Two-Step Membrane Binding of NDPK-B Induces Membrane Fluidity

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The two-step membrane binding of NDPK-B induces membrane fluidity decrease, changes in lipid lateral organization and protein cluster formation Liberty François-Moutal, Myriam Marie Ouberai, Ofelia Maniti, Mark E. Welland, Agnieszka StrzeleckaKiliszek, Marcin Piotr Wos, Slawomir Pikula, Joanna Bandorowicz-Pikula, Olivier Marcillat, and Thierry Granjon Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b03789 • Publication Date (Web): 06 Nov 2016 Downloaded from http://pubs.acs.org on November 10, 2016

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The two-step membrane binding of NDPK-B induces membrane fluidity decrease, changes in lipid lateral organization and protein cluster formation Liberty Francois-Moutal1, Myriam M. Ouberai2, Ofelia Maniti1, Mark E Welland2, Agnieszka Strzelecka-Kiliszek3, Marcin Wos3, Slawomir Pikula3, Joanna Bandorowicz-Pikula3, Olivier Marcillat1, and Thierry Granjon1 1

Organisation et Dynamique des Membrane Biologiques, Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, CNRS UMR 5246 ICBMS, Bâtiment Chevreul, 43 Bd du 11 Novembre 1918, 69622 Villeurbanne cedex, France. 2 University of Cambridge, Nanoscience Centre, 11 J.J. Thomson Avenue Cambridge, United Kingdom. 3 Department of Biochemistry, Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur Str., 02-093 Warsaw, Poland. Correspondence to: Dr. Thierry Granjon, Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, CNRS UMR 5246 ICBMS, 43 Bd du 11 Novembre 1918, 69622 Villeurbanne cedex, France. E-mail: [email protected]

Abstract Nucleoside diphosphate kinase (NDPK) are crucial elements in a wide array of cellular physiological or pathophysiological processes such as apoptosis, proliferation, or metastasis formation. Among NDPK isoenzymes, NDPK-B, a cytoplasmic protein, was reported to be associated with several biological membranes such as plasma or endoplasmic reticulum membranes. Using several membrane models (liposomes, lipid monolayers and supported lipid bilayers) associated with biophysical approaches, we show that lipid membrane binding occurs in a two-step process first initiated by a strong electrostatic adsorption process and followed by shallow penetration of the protein within the membrane. The NDPK-B binding leads to a decrease in membrane fluidity and formation of protein patches. NDPK-B ability to form microdomains at the membrane level may be related to protein-protein interactions triggered by its association with anionic phospholipids. Such accumulation of NDPK-B would amplify its effects in functional platform formation and protein recruitment at the membrane.

Key words: NDPK-B, plasma membrane, lipid monolayers, supported lipid bilayers, liposomes, fluorescence, infrared, Brewster angle microscopy, DPI, MIP Abbreviations: NDPK; nucleoside diphosphate kinase, NDPK-A; nucleoside diphosphate kinase isoenzyme A, NDPK-B; nucleoside diphosphate kinase isoenzyme B, DPI; Dual Polarization Interferometry, FTIR; Fourier Transformed Infrared, GUVs; giant unilamellar vesicles, LUVs; large unilamellar vesicles, MIP; maximum insertion pressure, MLV; multilamellar vesicles, PC; phosphatidylcholine, PE; phosphatidylethanolamine, PI; phosphatidylinositol, PS; phosphatidylserine, Chol; cholesterol, SLBs; supported lipid bilayers, RI; refractive index, TE; transverse electric, TM; transverse magnetic.

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Introduction Nucleoside diphosphate kinase (NDPK) are multifunctional proteins involved in a wide array of cellular physiopathological processes such as apoptosis, proliferation, or metastasis formation.1, 2, 3 NDPK proteins participate in the regulation of those multiple cellular processes via enzymatic and non-enzymatic functions. The major enzymatic function of NDPKs is the reversible phosphoryl transfer from nucleotides to nucleosides and generation of nucleoside triphosphates. Mechanisms behind the nonenzymatic NDPK functions are not clear but likely involve signaling roles of NDPK through protein-protein interactions.4 NDPKs have long been considered merely as housekeeping enzymes, but, with the finding that some of these proteins are important in pathological processes such as metastasis, the scientific community began to reevaluate their role in the cell and it is now well established that the NDPK family is more complex than first thought. Among the numerous NDPK isoenzymes NDPK-A, -B and –D, were reported to bind to membranes either in mitochondria, where NDPK-D influences cardiolipin lateral organization and is thought to be involved in apoptotic pathway, or in cytosol, where NDPK-A and -B membrane association was shown to influence several cellular processes like endocytosis, cellular adhesion, ion transport etc… This study focusses on the cytosolic NDPK-B, which also possesses a cell cycle dependent nuclear localization.5, 6 NDPK-B can act as a protein phosphotransferase, has an exonuclease activity and is an activating factor for c-myc among others.7, 8 NDPK-B, like its homolog NDPK-A, has been extensively studied in relation with cancer progression in cancer cell lines and model rodents.9, 10, 11, 12 NDPK-B was shown to be implicated in the suppression of cancer metastasis, but the mode by which it acts remains elusive, although a recent study reported the binding of NDPK-B to the promoter of the key focal adhesion factor, vinculin, reducing its subsequent expression and leading to diminished lung cancer cell dissemination.7 The list of NDPK-B interacting partners expands, and includes proteins involved in signal transmission, endocytosis, cell cycle.10, 13 Independent studies have shown a localization of NDPK-B at plasma membrane, where it is supposed to play a role in adhesion, signalling pathways, ion transport, or endoplasmic reticulum membrane, where it acts as a scaffold molecule via its symmetric structure.14, 15, 16, 17 Even if NDPK-B lacks defined sequences of membrane attachment and only exhibits few exposed hydrophobic regions, it is now accepted that NDPK-B is able to bind directly to lipids. Additionally, site-directed mutagenesis shows that membrane binding occurs mainly via interaction between anionic phospholipids, and positively charged residues (Lys56 and Arg58).17, 18, 19 In contrast, NDPK-A, despite having 88% sequence identity to NDPK-B, is not able to bind lipids and was suggested to be associated to membranes through protein scaffolds.19, 20 The mitochondrial homologue, NDPK-D, is able to bind lipid membranes containing mitochondrial anionic lipid cardiolipin, as well as zwitterionic lipid monolayers constituted of pure phosphatidylcholine.21 Even if the role of the interaction between NDPK-B cationic charged residues and membrane anionic phospholipid in the protein binding to membranes were already described, knowledge about the mechanisms and consequences of this binding are far from being understood and numerous points need to be enlightened. Consequently, the intriguing role of NDPK-B requires an in-depth study of its membrane binding mechanisms. The focus of our study is to further characterize NDPK-B membrane binding by applying different biophysical technics (ranging from Langmuir Isotherms, Dual Polarization Interferometry to FTIR and confocal microscopy) and several model membranes: liposomes, lipid monolayers and supported lipid bilayers (SLBs). Model membranes will allow us to use a limited number of membrane components and thus to bring forth new insights into the complex biological role of NDPK-B. In the present manuscript, we have not only confirmed the NDPK-B's ability to 2 ACS Paragon Plus Environment

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bind exclusively to anionic-containing membranes, but, using DPI measurements, we also reported that this interaction involves a two-step process, which is initiated by a strong electrostatic adsorption and followed by shallow insertion of the protein within the lipid layer. Moreover, FTIR and fluorescence spectroscopy measurements associated with confocal microscopy further indicated that this binding leads to a decreased fluidity of the membrane coupled with phospholipid lateral reorganization and accumulation of NDPK-B in protein patches at the membrane level.

