# Bacterial flotillins as destabilizers of phospholipid membranes

Ana Álvarez-Mena, Estelle Morvan, Denis Martinez, Melanie Berbon, Abigail Savietto Scholz, Axelle Grélard, Sarah Turpin, Erick Dufourc, Marc Bramkamp, Birgit Habenstein

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Ana Álvarez-Mena, Estelle Morvan, Denis Martinez, Melanie Berbon, Abigail Savietto Scholz, et al.. Bacterial flotillins as destabilizers of phospholipid membranes. Biochimica et Biophysica Acta:Biomembranes, 2024, 1867 (1), pp.184399. 10.1016/j.bbamem.2024.184399. hal-05383444

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# Bacterial flotillins as destabilizers of phospholipid membranes

Ana Álvarez-Mena<sup>a,b,1</sup>, Estelle Morvan<sup>b,1</sup>, Denis Martinez<sup>a</sup>, Melanie Berbon<sup>a,b</sup>, Abigail Savietto Scholz<sup>c</sup>, Axelle Grélard<sup>a,b</sup>, Sarah Turpin<sup>a</sup>, Erick J. Dufourc<sup>a,b</sup>, Marc Bramkamp<sup>c,d,*</sup>, Birgit Habenstein<sup>a,b,**</sup>

## ARTICLE INFO

Keywords:

Solid-state NMR

Flotillin

Membrane order

Membrane fluidity

Deuterium NMR

Phosphorus NMR

## ABSTRACT

From archaea to mammals evolutionary conserved flotillins are scaffolding proteins, recognized for their nandomain-segregating activity. Flotillins form basket-like oligomeric architectures on the membrane, based on a conserved secondary structure composition of the monomeric subunits: a membrane-targeting region, an SPFH domain and a coiled-coil "flotillin" domain. In *B. subtilis*, the two flotillins FloT and FloA are present, localizing mainly in distinct nanodomains and executing multiple cellular functions. We here use deuterium and phosphorus solid-state NMR to monitor the effect of the different flotillins FloT and FloA and their structural components on model membranes. We find a clear disordering effect of FloT and FloA on the membranes reaching the carbon positions in the centre of the membrane. This effect is imposed by the hydrophobic region and the adjacent SPFH domain and, surprisingly, further supported by the membrane-distant flotillin domain. Biological implications of this disordering action are discussed.

## 1. Introduction

Flotillins are scaffolding proteins involved in Alzheimer's and Parkinson's diseases [1] in humans, as well as in the maintenance of essential physiological conditions in bacteria [2]. They are conserved proteins associated with prokaryotic and eukaryotic membranes and are considered as membrane nanodomain markers because of their clear segregation into functional membrane domains (FMMs) [2,3]. The definition of membrane nanodomains is based on the longstanding concept of membrane "rafts", introduced by Simons and Ikonen[4], describing dynamically segregated membrane regions, enriched in specific lipids and proteins[5]. In prokaryotes, these membrane regions are highly dynamic and involved in adaptive cellular processes such as sporulation, biofilm formation and cell wall biosynthesis [3,6-8]. In contrast to eukaryotic flotillins, which associate with membrane domains due to posttranslational modifications such as myristoylation or palmitoylation, prokaryotic flotillins are non-acylated [9].

Research on flotillin proteins has predominantly focused on Gram-positive bacteria, while the role of these proteins and their involvement in lipid raft organization in Gram-negative species remains relatively unexplored. Most studies on bacterial flotillins have been conducted using the well-established model *Bacillus subtilis*, which produces two flotillins, FloA and FloT. Notably, the closest homologue to eukaryotic flotillins is *B. subtilis* FloT, which shares 35.4 % identity (67.1 % homology) with the *Mus musculus* flotillin (Suppl. Fig. 1) [10]. Both bacterial flotillins, FloT and FloA, segregate into 85-110 nm-sized domains and modulate lipid segregation; however, they do not co-localize *in vivo* and exhibit different protein domain architectures [11].

When comparing the conservation of FloT and FloA in phylogenetically diverse species (Suppl. Figs. 2 and 3), it is noteworthy that some species have both flotillins while others possess only one. This variation aligns with the differing lipid compositions within FMMs among bacteria, leading to significant differences in membrane organization. For example, the human pathogen *Bacillus cereus* and the oral commensal

<footnote><sup>a</sup> Institute of Chemistry & Biology of Membranes & Nanoobjects (UMR5248 CBMN), IECB, CNRS, Université Bordeaux, Institut Polytechnique Bordeaux, Pessac, France</footnote>

<footnote><sup>b</sup> Univ. Bordeaux, CNRS, Inserm, IECB, UAR3033, US01 Pessac, France</footnote>

<footnote><sup>c</sup> Institute for General Microbiology, University of Kiel, Am Botanischen Garten 1-9, 24118 Kiel, Germany</footnote>

<footnote><sup>d</sup> Centrale Microscopy, University of Kiel, Am Botanischen Garten 1-9, 24118 Kiel, Germany</footnote>

<footnote>* Corresponding author at: Institute for General Microbiology, University of Kiel, Am Botanischen Garten 1-9, 24118 Kiel, Germany.</footnote>

