Annexin A5 Binding and Rebinding to Mixed Phospholipid

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Chapter 19

Annexin A5 Binding and Rebinding to Mixed Phospholipid Monolayers Studied by SPR and AFM Xuezhong Du,1,3 David W. Britt,2 and Vladimir Hlady*,1 1Department

of Bioengineering, University of Utah, Salt Lake City, Utah 84112, U.S.A. 2Department of Biological Engineering, Utah State University, Logan, Utah 84112, U.S.A. 3Present address: School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, P.R. China *E-mail: [email protected]

Annexin A5 binding to the fluid and immobilized mixed monolayers of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylserine (DPPS) in the presence of Ca2+ ions was investigated using surface plasmon resonance (SPR) spectroscopy and atomic force microscopy (AFM). The amount of adsorbed annexin A5 found on the fluid DPPC-DPPS monolayer was almost twice as high as that found on the immobilized monolayer. The larger amount of protein bound to the fluid monolayer was likely due to the protein induced recruitment of negatively charged lipids and formation of specific binding patterns in the fluid monolayer. Bound annexin A5 could be completely desorbed in the presence of ethylenediaminetetraacetic acid (EDTA) from immobilized monolayer, but only partially removed from the fluid monolayer. AFM imaging suggested that adsorbed annexin A5 was confined to the polar lipid heads – subphase interface in surface patches of protein whose size correlated well with the size of paired annexin A5 trimers.

© 2012 American Chemical Society In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Introduction Annexin A5 belongs to a family of structurally homologous proteins that bind to phospholipid membranes in a calcium-dependent manner. Annexins are thought to participate in a variety of membrane-related processes, including exocytosis, endocytosis, vesicle trafficking, ion channel regulation and inflammation (1). Although the biochemical properties of annexins have been extensively investigated and their molecular structure in crystalline and membrane-bound forms has been elucidated in detail, their in vivo function still remains unclear (2). Annexin A5 is one of the most broadly distributed and abundant members of its family and has been implicated in blood coagulation where it competes for blood platelet phosphatidylserine binding sites with prothrombin and also inhibits the activity of phospholipase A1 in vitro (3). The crystal structure of annexin A5 reveals a tetrad of calcium binding site domains each containing a four-helix bundle with a fifth helix capping the binding site (4). The protein face which coordinates calcium ions is the feature which binds to phospholipid membranes via calcium bridges (5). Distinct binding sites for phospholipid head groups are known, including a novel, double-Ca2+ recognition site for phosphoserine that may serve as a phosphatidylserine receptor site in vivo (6). The annexin A5 molecular surface exhibits an overall flat topology with one convex and one concave side (Figure 1ab). The convex face of the molecule, opposite to the N terminus, is the area responsible for anchoring to phospholipid membranes. It contains two amino acid side chains (Trp 185 and Ala 101) that are found inserted into the phospholipid bilayer thus facilitating the formation of Ca2+-bridges (6). Annexin A5 in solution exists as a monomer, but in the presence of Ca2+ it can form trimers and 2-D crystals upon binding to phospholipid assemblies containing negatively charged phospholipids (7). A variety of techniques, such as fluorescence spectroscopy (8), electron paramagnetic resonance (EPR) (9), fourier transform infrared (FTIR) (10, 11), nuclear magnetic resonance (NMR) (12), electron microscopy (13), ellipsometry (14, 15), and atomic force microscopy (AFM) (16, 17) have been used to study annexin A5 binding to membranes containing negatively charged phospholipids. Adsorption of annexin A5 to phospholipid mono- and bilayers has also been studied using IR reflection adsorption spectroscopy and Brewster angle microscopy (18, 19), and with quartz crystal microbalance (20). Here we compare annexin A5 adsorption to fluid and to immobilized mixed dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylserine (DPPS) monolayers. The objective of the study, illustrated in Figure 1cd, was to find whether the annexin A5 binding to the fluid DPPC-DPPS monolayer at the air/water interface leads to the recruitment of lipid molecules and rearrangement of the monolayer which subsequently improves the affinity for re-binding of the same protein. Binding kinetics of annexin A5 were measured with an in situ surface plasmon resonance (SPR) spectroscopy. This SPR technique enables label-less, real-time recording of protein adsorption kinetics to monolayers at the air/water interface (21). SPR measures the changes in the refractive index in 420 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

the interfacial region and with the appropriate model these changes can provide information about the surface concentration of proteins (22, 23). The SPR experiments were complemented with AFM imaging to obtain information about the annexin A5 adsorption-desorption behavior and its spatial distribution at the phospholipid monolayer interface.