Experimental Section Materials Phosphatidylcholine (PC) (egg yolk, grade I), phosphatidylethanolamine (PE) (egg yolk, grade I), phosphatidylserine (PS) (bovine spinal cord, grade I) and phosphatidylinositol (PI) (wheat germ, grade I) were from Lipid Products. Laurdan was purchased from Fluka. Sheep whool cholesterol (Chol), >99% purity, lactate dehydrogenase and pyruvate kinase came from Sigma. NDPK-B (17 kDa) was purified as previously described.19 For confocal microscopy, labelling of NDPK-B was performed using Alexa 546-C5-maleimide (Molecular Probes). Purified NDPK-B in 20 mM Tris-HCl pH 7.4 buffer was treated with a 10-fold molar excess of Alexa 546-C5-maleimide (dissolved in 5 µl DMSO) for 90 min at room temperature. The labelled protein, called NDPK-Bred, was separated from the reagents using a Sephadex G25 column (GE Heathcare, France) eluted with 20 mM Tris-HCl pH 7.4 buffer. The labelling efficiency was estimated between 80-90%. Assay of NDPK activity NDPK activity was measured using a coupled lactate dehydrogenase/pyruvate kinase assay, with modifications.22 The assays were carried out at 25°C in a 1 mL reaction mixture containing 50 mM Tris-HCl, pH 7.4, 75 mM KCl, 5 mM MgCl2, 1 mM ATP, 0.1 mM NADH, 1 mM phosphoenolpyruvate, 1 mM TDP and 5 U of pyruvate kinase and lactate dehydrogenase. The results are representative of at least 3 independent measurements.

Preparation of liposomes Aliquots of the required lipids in chloroform solution were mixed in the desired molar ratio, i.e: PC-PE-PS-PI-Chol (12:35:22:9:22) which mimics the phospholipid composition of the inner leaflet of the plasma membrane PC (100), PC-PE-PS-PI (26:44:18:12), PC-PE-PSChol (12:35:31:22), PC-PE-PS (18:52:30), PC-PS (70:30).23 For Laurdan fluorescence experiments, Laurdan was added to lipid mixtures in chloroform at a 1/400 Laurdan/lipid molar ratio. LUVs were prepared by hydration and extrusion as previously described.24 Briefly, dry lipids were hydrated (20 mg/mL) in a 20 mM Tris-HCl, pH 7.4, 0.1 mM EDTA buffer and dispersed to produce MLV. The lipid suspension was subjected to 6 freeze/thaw cycles and then extruded 19 times through a polycarbonate membrane (Nucleopore) with 0.4 and 0.2 µm diameters pores using a mini-extruder.25 Interaction of NDPK-B with liposomes 14 µg of NDPK-B were incubated at 25°C during 20 min with 150 µg of liposomes. The mixture was then centrifuged at 160 000 g during 1 h using a Beckman airfuge. The supernatant was separated from the pellet which was then resuspended in 20 mM Tris-HCl, pH 7.4, 0.1 mM EDTA. In our conditions, NDPK activity does not differ in presence or in 3 ACS Paragon Plus Environment

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absence of liposomes, whatever the lipid composition. Consequently the percentage of binding was determined by using Eq (1) Binding % = activity in pellet/ (activity pellet + activity supernatant) x 100

(1)

Monomolecular film formation at air buffer interface and surface pressure measurements The film balance was built by Nima (UK) and equipped with a Wilhelmy-type surfacepressure measuring system. A circular Teflon trough with a surface of 27 cm2 was filled with 30 mL 20 mM Tris-HCl buffer, pH 7.4, 0.1 mM EDTA. Monolayers were formed on a clean air-buffer interface by spreading various phospholipids or phospholipid mixtures dissolved in chloroform-methanol (4:1) to attain the desired lateral surface pressure. After pressure stabilization, a final concentration of 4 nM of NDPK-B was injected in the subphase and the surface pressure was monitored. Dual polarization interferometry (DPI) The Analight 4D containing a silicon oxynitride FB80 AnaChip was used to perform all dual polarization interferometry measurements. An independent Harvard Apparatus PHD2000 programmable syringe pump was used to control the flow rate of bulk buffer. The bulk buffers were 25 mM Tris, pH 7.4, 137 mM NaCl, 2.7 mM KCl, for lipid bilayer formation and changed for 20 mM Tris, pH 7.4, 0.1 mM EDTA, 50 mM NaCl, for protein-lipid binding experiments. The chip was UV ozone cleaned previously to the experiment. The optical properties of the chip were then calibrated at 25°C using 80% (w/w) ethanol and water, followed by calibrations of the bulk buffer. The final liposome solution (0.2 mg.mL-1) was then injected at 15 µL/min, forming a stable bilayer at 25°C in the presence of 5 mM CaCl2. The bilayer was then left to stabilise for 30 min before changing the buffer for 20 mM Tris, pH 7.4, 0.1 mM EDTA, 50 mM NaCl. When the signal from the chip had stabilized, 125 µL of a protein solution was injected at 25 µL.min-1 in order to observe binding events followed by running buffer during 30 min. The protein was dissolved in bulk buffer and injected on to each bilayer type in successive experiments with the surface being cleaned with 2% SDS and ethanol and a new bilayer formed between experiments. The results are representative of at least 3 independent measurements. Calculation of mass per unit area for an adsorbed layer Two orthogonal polarizations are passed through the sensor chip creating two different waveguide modes, namely transverse electric (TE) and transverse magnetic (TM) waveguide modes. Each mode generates an evanescent field from the top sensing waveguide surface interacting with materials coming into contact with the sensor surface and resulting in a change in refractive index (RI). Thus, birefringence can be obtained with DPI by calculating the difference between two effective refractive indexes, namely RI of transverse magnetic (TM) waveguide mode (nTM) and RI of transverse electric (TE) waveguide mode (nTE). The mass per unit area of an adsorbed anisotropic layer can be calculated using the de Feijter formula: 26 (1) (2) where m lipid is the mass of lipid in the bilayer, m peptide is the mass of peptide bound to the bilayer, df is the thickness of the bilayer, niso is the average refractive index of the bilayer calculated from the experimentally obtained refractive index values nTM and nTE using the formula: 4 ACS Paragon Plus Environment