<footnote>** Corresponding author at: Institute of Chemistry & Biology of Membranes & Nanoobjects (UMR5248 CBMN), IECB, CNRS, Université Bordeaux, Institut Polytechnique Bordeaux, Pessac, France.</footnote>

<footnote>E-mail addresses: bramkamp@ifam.uni-kiel.de (M. Bramkamp), b.habenstein@cbmn.u-bordeaux.fr (B. Habenstein).</footnote>

<footnote n="1">¹ These authors contributed equally.</footnote>
*Streptococcus sanguinis*, conserve only a FloT-like flotillin (Suppl. Fig. 2), with approximately 63 % and 36 % of amino acid identity, respectively, compared to FloT from *B. subtilis*. In contrast, the Firmicutes *Listeria monocitogenes* (62 % amino acid identity) and *Staphylococcus aureus* (84 % amino acid identity) encode orthologs specifically for FloA flotillin (Suppl. Fig. 3). Conversely, the versatile opportunistic pathogen *Pseudomonas aeruginosa* encodes both FloA- and FloT-like proteins (Suppl. Figs. 2 and 3), suggesting a more complex organization.

In bacteria, peptidoglycan synthesis regulates the bacterial cell shape, making the temporal and spatial control of its synthesis essential during cell elongation and division. We have recently proposed that *B. subtilis* flotillins directly control membrane fluidity and that reduced fluidity in absence of flotillins reduces the mobility of the cytoskeleton protein element MreB and impacts on peptidoglycan synthesis. *In vivo*, cell membranes lose fluidity in the absence of FloT and FloA, while the fatty acid composition related to membrane fluidity remains identical, shifting peptidoglycan synthesis to the cell division site. Our results, supported by ²H-detected solid-state NMR data, suggested that the effect of flotillins on membrane fluidity should be direct [12].

FloT contains a 21-amino-acid membrane-targeting hydrophobic sequence, whereas FloA's sequence includes at least two membrane-targeting regions, separated by a α-helix breaking Proline. We have previously demonstrated that flotillins oligomerize into multimers of various sizes, driven by the multifunctional SPFH (Stominat, Prohibitin, Flotillin, and HflK/C) domain [2]. For eukaryotic proteins, SPFH domains have been proposed to facilitate protein-lipid interactions in addition to their role in SPFH oligomerization [13]. On the C-terminal end, both FloT and FloA encompass a conserved oligomerization domain adjacent to the SPFH-domain, also termed the flotillin domain. This domain, enriched in amino acids EA regions, is predicted to adopt an α-helical secondary structure [14]. Precise structures of bacterial flotillins are unknown; eukaryotic flotillins assemble into heteromeric oligomers, composed of Flot1 and Flot2, as proposed by two recent models (<4 Å resolution) derived from cryo-electron microscopy [15]. Based on the eukaryotic flotillin model, the coiled-coil motif, which varies in length between FloA and FloT, should therefore confer additional oligomerization properties [16].

We here aim at delineating the contribution of FloA and FloT and their common architectural components, SPFH and flotillin domain, to membrane-association and fluidity control. We have implemented the heterologous production and purification of *B. subtilis* FloT and FloA and truncated versions of FloT and FloA, lacking their flotillin domains. Changes in membrane fluidity were assessed by ²H and ³¹P NMR of multilamellar vesicles, probing both the hydrophobic zone at the core of the bilayer and the hydrophilic membrane surface. We show that both flotillins, FloT and FloA, increase the fluidity of phospholipid membrane vesicles, with FloT having a more pronounced fluidifying effect on the carbons along the acyl chain and a significantly greater impact on the membrane surface order. This membrane-fluidizing effect is controlled both by the flotillin domain of variable length and the membrane-binding domain size and composition.

# 2. Experimental section

## 2.1. Chemicals

1-palmitoyl-²H₃₁-2-oleoyl-sn-glycero-3-phosphocholine (²H₃₁-POPC) was purchased from Avanti Polar Lipids (Alabaster, Alabama, USA). Water deuterium depleted (H₂O, deuterium oxide ≤1 ppm) and CHCl₃ and methanol for liposome preparation were purchased from Sigma Aldrich Chemicals (Saint Quentin Fallavier, France).

## 2.2. Bacterial strains and culture conditions

The strain *Escherichia coli* DH5α was used for cloning and plasmid replication, and *E. coli* BL21(DE3) pLysS was used for protein expression

and purification. Both strains were grown in Luria-Bertani medium: 1 % tryptone (Oxoid; Thermo Fisher Scientific Waltham, MA, USA) 0.5 % yeast extract (Oxoid), and 0.5 % NaCl. The antibiotics used and their final concentrations were 100 µg/ml ampicillin and 50 µg/ml kanamycin.