Experimental Section Materials Dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylserine (DPPS) (both from Avanti Polar Lipids) were prepared as 0.5 mM stock solutions in chloroform (Spectrograde, Merck) and stored at 4°C prior to use. The DPPC−DPPS mixture (DPPC/DPPS molar ratio 4:1) was prepared volumetrically from the stock solutions. Annexin A5 (human, 33 kDa, 2.2 mg/ml, Sigma) was stored at −70 °C prior to use. Double-distilled deionized water (pH 5.5) was used for buffer preparation. N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid hemisodium salt (HEPES, >99.5%, Sigma), NaCl (99.5%, Sigma), CaCl2·2H2O (99%, Sigma), and ethylenediaminetetraacetic acid (EDTA, disodium salt dihydrate, (99%, Sigma) were used to prepare Ca2+ and EDTA-containing buffers: Ca2+ buffer contained 10 mM HEPES, 150 mM NaCl, 3 mM CaCl2, pH 7.4, and EDTA buffer contained 10 mM HEPES, 150 mM NaCl, 4 mM EDTA, pH 7.4. Langmuir Trough and Monolayer Preparation A small KSV−5000 Langmuir trough (36.5 cm × 7.5 cm, KSV Instruments) with symmetric compression barriers was used to construct surface pressure – molecular area (π – A) isotherms for the mixed lipid monolayers on water, HEPES, and HEPES + Ca2+ subphases. The trough was enclosed in a dust free cabinet on an anti-vibration table and maintained at ~20°C. A flame-cleaned Pt Wilhelmy plate, attached to a force transducer, was positioned in the middle of the trough. After aspirating the air/water interface during a blank compression cycle to remove any contaminants the DPPS-DPPC solution was spread dropwise at the air/water or air/buffer interface followed by 15 min for solvent evaporation, then compressed at a rate of 150 mm2/min until film collapse. For monolayer transfers compression was stopped at 20 mN/m. After holding the film at 20 mN/m for 1 h it was transferred onto an octadecyltrichlorosilane (OTS)-modified silicon wafer (24) by the Langmuir-Schaefer method: the hydrophobically modified Si wafer was horizontally pushed into the subphase through the interface and placed into a small beaker immersed in the subphase. The remaining lipids were aspirated from the interface, and the beaker containing the monolayer coated wafer was removed and maintained hydrated for subsequent AFM imaging. 421 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 1. (A-B) Top and side views of annexin A5. Cation binding sites and Trp 185 are outlined in lighter color. (C-D) Schematic illustration of annexin binding experiments to fluid and immobilized (D) monolayers. (C) The initial adsorption on fluid monolayer is carried out without the presence of SPR sensor. Adsorbed annexin recruits the DPPS (filled circles) into the binding site, thus improving the affinity for re-binding of the same protein. (D) Annexin binding to the monolayer immobilized by the contact with hydrophobically modified SPR sensor prior to the protein adsorption.