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(3) n buffer is the refractive index of the buffer, and (dn/dc)lipid and (dn/dc)peptide are the refractive index increments of the lipid and protein, using values of 0.135 and 0.185 mL/g for lipids and peptides, respectively.26, 27 Calculation of birefringence for an adsorbed layer The birefringence or optical anisotropy measures the difference in refractive index of the two orthogonal polarizations caused by the net alignment of the lipid molecules perpendicular to the waveguide surface that creates an anisotropic system. In this case, the measured birefringence provides information about the level of order or alignment in the plane perpendicular to the bilayer surface, whereas the mass is simply proportional to the thickness measured. Changes in the thickness and hence mass of the layer were determined by assuming a fixed refractive index of 1.47 for the bilayer. Mass and birefringence data were derived using the Analight® Explorer program as previously described.28 The deposition of the bilayers was monitored by the rate of TM and TE phase changes versus changes in mass together with the evolution of birefringence. The change in birefringence as a function of mass of protein bound was then used to characterize the effect of protein binding on membrane structure. DPI measures the lipid and protein mass per unit area (unaffected by surrounding buffer) at very high sensitivity and is also sensitive to the effect of alignment of the lipid (birefringence). Overall, therefore, we have used the thickness and birefringence to define the quality of our deposited membrane. We then focused on changes in birefringence during protein binding because this can reveal more details about the effect of the protein on the membrane structure than a change in thickness, and potentially allow a mechanism of action to be defined.29, 30, 31, 32 Infrared Spectra Liposomes were prepared as described above, using 20 mM Tris-HCl-deuterium oxide (2H2O) (p2H 7.4) buffer. The p2H was measured with a glass electrode and was corrected by a value of 0.4 according to Glasoe and Long. 33 For the assay with protein, the liposome suspension (150 µg) was mixed with 14 µg of enzyme in 20 mM Tris-HCl buffer, pH 7.4, and then incubated 20 min. Separation of liposome-bound and free protein was performed by centrifugation at 160 000 g for 40 min (Beckman Airfuge). The pellet was resuspended in 8 µL of 20 mM Tris-HCl-2H2O (p2H 7.4). Samples were loaded between two BaF2 circular cells, with a 56 µm Teflon spacer. Fourier Transformed Infrared (FTIR) spectra were recorded with a Nicolet iS10 FTIR spectrometer which was continuously purged with dry air; then 256 scans were collected and co-added per sample spectrum, and Fourier-transformed for each sample. The infrared spectra of buffer and residual water vapour were subtracted from the infrared spectrum of the sample. The FTIR spectra presented are average spectra of three independent measurements. Spectra were deconvoluted using PeakFit (Scientific Solutions, Switzerland). Fluorescence measurements Fluorescence measurements were performed with a Hitachi F4500 fluorometer (150 W Xe). The excitation and emission band-pass values were 5 nm. NDPK-B was incubated with liposomes during 20 min and was then centrifugated at 160 000 g during 1 hour using an Airfuge centrifuge. Spectra were recorded on the pellets using a 1 cm path length thermostated quartz cuvette. All fluorescence spectra were corrected for the baseline spectra of the buffer solution to remove the contribution of the Raman band. - Membrane fluidity characterisation: excitation Generalized polarization (ex GP): 5 ACS Paragon Plus Environment

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Experiments with Laurdan were conducted as follows: phospholipids and Laurdan in chloroform solution were mixed in a 400:1 molar ratio, and the liposomes were then prepared as previously described. Emission spectra were recorded from 420 to 550 nm at a fixed excitation wavelength of 360 nm at 37°C on 150 µg Laurdan-liposomes in the presence of 14 µg NDPK-B in a final volume of 150 µL. The excitation generalized polarization was calculated as: GPexc = (Ig- Il) / (Ig+Il) Where Ig and Il are the fluorescence intensities at the maximum emission wavelength in the gel and in the liquid crystalline phases 34 with an excitation wavelength fixed at 360 nm in our conditions. Delta GP was calculated as the difference between the GP of liposomes in the presence of NDPK-B minus the GP in the absence of NDPK-B. GP figure is a mean of three independent measurements. Preparation of giant unilamellar vesicles The GUVs consisted of PC (79.9%), PS (20%) and NBD-PE (0.1%). Lipid mixture was prepared in chloroform stock solution, at a total concentration of 1 mg.mL, with the appropriate lipid ratio. Vesicles were grown in 20 mM TrisHCl pH 7.4 containing 100 mM of sucrose (100 mOsm). For confocal microscopy, GUVs were prepared by the electroswelling method.35 We spread 5 µl of lipid mixture (1 mg/mL in chloroform) directly onto two homemade electrodes kept 1 cm apart in a swelling chamber. The chamber was filled with swelling solution (100 mM sucrose) and the wires were connected to a power generator. We applied a voltage of 2 V at 10 Hz for 1 h at room temperature, for the field-supported swelling of GUVs from the lipid films. The labelled liposomes, called GUVsgreen, were produced. Confocal microscopy 200 µl of GUVsgreen electroswollen in 100 mM sucrose were resuspended in 400 µl of 20 mM TrisHCl pH 7.4 50 mM NaCl (100 mOsm). Integrity, shape and size of the giant vesicles were always checked before addition of 30 µL of NDPK-Bred (2 µM) at room temperature. NDPK-Bred bound very rapidly, so measurements were made immediately under Leica SP5 confocal inverted DMI600 microscopy with environmental chamber and lasers: HeNe 633 nm and 594 nm; Argon 458 nm, 476 nm, 488 nm, 496 nm, and 514 nm; DPSS diode 561 nm; Pulsing solid state diode 635 nm. Each image is representative of at least three independent measurements.

Results and Discussion 1. Membrane binding characteristics of NDPK-B 1.1.