## 2.3. Plasmid construction

For the heterologous expression of the truncated variants FloT (1–196) and FloA(1–231), the regions containing the open reading frames of *floT* from amino acid 1 to 196 and *floA* from amino acid 1 to 231, respectively, were cloned and inserted into the plasmid pET24b. The gene products were amplified using the primers forward AAAAAACATATGACGATGCCGATTATCATGATTATCGGG and reverse AAAAAACTCGAGGCCCTGAAAATACAGGTTTTCAAGA-TAACCGTCTTATCGCGTAC for FloT(1–196), and the primers forward AAAAAACATATGGATCCGTCAACACTTATGATTCTGGC and reverse AAAAAACTCGAGGCCCTGAAAATACAGGTTTTTCGATGTTTTTGCCGATATCTACATCTGC for FloA(1–231). The PCR products and the pET24b vector were digested with NdeI (New England Biolabs) and XhoI (New England Biolabs) and subsequently cloned using Quick Ligase (New England Biolabs), following the instructions supplied by the manufacturer. The reaction mixtures were transformed into *E. coli* DH5α, and colonies were selected with 50 µg/ml kanamycin. After plasmid purification, the vectors were sequenced for confirmation.

## 2.4. Cell growth and protein expression

A single fresh colony of *E. coli* BL21(DE3) pLysS transformed with the plasmid pET52b containing FloT or FloA, as well as pET24 containing FloT(1–196) or FloA(1–231) (Suppl. Fig. 4A and B), was picked and cultured in 10 ml of LB medium with 100 µg/ml ampicillin for FloT and FloA, and 50 µg/ml for FloT(1–196) and FloA(1–231) at 37 °C under shaking conditions until reaching the exponential growth phase. A total of 10 % (v/v) of the preculture was used to inoculate 1 l of LB supplemented with antibiotics. The culture was incubated at 37 °C and 200 rpm until the optical density at 600 nm reached 0.6, then induced with 1 mM isopropyl β-D-1-thiogalactopyranoside. Best expression conditions for FloT and FloA were found at 37 °C for 3 h, whereas expression for FloT(1–196) and FloA(1–231) was highest at 30 °C for 3 h. Cells were harvested (6000 g, 30 min, 4 °C, JLA 9.1 rotor; Beckman Coulter, Brea, CA, USA) and frozen at –80 °C until purification.

## 2.5. Protein purification

Purification of FloT, FloA, and the truncated variants FloT(1–196) and FloA(1–231), was performed according to the protocol described by Bach and Bramkamp (2013) [17]. Cell pellets were resuspended in buffer A (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl₂) with a protease inhibitor cocktail (Roche), sonicated on ice (3 × 30 s, 37 %, Bandelin Sonopuls VS70T probe; Bandelin, Germany), and centrifuged (15,000 g, 60 min, 4 °C, F34–6–38 rotor; Eppendorf, Hamburg, Germany). The pellet was resuspended in buffer A supplemented with 1 % Triton X-100 and incubated at room temperature in a rotator tube for 1 h. To separate the solubilized proteins from the membrane and cell fractions, the samples were ultracentrifuged (42,000 rpm, 1 h, 4 °C, 50.2 Ti rotor; Beckman Coulter), and the resulting supernatants were loaded onto a HisTrap HP 5 ml column (GE Healthcare, Waukesha, WI, USA). For equilibration, buffer A with 20 mM imidazole was utilized, and for elution, buffer A with 500 mM imidazole was employed (Suppl. Fig. 4C). Following affinity chromatography, the proteins were loaded on a HiPrep 26/10 desalting column (GE Healthcare) to exchange the buffer to buffer A with 0.05 % of Triton X-100. Purification fractions were loaded onto a 15 % Tris-Tricine SDS-PAGE to check purity, using the protein marker Mark12 (Thermo Fisher Scientific) as a reference.
## 2.6. Reconstitution of flotillin constructs into membrane vesicles

Liposomes containing POPC-²H₃₁ were prepared by mixing 3 mg of lipid powders in organic solvents (chloroform/methanol, 2:1 ratio). Solvents were evaporated under a stream of air and the residual lipid film dispersed in 1 ml of Milli-Q filtered water and lyophilized overnight. The fluffy lipid powder was hydrated with 100 µl of buffer A containing 10% glycerol and homogenized by three cycles of vortexing, freezing (liquid nitrogen, -196 °C, 1 min) and thawing (50 °C in a water bath, 10 min). This protocol generated a milky suspension of micrometer-sized multilamellar vesicles (MLV), which were used as a control. FloT, FloT(1-196), FloA, and FloA(1-231) were solubilized in buffer A supplemented with 0.05 % Triton X-100, then added to preformed liposomes (molar ratio 1:25) and incubated for 1 h at room temperature. A dialysis step was performed for at 4 °C against buffer A with agitation to remove the detergent. The same protocol was applied for control samples, lacking the protein in the buffer A solutions. Proteoliposome solutions all showed a turbid aspect, indicating particle size of approx. 0.1–1 µm. Samples were ultracentrifuged (42,000 rpm, 1 h, 4 °C, TLA 120.1 rotor; Beckman Coulter) to pellet the proteoliposome. Reconstitution efficiency of the proteins into liposomes was measured by measuring the remaining soluble protein concentration in the supernatant (Suppl. Table 1).

