Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Podsumowanie

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Streszczenie

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni²⁺ coordinated by nitrilotriacetic acid (NTA(Ni²⁺)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Wprowadzenie

Studying membrane-proximal protein-protein interactions is a challenging endeavor due to difficulty in recapitulating the native environment of the integral membrane proteins involved¹. This is due to the necessity of detergent solubilization and the inconsistent orientation of proteins in proteoliposomes. In order to avoid these issues, we have employed a strategy whereby soluble domains of integral membrane proteins are expressed as His-tag fusion proteins, and these soluble fragments are anchored to scaffold liposomes via interactions with NTA(Ni²⁺) headgroups at the lipid surface. Using these scaffolds, lipid-proximal protein interactions can be investigated over a range of lipid and protein compositions.

We have effectively applied this method to investigate the critical protein-protein interactions that govern assembly of the mitochondrial fission complex and examine lipid interactions that modulate this process². During mitochondrial fission, a conserved membrane remodeling protein, called Dynamin-related protein 1 (Drp1)³, is recruited to the surface of the Outer Mitochondrial Membrane (OMM) in response to cellular signals that regulate energy homeostasis, apoptotic signaling, and several other integral mitochondrial processes. This large, cytosolic GTPase is recruited to the surface of mitochondria through interactions with integral OMM proteins^4-8. The role of one such protein, Mitochondrial Fission Factor (Mff), has been difficult to elucidate due to an apparent weak interaction with Drp1 in vitro. Nevertheless, genetic studies have clearly demonstrated that Mff is essential for successful mitochondrial fission^7,8. The method described in this manuscript was able to overcome previous shortcomings by introducing simultaneous lipid interactions that promote Drp1-Mff interactions. Overall, this novel assay revealed fundamental interactions guiding assembly of the mitochondrial fission complex and provided a new stage for ongoing structural and functional studies of this essential molecular machine.

To date, examination of interactions between Drp1 and Mff have been complicated by the inherent flexibility of Mff⁹, the heterogeneity of Drp1 polymers^2,10, and the difficulty in purifying and reconstituting full-length Mff with an intact transmembrane domain¹¹. We addressed these challenges by using NTA(Ni²⁺) scaffold liposomes to reconstitute His-tagged Mff lacking its transmembrane domain(MffΔTM-His₆). This strategy was advantageous because MffΔTM was extremely soluble when over-expressed in E. coli, and this isolated protein was easily reconstituted on scaffold liposomes. When tethered to these lipid templates, Mff assumed an identical, outward facing orientation on the surface of the membrane. In addition to these advantages, mitochondrial lipids, such as cardiolipin, were added to stabilize Mff folding and association with the membrane¹¹. Cardiolipin also interacts with the variable domain of Drp1^2,12 which may stabilize this disordered region and facilitate assembly of the fission machinery.

This robust method is widely applicable for future studies that seek to evaluate membrane-proximal protein interactions. Through the use of additional tethering/affinity interactions, the sophistication of these membrane reconstitution studies can be enhanced to mimic additional complexity found at the surface of membranes within cells. At the same time, lipid compositions can be modified to more accurately mimic the native environments of these macromolecular complexes. In summary, this method provides a means to examine the relative contributions of proteins and lipids in shaping membrane morphologies to during critical cellular processes.

Protokół

1. Scaffold Liposome Preparation

NOTE: Ideally, initial experiments should use a relatively simple and featureless scaffold (comprised of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine or PC) and DGS-NTA(Ni²⁺) (1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl](nickel salt)). Building off of these experiments, lipid charge, flexibility, and curvature can be introduced as individual factors with the potential to alter membrane-proximal interactions. These changes can be achieved by adding defined amounts of specific lipid constituents, including phosphatidylserine or cardiolipin (CL), phosphatidylethanolamine (DOPE or PE), or galactosyl(β) ceramide.