NDPK-B interaction with lipids of the plasma membrane inner leaflet

The ability of NDPK-B to interact with bilayers of different lipid composition in various conditions was tested using liposomes as membrane mimicking models and sedimentation assays. NDPK-B was incubated with liposomes and centrifuged for 1 hour at 160 000 g. The pellet was separated from the supernatant, and NDPK activity was assayed in each fraction as described in the experimental section. With zwitterionic liposomes made of PC, almost no binding of NDPK-B was observed (Fig. 1A). In those conditions NDPK-B binding was less than 5% of what was observed with anionic liposomes. When either PS or PI were 6 ACS Paragon Plus Environment

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interchanged from the liposome composition (PS being replaced by PI (Fig. 1A) or PI replaced by PS), most of NDPK-B (80 to 90%) associated to the membrane. This indicates that regardless of composition of the anionic of liposomes, the nature of the polar headgroup does not play a crucial role in NDPK-B adsorption to membranes. Absence of cholesterol or PE did not significantly influence the NDPK-B binding. To better define the nature of the interaction, the ability of NDPK-B to interact with liposomes was checked in the presence of increasing concentration of NaCl (Fig. 1B). The binding of NDPK-B on PC-PE-PS-PI-Chol (12:35:22:9:22) was not changed in the presence of 50 mM NaCl or 75 mM NaCl, indicating the presence of a non-ionic component. Addition of 100 mM NaCl decreased, whereas 150 mM NaCl prevented the NDPK-B binding. When the NaCl-free pellets were incubated and centrifuged in presence of 150 mM NaCl, NDPK-B was desorbed from membranes. As calcium may influence membrane binding and magnesium is implicated in NDPK activity, the same experiments were reproduced in presence of increasing amount of MgCl2 or CaCl2 and gave results similar to those obtained with NaCl (not shown).36,37 1.2.

NDPK-B is able to bind to PS and PI-containing monolayers

An interesting approach to study protein–membrane interactions consists of using lipid monolayers at the air/water interface as a model for a membrane leaflet. The lipid lateral packing density of the membrane-mimicking monolayer can be finely tuned by modulating the lateral surface pressure. The ability of NDPK-B to insert into various lipid monolayers spread at the air–water interface was monitored by measuring the variation in surface pressure after injection of the protein in the subphase at different initial pressures. Protein insertion provoked an increase in the surface pressure which was recorded until equilibrium was attained. Phospholipid increment in surface pressure (∆ П) was plotted versus the initial pressure (П i) and a linear ﬁt was used to measure the point of intersections of the lines with the axis (Fig. 2A). The Пi intercept indicates the maximum insertion pressure (MIP), that is the maximum surface pressure beyond which protein interaction no longer affects monolayer lateral surface pressure, which can be used to characterize protein adsorption and lipid specificity.38, 39 Fig. 2A and B show that NDPK-B injection beneath all phospholipid monolayers at low surface pressure, induced an increase (∆П) in the initial surface pressure. At high surface pressure this increase was observed only in the presence of negative charges. By comparing PC and PC-PE-PS-PI-Chol MIP, we tried to determine which phospholipid was responsible for the observed difference. Monolayer experiments permitted us to have stable monolayer single lipid composition. For each experimental set of data a confidence interval at a 95% confidence level was determined using Sigma Plot. This approach permitted us to obtain an estimated range of values in which the linear regressions in Fig. 2 are 95 % likely to be included (not shown) and to determine a confidence interval for each MIP (Fig 2B). For instance, a MIP of 28 mN.m-1 was determined for NDPK-B interaction with pure PS monolayers with [25 mN.m-1; 31 mN.m-1] 95 % confidence interval. Confidence intervals obtained with PS ([25; 31]), PI ([28; 34]) and PC:PE:PS:PI:Chol 12:35:22:9:22 mixture ([29; 37]) overlap, but are clearly not overlapping those obtained for PC ([18; 24]) or PE ([18; 24]) (pure zwiterionic monolayers). Our results do not allow us to evidence a difference in the interaction of NDPK-B with PS or PI, however a similar MIP value of 30 mN.m-1 was obtained for all tested compositions that contained 30 % or more negative charges (Fig. 2B and not shown), whereas a MIP around 20 mN.m-1 was measured for a pure PC or PE monolayer.


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MIP values between 30-40 mN.m-1 were described in literature for peptides binding to various monolayers such as surfactin, neuropeptides, dicyntaurin, LL-37, but also with proteins that extensively insert into monolayers such as apolipoproteins.39 Proteins shown to interact with membranes through electrostatic interaction and insert protein parts between lipids such as mitochondrial creatine kinase also present high MIPs for charged monolayers (32.6 mN.m-1 for PC-PE-CL (3:1:1)). 40, 41 Calcineurin, which interacts with membranes through positively charged patches was reported to have MIPs between 25 and 36 mN.m-1 for PS-containing monolayers. 42 For myristoylated proteins of VILIP family, which like NDPK-B, also possess positively charged patches on the protein surface, MIPs ranging from 23 to 27 mN.m-1 were recorded for PS-containing monolayers. 43, 44 For NDPK-B interacting with lipid monolayers mimicking the inner leaflet of the plasma membrane, the 30 mN.m-1 MIPs are in the range of the 30–35 mN.m-1. Thermodynamic analysis proved that the surface pressure in a lipid monolayer is related directly to the effective lateral pressure in a lipid bilayer and that, at the above-mentioned surface pressures, the monolayer properties correspond to the properties of bilayers. At 30-35 mN.m-1 surface pressure range, the area per lipid molecule in the monolayer corresponds to that in a bilayer, and the elastic compressibility modulus for a monolayer is comparable to that for a bilayer. So the above-found pressure of 30 mN.m-1 can be considered as a pressure mimicking the physiological conditions of the lipids in the biological context of the membrane. 39, 45, 46, 47, 48, 49 2. NDPK-B membrane binding : a two-step process Molecular mechanisms of the interaction were investigated using DPI and supported lipid bilayers (SLBs) as a model for the membrane. DPI is well established optical biosensing technique which allows simultaneous measurement in real time of mass and birefringence changes upon protein incubation with SLBs to characterize the multistep process of proteinlipid membrane interaction and in particular allows highlighting the ability of molecules to penetrate into a lipid membrane. 29, 30, 31, 27, 48, 50 SLBs were formed on silicon oxynitride DPI chip surfaces after adsorption and rupture of large unilamellar vesicles made of a phospholipid composition mimicking the inner leaflet of the membrane composed of PC-PE-PS-PI (26:44:18:12 ratio). 23 To evaluate the specificity of the binding of NDPK-B for this lipid composition, the study was performed in comparison with SLBs made of PC. The PC SLB is characterized by a thickness of 4.54±0.05 nm in good agreement with literature thickness and mass values (Table 1). 27, 51 DPI experiments gave a birefringence value of 0.0166±0.0002 for PC SLB significantly lower than the one for PC-PEPS-PI (0.0204±0.007) suggesting a lower level of order of the PC SLB than that of the membrane mimicking monolayer. We suggest that SLB made of PC/PE/PS/PI (26:44:18:12) is characterized by a better alignment of the lipid molecules in the plane perpendicular to the membrane surface, probably due to a geometry compensation of the lipids in the bilayer. The conical shape of PE may contribute to this compensation 51, altogether with lipid acyl chain length and unsaturation and polar head charge. NDPK-B was incubated during 5 min with the SLBs in order to characterize the rapid binding events upon protein incubation with lipid membranes, followed by rinsing time with proteinfree buffer for at least 30 min. The 5-min incubation allowed monitoring of the rapid binding steps and is a time scale commonly used for protein lipid interaction studies with DPI. The change in mass upon binding was monitored simultaneously to the change in birefringence. The change in protein/lipid mass ratio was then used to characterize the binding events (Fig. 3A). At the maximum binding recorded during the incubation time the mass and birefringence values were extracted to calculate molar ratios and the degree of perturbation of the bilayer order (percentage of birefringence change compared to the initial value) (Table 2). 8 ACS Paragon Plus Environment