## 2.7. Solid-state NMR

²H NMR experiments were performed on a Bruker Avance III spectrometer operating at 77 MHz (²H) with either a 4 mm double resonance HX-CPMAS probe or a 4 mm double resonance HFX CPMAS probe. A phase-cycled quadrupolar echo sequence (90°xτ-90°y-τ-acq) was used to perform the ²H NMR experiments [18].

Acquisition parameters were as follows: spectral window of 500 kHz, π/2 pulse width of 3.90 or 3.50 µs, interpulse delays (τ) of 40 or 50 µs, recycle delays of 1.3 or 2 s for ²H, and 3000 or 4096 scans. Samples were equilibrated for 20 min at a given temperature before data acquisition. The temperature was regulated to ± 1 °C.

All spectra were processed and analyzed using Bruker Topspin 4.0 software. For the ²H NMR spectra, Lorentzian line broadening of 300 or 500 Hz was applied before Fourier transformation, starting from the top of the echo.

Orientational carbon-deuterium order parameters ($S_{CD}$) were calculated from experimental quadrupolar splittings ($\Delta\nu_Q$) as described by Seelig in 1977 [19].

To record undistorted ³¹P ssNMR spectra, we applied a Hahn spin echo sequence, 90°-τ-180°-τ-acq at the ³¹P frequency of 162 MHz on a 400 MHz (9.4 T) Bruker Avance III HD spectrometer [20], with a 90° pulse of 8 µs, an echo delay of 40 µs, a recycle delay of 5 s, a spectral window of 64 kHz, and a number of scans ranging from 1024 to 2048. All spectra were processed and analyzed using Bruker Topspin 4.0 software. Spectra were processed using a Lorentzian line broadening of 100 or 300 Hz for ³¹P NMR spectra.

## 2.8. Simulation of ²H and ³¹P solid-state NMR spectra

Wide-line deuterium solid-state NMR spectra were simulated by calculation in the time domain (as free induction decays) and then Fourier transformed, according to a FORTRAN code developed by Erick Dufourc and implemented in a user-friendly graphical interface (Microsoft.NET) for Windows platforms by Arnaud Grélard. Each deuterated position of the perdeuterated lipid chain is described by its quadrupolar splitting ($\Delta\nu_Q$) experimentally estimated, its intrinsic line width, and the number of deuterons at a given acyl chain position according to the molecular structure. Liposome deformation, leading to non-spherical distributions of bilayer normals with respect to the magnetic field, was taken into account in the simulation (c/a parameter) [21]. A comparison between the experimental spectra and the calculated

spectra is made until satisfactory superimposition is obtained. Such a simulation leads to individual quadrupolar splittings for each labeled carbon position and, subsequently, to the corresponding $S_{CD}$ order parameters in bilayer membranes. The calculation of deuterated chain length and, consequently, bilayer thickness is performed according to Douliez et al. [22,23] by using the $S_{CD}$ order parameters obtained from simulations. Error bars for order parameters are estimated for liposomes without protein (Suppl. Table 1) and for liposomes with added proteins (Suppl. Table 2) from representative triplicate experiments, respectively. A similar approach is used for ³¹P NMR spectra, where the experimental parameters are the chemical shielding anisotropy ($\Delta\sigma$) and the intrinsic linewidth. Liposome deformation can also be taken into account.

## 2.9. Phylogenetic tree and amino acid alignment

For the study of the phylogenetic distribution of flotillin proteins in eukaryotes, prokaryotes, and archaea, amino acid sequences were retrieved from the UniProt database and aligned using MEGA software [24] with the ClustalW algorithm. Evolutionary distances were then calculated using the Maximum Likelihood statistical method.

To align the amino acid sequences of FloT- and FloA-like proteins in prokaryotes, a multiple sequence alignment was performed using T-Coffee [25], and the results were visualized with JalView [26], highlighting the residues that show a degree of conservation >30 %. The specific UniProt accession codes for each bacterial species are as follows: *B. subtilis*: O32076 and P54466, *B. cereus*: Q81161, *S. sanguinis*: A3CLK3, *P. aeruginosa*: Q9I666 and Q9HXR3, *S. aureus*: Q7A5C5, *L. monocytogenes*: Q92EP8, *C. botulinum*: B1KVL6, *C. crescentus*: Q9A331, *N. meningiditis*: A0A0U1RJ00, *C. jejuni*: Q0PBN7.

# 3. Results

## 3.1. Production and purification of specific flotillin constructs

The two distinct *B. subtilis* flotillins, FloT (509 amino acids; pI 5.14) and FloA (331 amino acids; pI 4.92), share a similar domain composition in their amino acid sequences; nevertheless, FloT has a larger flotillin domain and a higher number of both positively and negatively charged residues overall (Fig. 1).

To date, only low resolution structural data have been published for bacterial flotillins very recently [3], in contrast to eukaryotic flotillins, for which recent cryo-EM investigations have allowed to build a model [15,27]. However, for the SPFH domain and M region, these models are based on AlphaFold predictions [28].

