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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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 cardio....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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