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A slight initial, completely reversible, increase in mass was detected upon incubation of 1 µM NDPK-B with PC SLBs (Fig. 3A green line). This low binding is not perturbing the order of the lipid bilayer as no change or a limited increase of the initial value of the birefringence was monitored (Fig. 3B green line, Table 2). This initial binding is rapidly suppressed by washing the SLB with the buffer without NDPK. In contrast, a significant higher binding signal was observed in presence of PC-PE-PS-PI SLBs. The mass change shows a two-stage interaction process with an initial fast binding reaching almost the saturation value followed by a slower binding event (Fig. 3A black line). During rinsing with protein-free buffer partial desorption was observed. The change in birefringence related to these binding events shows three stages (Fig. 3B black line). These changes are composed of a first initial slight increase of the birefringence value followed by a sharp decrease and finally a last stage without any further variation until rinsing with buffer during which a total recovery of the birefringence was observed (Fig. 3B black line). The highest change of birefringence corresponds to 4% of variation of the initial value suggesting a limited perturbation of the SLB order. This slight perturbation is occurring at a molar ratio of 329±87 lipids per protein (Table 2). In order to further characterize the binding events, the birefringence changes as a function of the protein/lipid mass ratio changes was also plotted (Fig. 3C black line). The changes in slope along the interaction confirm the existence of different phases. The initial binding step is not causing any disruption of the SLB order (no change or slight increase of the birefringence along with a significant mass increase). Following this stage, a decrease of the birefringence is observed as more protein binding is occurring that levels off when reaching the saturation value. After rinsing, a complete recovery of the SLB order is observed even though a significant amount of protein remains bound to the SLB. This result suggests that the binding of NDPK-B is not permanently altering the SLB order and the membrane may rearrange to accommodate protein interaction. If a protein binds to top of the SLB (without any significant insertion) and this interaction does not affect the net alignment of the lipid molecules, the birefringence will not be affected. Therefore the first stage in NDPK-B binding can be attributed to a protein adsorption at the lipid polar headgroup. In a second stage, a decrease of the birefringence is indicative of lipid alignment perturbation and thus NDPK-B penetration into the membrane. As the SLB is a fluid and dynamic system the lipid molecules can adjust to the binding of a protein and the SLB can retain a high level of order. We used a positive control, melittin (Fig. 3D and supplementary information), a wellknown antimicrobial peptide, to compare effects induced by NDPK-B and to support the relationship between the change in birefringence and the binding mode. Melittin is known to interact with lipid membranes through a two state mechanism leading to the formation of toroidal pores. 52 Birefringence changes as function of protein/lipid ratio, displayed in Fig. 3D, show that the transmembrane insertion of melittin induces an important disruption of the lipid membrane order and integrity. The mass increase due to melittin binding to the PC-PEPS-PI (26:44:18:12) SLBs is associated with a continuous and significant decrease of the birefringence that levels off while reaching 40% change of the initial value (Table 2 and supplementary information). While rinsing, the mass removal is associated with a slight recovery of the birefringence. Melittin is thus altering significantly and permanently the SLB order upon binding, in good agreement with a transmembrane insertion of this peptide. 52 In contrast, the insertion of NDPK-B into the membrane, although significant, is much more moderate. An insertion deeper into the SLB core would, as in the case of melittin, have disrupted significantly the order of the acyl chains causing a higher variation of the birefringence. Our results would translate an interaction involving charged aminoacids at


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protein surface with a shallow penetration of the protein or parts of the protein into the monolayer. 3. Effect of NDPK-B binding to liposomes on the membrane fluidity and hydration Alterations in hydrogen bonding and differences in chain packing of phospholipids after NDPK-B binding to the phospholipid bilayer were further monitored at the lipid ester group and acyl chain stretching vibration level using infrared spectroscopy (Fig. 4).53, 54 The position of the ester bond stretching vibration is indicative of the hydration state of the lipid molecules. The large band present on the lipid spectrum of PC-PE-PS-PI, 26:44:18:12 liposomes ((Fig. 4A dotted line) between 1760 and 1700 cm-1 is composed of two contributions from hydrated (1728 cm-1) ester bonds and non-hydrated (1742 cm-1) ester bonds and is clearly different from the spectrum obtained in presence of NDPK-B (Fig 4A full line). 55 Deconvoluted spectra (Fig 4B and C) obtained in the presence (Fig. 4C) or absence (Fig. 4B) of NDPK-B showed that in the presence of NDPK-B (Fig. 4 C full line), the percentage of deshydrated absorption bands increases from 31 % to 53 % (Fig. 4F). This data indicates that protein binding induces perturbations at the ester bond level, with a decrease in the hydration state of lipid polar head. The location of the C-H asymmetric and symmetric stretching vibration provides information on the C-H bond motional freedom: the higher the wavenumber, the greater the acyl chain mobility. 54, 55, 51a This vibrational bands are highly sensitive to protein binding. 56 The adsorption of NDPK-B on PC-PE-PS-PI (26:44:18:12) vesicles led to a shift of symmetrical and asymmetrical CH2 bands from 2852 to 2850 cm-1 and from 2924 to 2918 cm-1 respectively (Fig. 4 D and E), in the absence of protein (dotted line) and in the presence of protein (full line). This shift is lowered (from 2925 to 2922 cm-1 (not shown)) by the presence of cholesterol in liposomes but is still present and is the sign of a decrease in the acyl chain mobility under protein binding. Protein binding to the lipid polar group may force lipid to gather in a more “ordered” organisation, thus decreasing membrane fluidity. NDPK-B effect on membrane fluidity was also checked using Laurdan fluorescence and PCPE-PS-PI-containing liposomes with or without cholesterol (composition which matches the plasma membrane lipid environment). Laurdan emission spectrum is highly sensitive to the probe surrounding solvent dipolar relaxation process. 57, 58 It displays a red shift in polar solvent related to the molecular dynamics of the water molecules at the hydrophobichydrophilic boundary of lipid bilayer. 58, 59 Thanks to Laurdan properties, the generalized polarization parameter (GP), determined as seen in the Experimental section, enabled us to characterize the physical state of the phospholid surrounding the probe. 58, 59 The GP parameter was calculated for liposomes before and after NDPK-B addition. In the presence of NDPK-B, liposome GP values clearly increased, with positive delta GPs, as plotted in Fig. 5. The increase in GP after protein addition reflects a decrease in the mobility of surrounding solvent molecules which can be related to a decrease in membrane fluidity. This effect is abolished in presence of 150 mM NaCl, known to desorb NDPK-B from those liposomes, indicating that the GP rise previously observed was indeed due to the NDPK-B binding to the liposome membrane. 4. Membrane-bound NDPK-B is organized in patches NDPK-B organization at the membrane level was visualized using giant unilamellar liposomes (GUVs) and confocal fluorescence microscopy (Fig. 6). A multicolour approach was used: the membrane was constituted of PC:PS (70:30) labeled with NBD-PE (0.1%) (green), whereas NDPK-B was labeled with Alexa 546 fluorophore (red) (Fig. 6). The images show the results for staining with the two dyes individually (Fig. 6A and B) and the 10 ACS Paragon Plus Environment