AlphaFold predicts that the highly hydrophobic M-region, located in the N-terminal domain of both proteins, is in an α-helical conformation (Fig. 2A). Interestingly, the significantly longer M-region of FloA is predicted to contain the beginning of a short α-sandwich extending into the SPFH domain, in contrast to FloT, which is predicted to have an SPFH domain with the generally reported sheet structure in the SPFH1 domain [29].

The similar segment (SPFH2 domain) between FloA and FloT within the SPFH domain is predicted to contain a β-sheet composed of several strands intercalated with α-helices, in agreement with previously solved structures [27,30,31] (Fig. 2A). Importantly, some regions are predicted with low confidence, and lipid interactions are not considered *per se*.

With the aim of comparing the effect of FloA and FloT on membrane association and fluidity, and distinguishing the impact of the M- and the SPFH-region and the flotillin domain on these effects, we successfully implemented the production of full-length FloT and FloA, and their truncated versions lacking the flotillin domain, FloT(1-196) and FloA (1-231), in *E. coli*. We adapted the purification method established in Zielińska et al. [12], achieving a production of 10–20 mg of pure FloT, FloA, FloT(1-196) and FloA(1-231) (Fig. 2B). Finally, we pre-formed POPC-²H₃₁ MLV (Fig. 2C) and incubated the proteins on preformed
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Strategic flotillin constructs including truncated versions lacking the flotillin domains. Schematic representation of the domain architectures of FloT and FloA compared to their truncated versions, FloT(1–196) and FloA(1–231). The membrane-targeting region (M) is shown in lavender, the SPFH domain in orange, and the flotillin domain in dark purple (upper panel). Amino acid sequences of FloT and FloA from *B. subtilis*, with positively charged residues marked in red and negatively charged residues marked in blue. Regions corresponding to each domain are highlighted with underlining, following the same color code as described above (lower panel). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

membranes at room temperature to reconstitute the different proteins in a lipid bilayer environment in the fluid lamellar phase (POPC-$^2$H$_{31}$ phase transition Tm is approx. -2 °C).

## 3.2. ssNMR report on flotillin-membrane interactions

The effect of FloT and FloA on MLVs is monitored by $^2$H and $^{31}$P ssNMR (Figs. 3 and 4). Deuterium NMR reports on the hydrophobic central region of the membrane, while phosphorus NMR reports on the membrane surface.

The spectrum of the control $^2$H$_{31}$-POPC shows the classical non-oriented axially symmetric spectrum in the fluid phase (also known as liquid-disordered, ld), where ~ 10 quadrupolar doublets can be detected. They correspond to differential ordering characteristics of the lipid chain. The widest splitting is assigned to the more rigid part of the chain, labeled positions 2 to 7, which are close to the very rigid glycerol backbone, whereas the smallest corresponds to position 16, the very disordered deuterated methyl group [19,32,33]. The central isotropic line corresponds to residual $^1$HO$^2$H, shifted from the spectrum center by 3–4 ppm ($^2$H), i.e., ~300 Hz. In the presence of FloT, the overall spectrum (Fig. 3A, center) is much narrower, and the quadrupolar doublets are less resolved. This indicates a very significant disordering effect of FloT on lipid chains (*vide infra*), consistent with previous results. The effect of the truncated form FloT(1–196) on membranes is also reflected in a reduction in the spectral width (Fig. 3A, center) of the control MLV spectrum, although to a lesser extent compared to the wild type.

FloA also has a fluidifying effect on $^2$H$_{31}$ POPC MLV (Fig. 3B, center), similar to that of FloT, though the reduction in width is notably less pronounced. The truncated form of FloA also reduces the spectrum width (Fig. 3B, top), but only marginally, showing a less significant effect than the wild-type form of FloA. These observations clearly indicate a membrane fluidizing effect of FloA, although less pronounced compared to that of FloT.

The $^{31}$P NMR spectra reflect interactions of the proteins at the membrane interface. The control spectrum displays axial symmetry, although the spectrum itself is asymmetric. This indicates that the phosphate head group is embedded in a lamellar fluid phase (inherently axially symmetric). The chemical shielding anisotropy ($\Delta\sigma$) is -46.6 ppm, reflecting a fairly large headgroup dynamics in a micrometer-sized MLV [34,35].

In the presence of FloT (Fig. 4A, center), the spectrum is composed of a powder pattern, reduced in width (-42.0 ppm), plus a small isotropic line (centered at 0 ppm), which corresponds to a few percent of the total spectral area. The latter indicates that a few percent of the system consists of vesicles of reduced size (hundreds of nm) that undergo isotropic tumbling, which reduces the chemical shielding anisotropy to zero [36,37]. The rest of the system remains in the form of micrometer-sized MLV. In the presence of the truncated form of FloT, the isotropic line is not present, and the $^{31}$P NMR powder spectrum shows minimal change in $\Delta\sigma$ (45.7 ppm) compared with the control.

