Lipid nanotechnologies for structural studies of membrane-associated proteins

Abstract

We present a methodology of lipid nanotubes (LNT) and nanodisks technologies optimized in our laboratory for structural studies of membrane-associated proteins at close to physiological conditions. The application of these lipid nanotechnologies for structure determination by cryoelectron microscopy (cryo-EM) is fundamental for understanding and modulating their function. The LNTs in our studies are single bilayer galactosylceramide based nanotubes of ~20 nm inner diameter and a few microns in length, that self-assemble in aqueous solutions. The lipid nanodisks (NDs) are self-assembled discoid lipid bilayers of ~10 nm diameter, which are stabilized in aqueous solutions by a belt of amphipathic helical scaffold proteins. By combining LNT and ND technologies, we can examine structurally how the membrane curvature and lipid composition modulates the function of the membrane-associated proteins. As proof of principle, we have engineered these lipid nanotechnologies to mimic the activated platelet's phosphtaidylserine rich membrane and have successfully assembled functional membrane-bound coagulation factor VIII in vitro for structure determination by cryo-EM. The macromolecular organization of the proteins bound to ND and LNT are further defined by fitting the known atomic structures within the calculated three-dimensional maps. The combination of LNT and ND technologies offers a means to control the design and assembly of a wide range of functional membrane-associated proteins and complexes for structural studies by cryo-EM. The presented results confirm the suitability of the developed methodology for studying the functional structure of membrane-associated proteins, such as the coagulation factors, at a close to physiological environment.

Dimeric Organization of Blood Coagulation Factor VIII bound to Lipid Nanotubes

“…The pFVIII-LNT SPT reconstruction at 20.5 Å confirms that the dimerization is not a result of imposing helical symmetry. The dimeric organization of the membrane-bound pFVIII is also not a result of the helical organization, as it is observed when FVIII is bound to lipid nanodiscs (with the same lipid composition as the LNT) when no helical order is imposed56. Finally, the cryo-ET and SPT reconstruction of FVIII free in solution confirms that FVIII remains monomeric in solution, which is consistent with our previously published dynamic light scattering data28.…”

“…The pFVIII-LNT SPT reconstruction at 20.5 Å resolution confirms that FVIII organizes as dimers when bound to the LNT, which was previously demonstrated by helical reconstruction5556. We are also showing a new pFVIII-LNT helical reconstruction resolved at 15.5 Å resolution.…”

“…Previously, we have demonstrated that several FVIII forms organize helically when bound to negatively charged LNT 37 55 and that the asymmetric unit of the human and porcine FVIII helically organized on LNT consists of two FVIII molecules 38 56 . To improve the fitting of the known FVIII structures within the cryo-EM map of the membrane-bound FVIII dimer we have calculated a new helical reconstruction of pFVIII-LNT at 15.5 Å resolution.…”

“…Imposing symmetry can also raise concerns regarding blurring out important features or giving rise to artifacts. To account for this, we have calculated the structure of pFVIII bound to LNT with a complementary approach for 3D reconstruction based on electron tomography of negatively stained helically ordered pFVIII-LNT tubes 55 56 combined with subtomogram volumes’ averaging and SPT reconstruction ( Fig. 3A ).…”

“…In this work we show the dimeric membrane-bound organization of blood coagulation Factor VIII bound to negatively charged lipid nanotubes (LNT) at close to physiological conditions. We have optimized single bilayer LNT to resemble the activated platelet surface and to bind FVIII with high affinity 37 53 54 55 , thus facilitating our structural and functional studies 38 54 55 56 . The pFVIII-LNT helical reconstruction calculated at 15.5 Å permits us to define the orientation of the FVIII monomers within the membrane-bound dimer.…”