422 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


SPR Measurement Compact SPR sensors (Spreeta, Texas Instruments) (25) were used for all SPR measurements. These devices combine the sensing Au surface with all the optic and electronic components required for SPR experiments in a compact and lightweight assembly with p-polarized near-infrared light (840 nm) as the light source. The Au surfaces of the SPR sensors were first cleaned with O2 plasma for 1 min (Plasmod, Tegal Corp.), and then rendered hydrophobic with 2 mM octadecanethiol (ODT, >95% GC, Fluka) in absolute ethanol for 10 min followed by rinsing with absolute ethanol and double-distilled water, respectively. A dual microtrough-SPR set-up, schematically shown in Figure 2, was used to measure annexin A5 adsorption/desorption cycles from the mixed lipid monolayers. Each PTFE microtrough had an undercut perimeter in order to eliminate meniscus and obtain a flat air/water interface. One microtrough contained the KSV-surface pressure transducer and was used to measure the volume of DPPC-DPPS chloroform solution required to obtain the surface pressure of ~25 mN/m during film spreading as no compression barriers were used. The same volume of DPPC-DPPS solution was then spread at the interface of the second microtrough for protein adsorption/desorption experiments. A slow dropwise spreading technique with additional time for chloroform evaporation was used to reach the target surface pressure. The annexin A5 solution (5 μL) was injected into the subphase by microsyringe (Hamilton, Gastight 10 μL) to achieve a final concentration of 1 μg/ml; in the desorption cycle protein-free solution was pumped into one end of the microtrough while the equivalent volume was pumped out of the opposite end to exchange the subphase several times (~5) over. The SPR sensor was brought into contact with the DPPC−DPPS monolayer, which immobilized the monolayer onto the ODT-modified surface of the SPR sensor. As depicted in Figure 1, when the hydrophobically modified SPR sensor contacted the mixed lipid monolayer after the first cycle of annexin A5 adsorption took place, the experiment is referred to as “binding to fluid monolayer” (i.e., annexin A5 adsorption took place before the SPR sensor immobilized the DPPC-DPPS monolayer). When the SPR sensor contacted the DPPC-DPPS monolayer before annexin A5 was injected into subphase to initiate the first adsorption-desorption cycle the experiment is referred to as “binding to an immobilized monolayer” (i.e., the monolayer was immobilized by the ODT-modified surface of the SPR sensor before any protein adsorption took place). In each case after the first adsorption-desorption cycle, a re-binding of annexin A5 was measured using SPR by repeating the adsorption-desoprtion cycle one more time. AFM Measurement AFM imaging was performed in a contact mode under buffer solution using a Nanoscope II SPM (Digital Instruments Inc.) equipped with a “D” scanner (10 μm × 10 μm). A low force constant cantilever (0.01 N/m, Park Scientific Instruments) was used to minimize manipulation of the monolayer by the tip. The AFM tip was first cleaned under a high-intensity UV lamp for ~15 min just prior to imaging to remove organic contaminants from the tip surface. An O-ring was used to seal the 423 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


flow cell of the AFM to keep the sample in buffer solution during the transfer and imaging. After the mixed phospholipid monolayer was imaged under solution, 1 μg/ml annexin A5 solution in the Ca2+-containing buffer was slowly injected into the AFM flow cell. The annexin-bound monolayer was imaged after 30 min incubation.

Figure 2. Schematic representation of dual Langmuir microtrough−SPR setup for protein adsorption to and desorption from the lipid monolayer. Left trough was used to find the amount of spread lipids to achieve surface pressure, π = 20 mN/m. Right trough was used for protein binding experiments. Protein was injected into the subphase using microsyringe and adsorption took place from unstirred solution. In the desorption step, aqueous subphase was exchanged several times with protein-free solutions.

Results and Discussion Figure 3 shows the π − A isotherms measured for the 4:1 DPPC-DPPS monolayers on several different subphases. The monolayer on pure water showed a transition from liquid-expanded to liquid-condensed phase without any obvious plateau. In the absence of Ca2+ in the buffer, the monolayer displayed an isotherm similar to that on pure water but with increased molecular areas, attributed to the influence of HEPES and NaCl in the buffer subphase. In the presence of Ca2+ in the buffer, the monolayer was more expanded at low surface pressures (i.e. larger molecular areas) but was more compact (i.e. smaller molecular areas) at high 424 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


surface pressures with a cross-over point at ~15 mN/m. It has been shown that Ca2+ ions mediate binding of proteins to lipid membranes by direct interaction with the phosphoryl moiety of the phospholipid headgroup (26), which gives rise to a change in headgroup orientation at the low and high surface pressures. The effects Ca2+ ions exerted on dioleoylphosphatidylserine (DOPS) included the change in the surface area per molecule from 0.675 to 0.625 nm2 upon Ca2+ binding (27, 28), possibly a partial or complete neutralization of negative charges on the phosphatidylserine (PS) headgroups, as well as its dehydration and consequent conformational changes (29).

Figure 3. Surface pressure − area isotherms of DPPC−DPPS monolayers (DPPC/DPPS, molar ratio 4:1) on various subphases measured with the compression rate of 150 mm2/min.