For FloA (Fig. 4B, center and top), the $^{31}$P spectrum shows an isotropic line of a few percent and a powder pattern of reduced width
<figure><img src="image_3.png" /></figure>

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

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

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

<figure><img src="image_7.png" /><figcaption>**Fig. 2.** A AlphaFold [28] structure prediction models for FloT and FloA, with color patterns based on the pLDDT confidence score for each residue (upper panel). AlphaFold structure predictions are colored according to domain distribution, following the same color pattern as depicted in Fig. 1 (lower panel). B) Bands on a 15 % SDS-PAGE gel corresponding to the elution fractions from the purification of FloT, FloT(1–196), FloA, and FloA(1–231) under soluble conditions using affinity chromatography. C) Chemical structure of the lipid POPC-$^2$H$_{31}$, used to reconstitute MLVs. For $^2$H solid-state NMR, the palmitic chain, containing 14 methylene groups (C-$^2$H$_2$) and one methyl group (C-$^2$H$_3$), is labeled.</figcaption></figure>

($-42.0$ ppm) compared with the control. Only slight spectral differences are observed between the full-length and truncated form of FloA ($-43.2$ ppm).

In summary, the disordering action detected by $^2$H NMR is also observed in the head group. Additional information is that a few percent of the system consist of small vesicles, an information that is difficult to obtain with $^2$H NMR due to the large number of splittings and a loss of resolution. $^{31}$P NMR complements carbon-deuterium order parameter analysis because the $^{31}$P lineshape depends on interactions with molecular partners and does not report on dynamics only, which we intend deriving from the carbon-deuterium order parameter. For example, the lesser fluidifying effect of FloT(1–196) on the $S_{CD}$ near to the glycerol backbone might arise from less fluidifying interactions of the FloT (1–196) complex structure with the acyl chains locally while no such effect is observed on the phosphorus headgroups.

### 3.3. Impact of FloA and FloT on membrane ordering

The reduction in spectral width observed in both deuterium and $^{31}$P NMR clearly reflects a disordering effect promoted by flotillins. Spectral simulations can be used to quantify this effect. Phosphorus-31 spectra show a slight reduction in $\Delta\sigma$, which can be interpreted as an increase in headgroup dynamics and/or a change in the orientation of the lipid headgroup. In view of the above, the disordering of the lipid head group appears to be the most probable. While the distinct effects of flotillin constructs on lamellar membranes reflect their structural behavior, an additional parameter contributing to the effect is related to the efficiency with which flotillin constructs can integrate the membrane. We therefore assessed the efficiency by measuring the relative protein quantity in the supernatant after recovering the pellet for solid-state NMR analysis, indicating 10–14 % of the proteins in the supernatant. Considering the small differences of incorporation efficiencies into

liposomes between the constructs (Suppl. Table 1), these differences could not account for the differences between the effects of the proteins on liposome fluidity. Deuterium NMR provides a detailed description of the fatty acyl chain ordering, i.e., the time and space angular fluctuations of C—D bonds. Fig. 5 shows a plot of the deuterium carbon order parameters, $|S_{CD}|$, as a function of the position of the labeled carbon, from position 2, close to the glycerol backbone, to position 16, in the center of the bilayer. For convenience, $2*|S_{CD}|$ is plotted. A value of 1 indicates full rigidity, meaning the absence of intermolecular or molecular motions (with C—D planes oriented at 90° with respect to the normal of the bilayer), whereas a value of 0 depicts a situation similar to a liquid environment, where all chain conformers are equally probable [19,22,33]. Fig. 5 shows a decrease of C—D order parameters from the glycerol backbone to the center of the bilayer for all systems. The decrease is not linear, as a “plateau” (quasi-constant order) of relatively high order parameters is observed down to position 7–9. This reflects the tight packing of lipids chains due to favorable van der Waals interactions, conferring a fluid (order below 1) and stable character to the membrane. Moving towards the center of the membrane, down to position 16, the order decreases considerably, indicating a highly dynamic membrane center.

In the presence of full-length FloT (Fig. 5A), $|S_{CD}|$ for all labeled positions shows an overall decrease of around 23 %, indicating that the entire aliphatic chain becomes highly disordered. The truncated version of FloT exerts almost the same disordering action, with a lesser impact on positions close to the glycerol backbone.

For FloA (Fig. 5B), the full-length protein also exerts a disordering effect on all labeled positions, amounting to around 15 %. Here again, the truncated form of FloA is much less effective at inducing chain disorder near the glycerol backbone and remains less effective moving towards the center of the membrane.
<figure><img src="image_8.png" /><figcaption>Fig. 3. Comparison of $ ^{2}H $ quadrupolar spin echo spectra of $ ^{2}H_{31}$-POPC at 298 K, analyzing the absence and presence of flotillin proteins and their truncated variants at a 1:25 protein-to-lipid ratio. Panel A, from bottom to top: $ ^{2}H_{31}$-POPC MLV $ ^{2}H$ NMR spectra, control and in the presence of full-length FloT and truncated FloT (1–196). Panel B, from bottom to top: $ ^{2}H_{31}$-POPC MLV $ ^{2}H$ NMR spectra, control and in the presence of full-length FloA and truncated FloA(1–231). Spectral simulations are represented by a red dashed line. Vertical black dashed lines serve as guides, aligned with the widest splitting of the control spectrum. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