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simultaneously obtained overlay image (Fig. 6C). NDPK-Bred did not bind to pure PC GUVs, but was still able to bind PC:PE:PS:PI:Chol liposomes with the same binding percentage (not shown) meaning that the red staining did not affect NDPK-B binding to membranes. Fig. 6, shows that membrane bound NDPK-Bred is not distributed homogeneously at the PScontaining GUVs surface and formed protein patches at the membrane level.

Summary and Conclusions NDPK-B, a cytosolic and nuclear isoform of nucleoside diphosphate kinase, has been implicated in several physio-pathological processes such as apoptosis or formation of metastasis. 60, 61 Although its role in these processes is not fully understood, it is now established that its NDPK enzymatic activity is not mandatory. 62 NDPK-B is able to interact with multiple partners that have impacts on various physiological and pathological processes and is retrieved bound to the plasma membrane. As stated by Li et al., a limitation of the complexity of the system, by using a small number of hypothetical partners, may facilitate data interpretation. 10 Consequently, we analysed its interaction with phospholipids using liposomes, monolayers and supported lipid membranes as biomimetic tools. The lipid binding mode not only determines specificity and affinity for the membrane, but also the possible effects of the protein on membrane morphology and dynamics and may have major biological consequences. 63 NDPK-B binding on liposomes in the presence or absence of NaCl was assessed here (Fig. 1). NDPK-B was only able to bind to anionic membranes (made of PC, PE, PS, PI, Chol) suggesting an important electrostatic component to binding of NDPK-B, as previously described. 17, 19 Additionally, we showed that the nature of the negatively charged polar head itself does not determine the adsorption process as the same protein binding proportion was obtained whatever the presence of PS or PI in the bilayer composition. The presence or absence of either PE and/or cholesterol did not either significantly modify NDPK-B binding percentage. Langmuir monolayer experiments were used to determine NDPK-B maximum insertion pressure (MIP) within the monolayers depending on the monolayer lipid composition. Two distinct behaviours, confirmed by statistical analysis of the confidence intervals at 95 % confidence levels, were observed on Fig. 2A: a first set of linear regressions obtained with zwitterionic phospholipids (either PC or PE), crosses the abscisse axe at 20 mN.m-1 MIP, and the second set of linear regressions observed with anionic phospholipid composition (PI; PS; or PC-PE-PS-PI-Chol), crosses the abscisse axe around 30 mN/m MIP. The negative charge density (100% for PI and 30 % for PC-PE-PS-PI-Chol monolayers) does not modify the MIP. By comparing MIP values obtained for other proteins previously described 39, 40, 41, 42, 43, 44, present results indicate that NDPK-B is able to bind anionic lipid membranes under surface pressure conditions considered to mimic the conditions of the biological membranes. 39, 45, 46, 47, 48 DPI measurements unravelled a two-step process (Fig. 3): first an adsorption of NDPKB at the anionic SLB surface followed by a shallow penetration of the protein between the lipids. This interaction may be modulated by external factors such as local concentrations of electrolytes and may contribute to additional regulatory processes. Infrared measurements showed that binding of NDPK-B to anionic membranes triggers dehydration and loss of hydrogen bonding at the level of ester bonds (Fig. 4). Of note, CH2 stretching vibration band positions were shifted towards lower wavenumbers which is characteristic of a decrease in acyl chain mobility. 54, 55 Binding of NDPK-B to the lipid membrane involves at least 6 charged residues per hexamer 17 and may bring together lipid molecules leading to a decrease in their capacity to freely diffuse within the bilayer leaflet. Laurdan experiments supported the decrease in membrane fluidity induced by NDPK-B 11 ACS Paragon Plus Environment

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binding as they showed an increase in GP values indicative of a decrease in the mobility of solvent molecules surrounding the probe when NDPK-B was bound to the membrane (Fig. 5). Those effects are undoubtedly due to NDPK-B binding to the membrane as they were not observed in the presence of 150 mM NaCl which induces the protein desorption. Thus, the fact that, NDPK-B binding to membranes has effects on membrane fluidity (Fig. 4 and 5) on one hand, and, that it induces some clusters formation on the other hand (Fig. 6) may have some important physiological consequences as significant membrane proteins involved in diverse physiological functions are argued to be affected by membrane rigidity. Indeed, we may hypothesize that the presence of NDPK-B-enriched domains at the plasma membranes offers an anchorage point to these proteins or molecules. They also suggest that these proteins will be also enriched at these points at the plasma membrane and putatively their physiological activity will be enhanced. A plethora of molecules are, at some point in their physiological roles, found to be associated to NDPK-B. For example, at the membrane level, NDPK-B regulates cAMP signaling by forming a complex with G proteins and is required in the plasma membrane content of G(s) proteins, NDPK-B also activates Ca2+ flux by activating TVRP5, a Ca2+-permeable channel that regulates urinary Ca2+ excretion. 64 NDPK-B interacts with MDM2 (a protooncogene best understood for its critical role as negative regulator of the p53 tumor suppressor). NDPK-B has also been reported to be membrane-associated and influence several membrane-linked cellular processes: endocytosis, cellular adhesion, ion transport, receptor endocytosis and desensitization by agonists, modulation of phagocyte NADPH oxidase activity. NDPK-B is known to interact with intrinsic or peripheral membrane proteins such as integrin cytoplasmic domain-associated protein 1α (ICAP-1 α) or thromboxane A2 receptor and was recently reported to be associated with dynamin superfamily proteins. 65, 15, 66 Accumulating evidence suggests that NDPK interacts with and affects various components and regulators of the cytoskeleton, including actin-binding proteins, intermediate filaments, and cytoskeletal attachment structures (adherence junctions, desmosomes, and focal adhesions). 4 Moreover, proteo-lipid domains observed by confocal microscopy may contribute to the scaffolding role attributed to NDPK-B 17, 67, 68: such complexes may be promoted in vivo by NDPK-B in order to stabilize interactions and/or to provide nucleotides such as GTP to a specific set of proteins bound to this particular region of the membrane. NDPK-B could provide additional regulation cascades to finely tune physiological processes in the vicinity of the membrane. Acknowledgements: This work was supported by Université Claude Bernard Lyon 1 (France), CNRS (France), by a POLONIUM grant (27727TA), the EU FP7 Project BIOIMAGing in research INnovation and Education, GA No. 264173 (BIO-IMAGINE), NIEB PAS (Poland), and by the BBSRC (No. BB/H003843/1). We are very grateful to Dr. May Khanna from University of Arizona, Tucson for manuscript reading, fruitful suggestions and English correction. References : 1. Boissan, M.; Lacombe, M. L. Learning about the functions of NME/NM23: lessons from knockout mice to silencing strategies. Naunyn Schmiedebergs Arch Pharmacol 2011, 384 (4-5), 421-31. 2. Mehta, A.; Orchard, S. Nucleoside diphosphate kinase (NDPK, NM23, AWD): recent regulatory advances in endocytosis, metastasis, psoriasis, insulin release, fetal erythroid lineage and heart failure; translational medicine exemplified. Mol Cell Biochem 2009, 329 (12), 3-15.


