham and PBP. In study 2, we observe the consumption of the PBP in a natural environment and choice between keep or quite. 38% keep the PBP product. Some additional characteristics (origin, organic or not, nutritional facts, environmental impact) could be of interest for consumers. We do not observe significant effects of traditional socio-demographics. In conclusion, even when the produce is consumed, a market exits, with flexitarians and with omnivores too.