## 4. Discussion

FloT and FloA are intimately associated with numerous bacterial cell functions. While mainly localizing in distinct membrane nanodomains, the structures and functional mechanisms of flotillins are still unclear [12]. In Zielinska et al.[12], we have shown that in fast growing cells (B. subtilis) the absence of FloT and FloA engenders decreased membrane fluidity and a reduction in elongasome-mediated peptidoglycan synthesis. Based on an initial $ ^{2}H $ analysis, we have suggested that FloT has a direct effect on membrane fluidity. Here, we achieved the production of milligram-quantities of pure B. subtilis flotillins FloT and FloA, and their truncated versions FloT(1–196) and FloA(1–231), lacking the so-called flotillin domain. We show that all flotillin constructs disorder the already highly fluid POPC membrane, further revealing the contributions of the flotillin domains in FloT and FloA. Furthermore, our data show that flotillins can induce an isotropic phase, as reflected in $ ^{31}P$ NMR spectra, possibly reporting on the creation of small vesicle by flotillin oligomers. All of the above-mentioned points will be discussed below, and set in the context of their structural and functional implications.

### 4.1. *The membrane-disordering action of flotillins*

The most important discovery is the significant disordering effect of all flotillins, FloA, FloT, FloA(1–231) and FloT(1–196) on POPC MLV,

with FloA(1–231) showing a much less important impact on membrane order. The full-length FloT and FloA exert the greatest disordering effect, with a 23 % and 15 % decrease in order, respectively. This effect is observed for all methylene groups in the chain. This effect is synonymous with membrane shrinkage [22,23], i.e., in the presence of flotillins, the lipid bilayer becomes thinner. It should be noted that POPC at 298 K is already in a very fluid phase. This indicates that all flotillin constructs can be considered as powerful membrane destabilizers. Such an effect has already been observed with small amphipathic peptides of 20–30 amino acids, which act as membrane wedges [38,39], i.e., by intercalating between lipids, giving aliphatic segments more room to fluctuate and thereby lowering the order. The N-terminal amino acids of FloT and FloA are hydrophobic, and the putative helical segment would be a good candidate for initiating such a wedge. When considering the structures predicted by Alphafold, FloT contains a small membrane-adjacent β-sheet, the SPFH1 domain, recently reported to interact with the membrane for a distinct SPFH domain containing protein [29]. Indeed, in FloT, this region contains several hydrophobic residues which are potential membrane-affine candidates. Such insertion of these residues could further increase a wedge effect on the membrane surface. When considering the potential wider implication of SPFH domain containing proteins, which might generally follow similar principles of structuration and assembly, the membrane-fluidifying effect could reflect a broad feature of SPFH-domain proteins [29]. However, the situation for FloA is very different when considering the membrane-
<figure><img src="image_9.png" /><figcaption>**Fig. 4.** $ ^{31}P $ solid-state NMR analysis (black line) of POPC-$ ^{2}H_{31} $ liposomes, with or without *B. subtilis* flotillin proteins: FloT and FloT(1–196) in A, and FloA and FloA (1–231) in B, at a lipid-to-protein molar ratio of 25:1, performed at 298 K. The red dashed line corresponds to the simulation performed for each experimental condition. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

adjacent domain. While to be considered with caution because of the low confidence score, AlphaFold predictions of FloA indicate the formation of a distinct structural motif including α-helical segments on the membrane surface (Fig. 2A), potentially responsible for the minor disordering effect of FloA(1–231).

By removing the flotillin domain (residues 196–509) of FloT, potentially contributing to flotillin oligomerization properties, the disordering effect is still present but less pronounced on the membrane surface. However, when considering the regions in the membrane center, the effect of the flotillin domain to disordering is negligible; that is the effects of FloT(1–196) and full-length FloT are comparable. It seems that this highly hydrophilic part, although not expected to interact with membranes, contributes to the disordering effect. A possible explanation could lie in the more stable high-order oligomeric, possibly cone-like, assembly [15].

FloA is much less active, despite having around 49 hydrophobic amino acids at the N-terminal beginning of its sequence. The putative membrane part may not have a similarly efficient conformation to penetrate the membrane core and act as a wedge. Interestingly, the flotillin domain (231–331) of FloA is very hydrophilic, as in FloT, and is necessary for creating more disorder in the membrane. FloA(1–231), dissimilar to FloT(1–196), is consequently creating less disorder along the acyl chain in contrast to the full-length protein. These results are consistent with both the difference in length of the flotillin domain, which results in a less pronounced effect in FloA containing the significantly shorter flotillin domain (by 213 amino acids), and the suggested differences in the structure and conformation of the M-region adjacent

to the first predicted helical segment (SPFH1-domain). Nevertheless, it seems that the common disordering action of FloT and FloA is fostered by the hydrophobic part and, somewhat counterintuitively, by the flotillin segment. Possible future studies could aim at detecting any synergistic action, when incorporating both FloT and FloA in MLVs simultaneously.