Figure 4a shows the typical SPR sensorgrams of annexin A5 binding to the DPPC−DPPS monolayers on Ca2+-containing buffer subphases. In the case of binding to the fluid monolayer the SPR sensor was brought in contact with the monolayer just prior to the onset of the first desorption cycle with EDTA-containing buffer. After the desorption cycle was over and the Ca2+-containing buffer was restored, the adsorption/desorption cycle was repeated and monitored with SPR. The results showed that the re-binding of annexin A5 to a previously fluid monolayer resulted in almost twice as high adsorbed 425 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


amount as that found on the immobilized monolayer. Namely, the refractive indices of the two types of DPPC−DPPS monolayers at the initial conditions (i.e. fluid vs. immobilized) and the initial SPR baselines should have been, in principle, identical before first adsorption because the same volumes of mixed lipids solution were spread onto the identical trough areas. Increased annexin A5 adsorption to the fluid phospholipid monolayer was likely due to the increased number of interactions between protein and DPPS head groups via Ca2+ bridges generated by recruitment and lateral diffusion of DPPS within the fluid monolayer. The recruitment of DPPS into the protein binding site is equivalent to a template-induced affinity increase and explains the higher amount of bound annexin A5. The templating effect was confirmed by measuring annexin A5 re-binding in the second adsorption/desorption cycle which resulted in an even higher adsorbed amount. Such a build-up of annexin A5 was probably the results of more efficient final 2-D packing of the protein molecules at the interface. Upon desorption with the EDTA-containing buffer, followed by Ca2+-containing buffer, the adsorbed annexin A5 was only partially removed from the previously fluid monolayer indicating that a favorable matching of lipids to protein combined with the lateral protein-protein interactions have strengthened the adsorbed layer. The occurrence of EDTA desorption resistant forms of blood platelet membrane bound annexin has been described in the literature (30, 31). In the case of annexin A5 adsorption to immobilized DPPC−DPPS monolayer the amount of the protein adsorbed in the initial adsorption cycle was typically ~ 45% lower than that found for the fluid monolayer: the baseline refractive index (RI) of 1.33745 increased to RI ~ 1.33773 during for adsorption on immobilized DPPC−DPPS monolayer vs. the final RI of ~ 1.33807 on the fluid monolayer. The lower protein affinity to the immobilized monolayer was futher indicated in the desorption step during which bound annexin A5 was completely desorbed from the surface in the presence of EDTA. In the second adsorption/desorption cycle annexin A5 re-binding to immobilized monolayer was almost identical to that found in the initial adsorption/desorption cycle. In contrast, EDTA desorbed only ~1/3 of the bound protein from the fluid monolayer, and the second adsorption/ desorption cycle resulted in another ~30% increase of bound annexin A5 amount above what was found prior to the first desorption step. Figure 4b shows the change of the refractive index unit (RIU) with time, d(RIU)/dt, (i.e. therefore proportional to the binding rate) as a function of the refractive index. In the second adsorption step, the binding rate was significantly faster in the case of (previously) fluid monolayer when compared with those found for immobilized monolayer. From the differences in protein re-binding to fluid and immobilized monolayers we infer that the effect was due to the protein-induced templating of the DPPC-DPPS monolayer during which DPPS molecules were recruited into spatial patterns that facilitated stronger binding of annexin A5. Similar monolayer templating effects have been already observed in our lab with ferritin (21, 32). It is known that Ca2+ concentration during binding determines the manner in which annexin A5 binds to membranes (2, 31). The present study suggests that the fluidity of the lipid layer might have also caused differences between the forms of bound annexin (31, 33, 34). 426 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

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Figure 4. (A) SPR sensorgrams of annexin A5 binding to fluid and immobilized DPPC−DPPS monolayers (DPPC/DPPS, molar ratio 4:1) using solution annexin A5 concentration of 1 μg/ml; (B) change of the refractive index unit (RIU) with time (proportional to the protein binding rate) as a function of the refractive index (proportional to the protein adsorbed amount).

Figure 5. Lower resolution AFM topography of DPPC−DPPS monolayer (DPPC/DPPS, molar ratio 4:1) transferred at the surface pressure 20 mN/m prior to (a, c) and after annexin A5 binding from 1 μg/ml solution (b,d). Scale bars: 500 nm (a,b) and 100 nm (c,d).