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53. Arrondo, J. L.; Goni, F. M.; Macarulla, J. M. Infrared spectroscopy of phosphatidylcholines in aqueous suspension. A study of the phosphate group vibrations. Biochim Biophys Acta 1984, 794 (1), 165-8. 54. Gericke, A.; Smith, E. R.; Moore, D. J.; Mendelsohn, R.; Storch, J. Adipocyte fatty acid-binding protein: interaction with phospholipid membranes and thermal stability studied by FTIR spectroscopy. Biochemistry 1997, 36 (27), 8311-7. 55. Lewis, R. N.; McElhaney, R. N.; Pohle, W.; Mantsch, H. H. Components of the carbonyl stretching band in the infrared spectra of hydrated 1,2-diacylglycerolipid bilayers: a reevaluation. Biophys J 1994, 67 (6), 2367-75. 56. Granjon, T.; Vacheron, M. J.; Vial, C.; Buchet, R. Mitochondrial creatine kinase binding to phospholipids decreases fluidity of membranes and promotes new lipid-induced beta structures as monitored by red edge excitation shift, laurdan fluorescence, and FTIR. Biochemistry 2001, 40 (20), 6016-26. 57. Bagatolli, L. A.; Maggio, B.; Aguilar, F.; Sotomayor, C. P.; Fidelio, G. D. Laurdan properties in glycosphingolipid-phospholipid mixtures: a comparative fluorescence and calorimetric study. Biochim Biophys Acta 1997, 1325 (1), 80-90. 58. Parasassi, T.; De Stasio, G.; Ravagnan, G.; Rusch, R. M.; Gratton, E. Quantitation of Lipid Phases in Phospholipid-Vesicles by the Generalized Polarization of Laurdan Fluorescence. Biophys J 1991, 60 (1), 179-189. 59. Parasassi, T.; De Stasio, G.; d'Ubaldo, A.; Gratton, E. Phase fluctuation in phospholipid membranes revealed by Laurdan fluorescence. Biophys J 1990, 57 (6), 11791186. 60. Kang, Y.; Lee, D. C.; Han, J.; Yoon, S.; Won, M.; Yeom, J. H.; Seong, M. J.; Ko, J. J.; Lee, K. A.; Lee, K.; Bae, J. NM23-H2 involves in negative regulation of Diva and Bcl2L10 in apoptosis signaling. Biochem Biophys Res Commun 2007, 359 (1), 76-82. 61. Miyazaki, H.; Fukuda, M.; Ishijima, Y.; Takagi, Y.; Iimura, T.; Negishi, A.; Hirayama, R.; Ishikawa, N.; Amagasa, T.; Kimura, N. Overexpression of nm23-H2/NDP kinase B in a human oral squamous cell carcinoma cell line results in reduced metastasis, differentiated phenotype in the metastatic site, and growth factor-independent proliferative activity in culture. Clin Cancer Res 1999, 5 (12), 4301-7. 62. MacDonald, N. J.; De La Rosa, A.; Benedict, M.; Freije, J.; Krutsch, H.; Steeg, P. A serine phosphorylation of Nm23, and not its nucleoside diphosphate kinase activity, correlates with suppression of tumor metastatic potential. J Biol Chem 1993, 268 (34), 25780-25789. 63. Lemmon, M. A. Membrane recognition by phospholipid-binding domains. Nat Rev Mol Cell Biol 2008, 9 (2), 99-111. 64. Cai, X.; Srivastava, S.; Surindran, S.; Li, Z.; Skolnik, E. Y. Regulation of the epithelial Ca2+ channel TRPV5 by reversible histidine phosphorylation mediated by NDPK-B and PHPT1. Mol Biol Cell 2014, 25 (8), 1244-1250. 65. Fournier, H. N.; Dupe-Manet, S.; Bouvard, D.; Lacombe, M. L.; Marie, C.; Block, M. R.; Albiges-Rizo, C. Integrin cytoplasmic domain-associated protein 1alpha (ICAP-1alpha ) interacts directly with the metastasis suppressor nm23-H2, and both proteins are targeted to newly formed cell adhesion sites upon integrin engagement. J Biol Chem 2002, 277 (23), 20895-902. 66. Boissan, M.; Montagnac, G.; Shen, Q.; Griparic, L.; Guitton, J.; Romao, M.; Sauvonnet, N.; Lagache, T.; Lascu, I.; Raposo, G.; Desbourdes, C.; Schlattner, U.; Lacombe, M.-L.; Polo, S.; van der Bliek, A. M.; Roux, A.; Chavrier, P. Nucleoside diphosphate kinases fuel dynamin superfamily proteins with GTP for membrane remodeling. Science 2014, 344 (6191), 1510-1515.


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67. Hippe, H.-J.; Abu-Taha, I.; Wolf, N. M.; Katus, H. A.; Wieland, T. Through scaffolding and catalytic actions nucleoside diphosphate kinase B differentially regulates basal and β-adrenoceptor-stimulated cAMP synthesis. Cell Signal 2011, 23 (3), 579-585. 68. Hippe, H.-J.; Wolf, N. M.; Abu-Taha, H. I.; Lutz, S.; Le Lay, S.; Just, S.; Rottbauer, W.; Katus, H. A.; Wieland, T. Nucleoside diphosphate kinase B is required for the formation of heterotrimeric G protein containing caveolae. Naunyn-Schmiedeberg's Arch Pharmacol 2011, 384 (4), 461-472. a

TABLE 1. Structural parameters of supported lipid bilayers. The values are the means of three determinations ± standard deviation. 2

Lipid composition RI Thickness (nm) Mass (ng/mm ) Birefringence PC/PE/PI/PS 1.47 5.7±0.8 5.7±0.8 0.0204±0.0007 (26:44:18:11) PC 1.47 4.54±0.05 4.59±0.05 0.0166±0.0002 a The SLB thickness and birefringence were calculated from the TM and TE phase changes using the assumed isotropic refractive index (n) 1.47. The mass of SLBs was calculated from the thickness and RI using the dn/dc value for lipids of 0.135 g/mL.