1. Combine lipids dissolved in chloroform in a clean glass test tube. Evaporate the solvent with dry nitrogen gas while rotating the tube to form a thin lipid film. Remove residual solvent with a centrifugal evaporator for 1 h at 37 °C.
   NOTE: Various liposome formulations are used in the protocols described below: scaffold liposomes (3.3 mol% DGS-NTA(Ni²⁺) / 96.7 mol% DOPC), scaffold liposomes with cardiolipin (3.3 mol% DGS-NTA(Ni²⁺) / 10 mol% cardiolipin / 86.7 mol% DOPC), flexible scaffold liposomes with cardiolipin (3.3 mol% DGS-NTA(Ni²⁺) / 10 mol% cardiolipin / 35 mol% DOPE / 51.7 mol% DOPC), and enriched scaffold liposomes (10 mol% DGS-NTA(Ni²⁺) / 15 mol% cardiolipin / 35 mol% DOPE / 40 mol% DOPC).
2. Add Buffer A (25 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 150 mM KCl, pH adjusted to 7.5 with KOH) preheated to 37 °C such that the final lipid concentration is 1 - 2 mM. Incubate 30 min at 37 °C with occasional vortexing to fully resuspend the lipid mixture (Figure 1a).
3. Transfer to a plastic test tube, place the tube in liquid nitrogen until completely frozen (roughly 30 s), and place in a 37 °C water bath until fully thawed (roughly 1 - 2 min). Repeat for a total of 4 freeze-thaw cycles (Figure 1b).
4. Prepare a lipid extruder by soaking 4 filter supports and a polycarbonate filter in buffer and assembling the extruder according to manufacturer instructions. Extrude the lipid solution through the filter 21 times. Use gentle, constant pressure to ensure a homogenous size distribution (Figure 1c).
   NOTE: For all experiments described in this protocol, a 1.0 µm polycarbonate filter was used for extrusion. Drp1 interaction with anionic lipids can be observed with a variety of liposome diameters ranging from 50 nm to 400 nm¹² or larger¹³. Hence, the filter size of 1 µm was chosen to be ideal for both GTPase activity and for electron microscopy. If other liposome diameters are desired, preparation of giant unilamellar vesicles^14,15 (GUVs) or small unilamellar vesicles¹⁶ (SUVs) can be used. Dynamic light scattering can be used to assess liposome size heterogeneity¹³.
5. Store extruded liposomes at 4 °C and discard after 3 - 5 d.

2. Use of Scaffold Liposomes for Protein Binding Analysis

1. Sample Preparation
   1. Incubate His-tagged MffΔTM (5 µM final) with scaffold liposomes (40 mol% PC / 35 mol% PE / 15 mol% CL / 10 mol% DGS-NTA(Ni²⁺); 50 µM final) for at least 15 min at RT in Buffer A + BME (25 mM HEPES, 150 mM KCl, 10 mM β-mercaptoethanol (BME), pH adjusted to 7.5 with KOH). For an Mff-free control, incubate liposomes with a his-tagged control protein (such as GFP) to bind and shield exposed NTA(Ni²⁺).
      NOTE: MffΔTM was expressed and purified as described in a previous study². GFP was purified in a similar manner, but the ion-exchange step was omitted. BME was required for these experiments because Drp1 is sensitive to oxidation, which can alter its activity and assembly properties.
   2. Add Drp1 (2 µM final) and incubate for 1 h at RT.
      NOTE: Drp1 was expressed and purified as described in a previous study². After incubation with Drp1, the effect of nucleotide binding on membrane deformation can be investigated by incubating one additional hour with 2 mM MgCl₂ and either 1 mM GTP, 1 mM GMP-PCP, or Buffer A + BME.
2. Negative Stain Transmission Electron Microscopy (EM) Analysis
   1. Transfer 5 µL of sample to a sheet of laboratory film, and lay a carbon-coated Cu/Rh grid on the sample. Incubate the grid 1 min on the sample, blot away excess liquid on filter paper, and transfer to a drop of 2% uranyl acetate. Incubate 1 min, blot excess stain on filter paper, and transfer to a grid box. Store under vacuum O/N to ensure full desiccation.
   2. Image samples using a transmission electron microscope at 18,500 - 30,000X magnification to observe ultrastructural changes in protein and liposome morphologies¹⁷. 
      Note: Ultrastructural changes can be quantified using image analysis software, such as ImageJ¹³ (http://imagej.nih.gov/ij/). Protein decoration can be measured when compared with naked lipid templates. Additionally, the diameters of tubular segments can be measured from the outermost portion of the assemblies¹³. A more detailed analysis can be performed using cryo-electron microscopy¹⁷. This method can be used to image native protein-lipid assemblies in solvent without the use of heavy metal stains that coat the sample. In this way, detailed structural features not apparent in negative stain, including changes in the underlying lipid morphology, can be examined and quantified.

3. Use of Scaffold Liposomes for Enzymatic Assay

Note: A colorimetric GTPase assay¹⁸ was used to measure phosphate liberation via GTP hydrolysis. Alternative GTPase assays are available¹⁹ and can be implemented as needed.