Figure 5 shows AFM topographical images of DPPC−DPPS monolayer before (Figure 5a,c) and after (Figure 5b,d) annexin A5 adsorption in the presence of Ca2+-containing buffer solution. The DPPC-DPPS monolayer was transferred at the surface pressure 20 mN/m, and the corresponding AFM images show the transferred monolayer as composed of small and isolated domains with with 428 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


heights ~ 2.0 nm with respect to the underlying OTS-silicon. The theoretical length of a DPPC molecule was reported to be at about 2.8 nm (35). Taking into the account the phase of the monolayer with tilted chains at 20 mN/m, it is reasonable to expect that the transferred phospholipid monolayer to have the roughness near 2.0 nm. This roughness is the result of sub-100 nm sized domains formed through calcium condensed lipid aggregates. Unlike pure DPPC monolayers that exhibit micron-sized domains, none are observed for the mixed DPPC-DPPS system at 20 mN/m on the calcium containing HEPES buffer subphase. After annexin A5 binding to the monolayer for 30 min, the lower resolution AFM images revealed a surface topography consistent with a nearly uniform, featureless monolayer with only a few defect sites (Figure 5b,d). It has been shown that annexin A5 does not interact or penetrate into the mixed DPPC−DPPS monolayer in the absence of Ca2+ even at the low surface pressure of 10 mN/m (36). Previous studies by ellipsometry (14), low-angle neutron scattering (37), and NMR (38) also confirmed a peripheral binding of annexin A5 without substantial penetration into the phospholipids monolayers (39).

Figure 6. Higher resolution AFM topography of different DPPC−DPPS monolayer areas with adsorbed annexin A5 layer. The DPPC−DPPS monolayer (DPPC/DPPS, molar ratio 4:1) was transferred at the surface pressure 20 mN/m. The adsorption of protein took place from 1 μg/ml annexin A5 solution in the Ca2+-containing buffer for 30 minutes before the AFM imaging. Scale bars: 50 nm (a,b) and 20 nm (c).

Higher resolution AFM images revealed features with lateral dimensions near 10 nm, which might correspond to protein assemblies (Figure 6). The trimers of annexin A5 are the basic building blocks of protein 2-D crystals (16, 41), and are observed to be 10 nm in diameter by electron microscopy (41) and 14 nm by AFM (increase due to tip-sample convolution) (42). Based on the size, the ellipsoidal-shaped objects in Figure 6 (size ~10 nm, height ~3.0 nm) correlate well with the size and height of paired annexin A5 trimers (26). Overall, the AFM images supported the 2-D nucleation model proposed by Brisson et al (40). First annexin A5 molecules bind to several DPPS molecules in a Ca2+-dependent manner, then these bound molecules serve as a 2-D nucleus for binding of other 429 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


annexins (38). The protein−protein interactions then propagate and ultimately result in the formation of 2-D crystals of annexin A5 with the protein−lipid interactions acting as anchoring points of the 2-D arrays with the membrane (40). However, Fourier analysis of the present AFM images did not reveal long range order. Given the difference between annexin A5 adsorption on fluid vs. immobilized lipid monolayers (Figure 4), the absence of the long range order might not be unexpected since AFM was used to image protein adsorbed to the immobilized mixed lipids monolayer.

Summary In this study we investigated the effect of DPPC-DPPS monolayer fluidity on annexin A5 binding and re-binding. The difference between the amount of protein adsorbed to fluid vs. immobilized monolayers could be explained by protein-induced monolayer templating during which the local recruitment of DPPS molecules improves the protein binding affinity. The increased annexin A5 affinity to previously fluid and templated monolayer was confirmed by improved protein re-binding to the templated film. Bound annexin A5 could be completely desorbed in the presence of ethylenediaminetetraacetic acid (EDTA) from immobilized monolayers, but only partially removed from the monolayer that was fluid during the initial protein adsorption step. AFM imaging revealed features that could be attributed to unordered 2-D protein-patches built up of dimers of the annexin A5 trimers.

Acknowledgments This study was supported by NIH grant RO1 HL84586.

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Annexin A5 Binding and Rebinding to Mixed Phospholipid

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