TABLE 2. Experimental parameters of protein-supported lipid bilayer interactions. The values are the means of three determinations ± standard deviation.

NDPK-B

∆Mass 2 ng/mm 2.3±1.2

Protein/lipid a mass ratio 0.4±0.1

Lipid/protein molar ratio 329±87

NDPK-B

0.12±0.01

0.027±0.003

5060±661

SLB

Protein

PC/PE/PI/PS (26:44:18:11) PC

∆Birefringence

Disruptionb

-0.0009±0.0006

- 4%

+0.00006±0.00003

-

a

Protein/lipid mass ratio values were calculated using the mass values of the corresponding SLBs prior to protein injection, together with the additional mass gain after protein injection. The mass changes were determined using the dn/dc values for proteins and lipids of 0.182 and 0.135 g/mL respectively 27. b Percentage of birefringence change compared to the initial value.


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100 90 80 70 60 50 40 30 20 10 0

B Percentage of bound NDPK-B (%)

A Percentage of bound NDPK-B (%)

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100 80 60 40 20 0

0

25

50

75

[NaCl] (mM)

100

125

150

Figure 1. Binding of NDPK-B to liposomes. (A) Binding assays were carried out with 150 µg of liposomes made either of PC, PC-chol (78:22), PC-PS (70:30), PC-PS-chol (48:30:22), PC-PE-PS (18:52:30), PC-PE-PS-chol (12:35:31:22), PC-PE-PS-PI (23:46:22:9), PC-PE-PS-PI-chol (26:44:18:12), PCPI (70:30) or PC-PE-PI (18:52:30) and 14 µg of NDPK-B in 200 µL of 20 mM Tris-HCl, pH 7.4. (B) Binding assays were carried out in the presence of increasing concentrations of NaCl (0, 50, 75, 100 and 150 mM), with 150 µg of PC-PE-PS-PI-chol (26:44:18:12) liposomes and 14 µg of NDPK-B in 200 µL of 20 mM Tris-HCl, pH 7.4. Data represent mean ± SD (n = 3 per condition).

ACS Paragon Plus Environment

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A

16

PC

14

PI

12 PS

10

∆π (mN/m)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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PC-PE-PS-PI-Chol (12:35:22:9:22)

8 6

PE

4 2 0 0

10

20

30

πi (mN/m)

B Monolayer MIP (mN/m) Confidence interval (95% )

PC

PE

PS

PI

PC-PE-PS-PI-ch

20

20

28

30

31

[18; 24]

[18; 24]

[25; 31]

[28; 34]

[29;37]

Figure 2. Effect of NDPK-B on monolayer surface pressure. (A) Plot of the surface pressure increase (∆П) as a function of the initial surface pressure (Пi) to determine the maximal insertion pressure (MIP). Monolayers were made of PC-PE-PS-PI-chol (12:35:22:9:22) (black squares), PE (empty squares), PC (black diamonds), PI (grey triangles), PS (crosses) and spread over a buffer of 0.1 mM EDTA, 20 mM Tris, pH 7.4 at a temperature of 21 °C. (B) Maximal insertion pressure (MIP) of NDPK-B into various monolayers and 95% confidence interval (mN.m-1) obtained using Sigma Plot for each set of data.


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D

0.0004

Protein/lipid ratio changes

0.0002

-0.001

-1E-18 -0.0002 -0.0004 -0.0006 -0.0008 -0.001

Protein/lipid ratio changes

0 0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

Birefringece changes

C Birefringence changes

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0

0.1

0.2

0.3

0.4

0.5

0.6

-0.002 -0.003 -0.004 -0.005 -0.006 -0.007 -0.008

Figure 3. Effect of NDPK-B binding on supported lipid bilayers (SLB). Plots of the protein/lipid mass ratio changes as a function of time (A) and plots of the birefringence changes as a function of time (B) in 20 mM Tris, pH 7.4, 50 mM NaCl, 0.1 mM EDTA. SLBs were composed of PC-PE-PI-PS (26:44:18:12) (black line), PC (green line). Protein injections (NDPK B 1 µM) were performed during 5 min followed by rinsing time with buffer for at least 30 min. Plots of the birefringence changes as a function of the changes in protein/lipid mass ratio (C) in 20 mM Tris, pH 7.4, 50 mM NaCl, 0.1 mM EDTA. SLBs were composed of PC-PE-PI-PS (26:44:18:12) (black line for NDPK B and blue line for melittin), PC (green line). Protein injections (NDPK B 1 µM and melittin 2 µM) were performed during 5 min followed by rinsing time with buffer for at least 30 min, indicated by the arrow heads. Protein/lipid mass ratio values were calculated using the mass values of the corresponding SLBs prior to protein injection, together with the additional mass gain after protein injection. The mass changes were determined using the dn/dc values for proteins and lipids of 0.182 and 0.135 g/mL respectively.


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Figure 4. Effect of NDPK-B binding on lipid infrared spectra. Infrared spectra of 150 µg of PC-PE-PSPI (23:46:22:9) alone (dotted line) or with (full line) 14 µg of NDPK-B. Samples were resuspended in 8 µL of 20 mM Tris-HCl-2H2O, p2H 7.4, as described in Materials and Methods. (A) Infrared spectra in the region of the ester stretching vibration. (B) Deconvolution of ester stretching vibration in the absence of NDPK-B (C) Deconvolution of ester stretching vibration in the presence of NDPK-B Deconvoluted spectra were obtained using PeakFit software by the least residual method. Black experimental spectra and grey - fitted spectra, with r²=0.9996, Fit sdt=0.00017; for (B) and r²=0.9937 Fit sdt=0,00051 for (C). (D) Infrared spectra in the region of the symmetric CH stretching vibration. (E) Infrared spectra in the region of the asymmetric CH stretching vibration. (F) Summary of the maximum wavenumber (cm-1) in each infrared band in presence or absence of NDPK-B.


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0.02

0.015

Delta GP

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.01

0.005

0

Figure 5: GP variations of Laurdan in PC-PE-PS-PI and PC-PE-PS-PI-chol liposomes after NDPK-B binding. Delta GP was calculated as GP in presence of 14 µg NDPK-B minus GP in the absence of NDPK-B.


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Figure 6. Organization of NDPK-B at the membrane level of giant liposomes PC:PS (70:20). (A) GUV membrane was labelled with 0.1 % of NBD-PE (shown in green). (B) NDPK-B was labelled with Alexa546 (shown in red). (C) Merge of green and red images. Control liposomes in absence of NDPK are shown in the left column, while effects of protein binding are shown in the right column.


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

- - - - -+ +-+ +++ +++

-


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Two-Step Membrane Binding of NDPK-B Induces Membrane Fluidity

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