## 4.2. Are flotillins inducing non-lamellar phases?

We have shown that the addition of FloA and FloT to MLV has produced isotropic lines in $ ^{31}P$-ssNMR spectra. Although the amount is only a few percent relative to the total spectral area (NMR is quantitative), this is a significant feature. The characteristics can be attributed to non-lamellar phases or to very small vesicles that average to zero the chemical shielding anisotropy either through rapid diffusion of individual molecules over a highly curved surface or through isotropic tumbling of the small objects themselves (as in micelles). Interestingly, in contrast to FloT(1–196), the truncated FloA(1–231) still evokes a certain percentage of a distinct membrane state (non-lamellar or small vesicles).

## 4.3. Potential biological implications

Taken together, our results demonstrate a conserved fluidifying effect of flotillin homologues on model membranes in the liquid disordered lamellar phase. Because our recent results indicated an *in vivo* validity of membrane fluidity-increasing effect of FloT in *B. subtilis* [12],
<figure><img src="image_10.png" /><figcaption>**Fig. 5.** Carbon-deuterium order parameters of $^2$H$_3$-POPC MLV in the absence and presence of flotillins, at *T* = 298 K. Panel A: FloT full-length and truncated. Panel B: FloA full-length and truncated. Flotillins:lipid molar ratio is 1:25. Error bars represent the standard deviation estimated for each carbon position (Suppl. Fig. 5, Suppl. Table 2 and 3).</figcaption></figure>

both fluidizing effects and their *in vivo* validity are likely relevant for the
here-investigated FloA and bacterial flotillins in general. It has recently
been proposed that proteins containing SPFH domains generally oligo-
merize into basket-like multimeric structures with similar structural
features [29], further supporting a hypothesis that flotillins might have
similar effects on membranes. This line of reasoning is more challenging
to be extended to eukaryotic flotillins because of their acylation sites,
potentially conferring distinct effects on the membranes. Because of
their very important effect on membrane fluidity and their capacity of
creating a non-lamellar membrane phase or small vesicles, it is tempting
to hypothesize that also bacterial flotillins could be involved in mem-
brane shaping or the coordination of intracellular compartments as
observed for the eukaryotic flotillins reported which shape the mem-
branes such as during endocytosis [40] and exosome secretion [41].

A basket-like structure with the smaller opening towards coiled-coil
flotillin domain, would explain the quantitative difference in the effects
of FloA and FloT on the membrane, due to the reinforced closing in FloT.
However, simply the size of FloT oligomers *versus* FloA might explain
this quantitatively distinct but qualitatively similar impact.

When considering solely the truncated versions FloT(1–196) and
FloA(1–231), lacking the flotillin domain, FloT(1–196) maintains its
disordering impact towards the inner membrane while this is signifi-
cantly reduced on the membrane surface, suggesting that the disorder-
ing effect on the inner membrane positions depends rather on the SPFH
domain and the M-region. Consistent with a different predicted structure
of the M-region and SPFH1 domain in FloA, the global disordering effect
of FloA depends on all domains, including the flotillin domain on all

positions over the membrane.

Interestingly, FloA(1–231) still demonstrates activity in forming a
non-lamellar phase or small vesicles in contrast to FloT(1–196). This
might further support the potentially different roles and functional
states of both flotillins, in which cellular conditions having a destabi-
lizing effect on the flotillin domain, such as local pH, ion concentration
or molecular partners, would differently affect FloA and FloT.

CRediT authorship contribution statement

**Ana Álvarez-Mena:** Writing – review & editing, Writing – original draft, Investigation, Formal analysis, Data curation. **Estelle Morvan:** Writing – review & editing, Writing – original draft, Investigation, Formal analysis, Data curation. **Denis Martinez:** Writing – review & editing, Investigation, Formal analysis, Data curation. **Melanie Berbon:** Writing – review & editing, Methodology, Investigation. **Abigail Savietto Scholz:** Writing – review & editing, Investigation. **Axelle Grélard:** Writing – review & editing, Investigation, Formal analysis, Data curation. **Sarah Turpin:** Writing – review & editing, Investigation. **Erick J. Dufourc:** Writing – review & editing, Writing – original draft, Investigation, Formal analysis, Data curation. **Marc Bramkamp:** Writing – review & editing, Writing – original draft, Validation, Formal analysis, Conceptualization. **Birgit Habenstein:** Writing – review & editing, Writing – original draft, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.

Declaration of competing interest

The authors declare the following financial interests/personal re-
lationships which may be considered as potential competing interests:

Birgit Habenstein reports financial support was provided by French National Research Agency. Birgit Habenstein reports financial support was provided by Foundation of the House of Chemistry. Marc Bramkamp reports financial support from the Deutsche Forschungsgemeinschaft. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the French National Research Agency ANR (BH, grant No. ANR-19-CE13-0021 and BH, grant number ANR-23-CE11-0005-01). The project has benefitted from the support of the Centre National de la Recherche Scientifique CNRS Momentum (BH), the "Fondation de la Maison de la Chimie" (BH) and the Biophysical and Structural Chemistry Platform at IECB, CNRS UAR 3033, INSERM US001. We acknowledge financial support from the Deutsche Forschungsgemeinschaft (grant BR 2915/7-1 to Marc Bramkamp).

**Appendix A. Supplementary data**

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bbamem.2024.184399.

Data availability

Data will be made available on request.

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