1. Incubate His-tagged MffΔTM (Mff), Fis1ΔTM(Fis1), or GFP (5 µM final for all) with scaffold liposomes (150 µM final) for 15 min at RT in Buffer A + BME (volume = 30 µL). Add Drp1 (500 nM final) and incubate an additional 15 min at RT (volume = 80 µL).
   NOTE: Fis1 was purified in a similar manner to Mff², but the ion-exchange chromatography step was omitted. The purpose of His-tagged GFP is to shield the NTA(Ni²⁺) headgroups and prevent nonspecific charge interactions with other proteins. If no effect is observed in the absence of GFP, then this control may not be required. Alternative blocking proteins (of comparable size to the protein of interest) can be used as well, but GFP allows for direct visualization of the interactions with scaffold liposomes.
2. Transfer tubes to a thermocycler set to 37 °C, and initiate reactions by addition of GTP and MgCl₂ (1 mM and 2 mM final, respectively; volume = 120 µL).
3. At desired time points (i.e. T = 5, 10, 20, 40, 60 min), transfer 20 µL of reaction to wells of a microtiter plate containing 5 µL of 0.5 M EDTA to chelate Mg²⁺ and stop the reaction.
4. Prepare a set of phosphate standards by diluting KH₂PO₄ in Buffer A + BME to calibrate results. A useful set of standards is 100, 80, 60, 40, 20, 10, 5, and 0 µM. Add 20 µL of each to wells containing 5 µL of 0.5 M EDTA.
5. Add 150 µL of Malachite green reagent (1 mM malachite green carbinol, 10 mM ammonium molybdate tetrahydrate, and 1 N HCl) to each well, and read OD₆₅₀ 5 min after addition.
   NOTE: GTP is acid labile, and will hydrolyze in the presence of malachite green reagent. Ensure that the time between adding malachite reagent and reading is constant to ensure reproducible results.
6. Generate a standard curve by plotting OD₆₅₀ of the standards as a function of phosphate concentration. Use linear regression to determine the relationship between OD₆₅₀ and phosphate concentration in a sample.
7. Using the linear regression, convert the OD₆₅₀ of the protein reaction samples to µM phosphate. Determine the rate of phosphate generation for each reaction mixture by plotting phosphate concentration as a function of time, and convert to k_cat by dividing the rate by the Drp1 concentration (0.5 µM).
   NOTE: Only the initial linear rate should be used to determine the rate of phosphate generation, and a minimum of 3 data points must be used. If the rate of reaction is sufficiently rapid that the first three data points are not linear (i.e. the r² of the linear fit is less than 0.9) a significantly shorter time course with at least 3 time points should be performed.

Wyniki

While the interaction between Drp1 and Mff has been demonstrated to be important for mitochondrial fission, this interaction has been difficult to recapitulate in vitro. Our goal was to better emulate the cellular environment wherein Drp1 and Mff interact. To this end, liposomes containing limiting concentrations of NTA(Ni²⁺) headgroups were prepared by rehydrating a lipid film as described above. The lipid solution initially consists of unilamellar and multilamellar v...

Dyskusje

This protocol offers a method for investigating protein-protein interactions involving integral membrane proteins. By utilizing a modular liposome scaffold, investigators are capable of assessing the activity of one or more proteins in a lipid-proximal environment. Previous studies have demonstrated a similar method for receptor enzymes of the plasma membrane^24-26. We have expanded this method to incorporate lipid cofactors and explore interactions between proteins that make up the mechan...

Materiały

Name	Company	Catalog Number	Comments
Phosphatidylcholine (DOPC)	Avanti Polar Lipids	850375
Phosphatidylethanolamine (DOPE)	Avanti Polar Lipids	850725
DGS-NTA(Ni2+)	Avanti Polar Lipids	790404
Bovine Heart Cardiolipin (CL)	Avanti Polar Lipids	840012
Chloroform	Acros Organics	268320010
Liposome Extruder	Avanti Polar Lipids	610023
Cu/Rh Negative Stain Grids	Ted Pella	79712
Microfuge Tube	Beckman	357448
GTP	Jena Biosciences	NU-1012
GMP-PCP	Sigma Aldrich	M3509
Microtiter Plate strips	Thermo Scientific	469949
EDTA	Acros Organics	40993-0010
Instant Blue Coomassie Dye	Expedeon	ISB1L
HEPES	Fisher Scientific	BP310
BME	Sigma Aldrich	M6250
KCl	Fisher Scientific	P330
KOH	Fisher Scientific	P250
Magnesium Chloride	Acros Organics	223211000
4 - 20% SDS-PAGE Gel	Bio Rad	456-1096
4x Laemmli Loading Dye	Bio Rad	161-0747
HCL	Fisher Scientific	A144S
Malachite Green Carbinol	Sigma Aldrich	229105
Ammonium Molybdate Tetrahydrate	Sigma Aldrich	A7302
Laboratory Film	Parafilm	PM-996
Uranyl Acetate	Polysciences	21447
Tecnai T12 100 keV Microscope	FEI
Optima MAX	Beckman
TLA-55 Rotor	Beckman
Refrigerated CentriVap Concentrator	Labconico
Mastercycler Pro Thermocycler	Eppendorf
VersaMax Microplate reader	Molecular Devices