Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes

Summary

We present a combination of Cryo-electron microscopy, lipid nanotechnology, and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described.

Abstract

Cryo-electron microscopy (Cryo-EM)¹ is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and membrane environment².

Coagulation Factor VIII (FVIII)³ is a multi-domain blood plasma glycoprotein. Defect or deficiency of FVIII is the cause for Hemophilia type A - a severe bleeding disorder. Upon proteolytic activation, FVIII binds to the serine protease Factor IXa on the negatively charged platelet membrane, which is critical for normal blood clotting⁴. Despite the pivotal role FVIII plays in coagulation, structural information for its membrane-bound state is incomplete⁵. Recombinant FVIII concentrate is the most effective drug against Hemophilia type A and commercially available FVIII can be expressed as human or porcine, both forming functional complexes with human Factor IXa^6,7.

In this study we present a combination of Cryo-electron microscopy (Cryo-EM), lipid nanotechnology and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described. The representative results demonstrate that our approach is sufficiently sensitive to define the differences in the helical organization between the two highly homologous in sequence (86% sequence identity) proteins. Detailed protocols for the helical organization, Cryo-EM and electron tomography (ET) data acquisition are given. The two-dimensional (2D) and three-dimensional (3D) structure analysis applied to obtain the 3D reconstructions of human and porcine FVIII-LNT is discussed. The presented human and porcine FVIII-LNT structures show the potential of the proposed methodology to calculate the functional, membrane-bound organization of blood coagulation Factor VIII at high resolution.

Introduction

Blood coagulation Factor VIII (FVIII) is a large glycoprotein of 2,332 amino acids organized in six domains: A1-A2-B-A3-C1-C2 ³. Upon Thrombin activation FVIII acts as the cofactor to Factor IXa within the membrane-bound Tenase complex. Binding of activated FVIII (FVIIIa) to FIXa in a membrane-depending manner enhances FIXa proteolytic efficiency more than 10⁵ times, which is critical for efficient blood coagulation⁴. Despite the important role FVIII plays in coagulation and the Tenase complex formation, the functional membrane-bound FVIII structure is yet to be resolved.

To address this, single lipid b....

Protocol

1. Sample Preparation

1. Buffer exchange human FVIII-BDD¹⁴ and porcine FVIII-BDD¹⁵ against HBS-Ca buffer (20 mM HEPES, 150 mM NaCl, 5 mM CaCl₂, pH = 7.4) and concentrate to 1.2 mg/ml. Keep the protein solution at -80 °C.
2. Prepare lipid nanotubes (LNT) by mixing GalactosylCeramide (GC) and phosphatidylserine (PS) at 1:4 w/w ratio in chloroform. Evaporate the chloroform under argon and solubilize the lipids in HBS buffer to 1 mg/ml. Keep the LNT solution at 4 °C.

2. Cryo-electron Microscopy of FVIII-LNT

1. Cryo-EM Sample Preparation
   1. Glow discharge the 300....

Results

Recombinant human and porcine FVIII were successfully organized helically on negatively charged single bilayer LNT, resembling the activated platelet surface. The helical organization of the human and porcine FVIII-LNT was consistent through the collected digital micrographs (Figure 2).  The control LNT and the human and porcine FVIII-LNT helical tubes were selected and segmented with the e2helixboxer.py GUI and initial data sets created with the e2workflow.py GUI, Single particle option.......

Discussion

In this work a methodology is presented to differentiate between two membrane-bound organizations of highly homologous proteins: human and porcine FVIII self-assembled on lipid nanotubes in the conditions encountered in the human body.

In the described procedure, human and porcine FVIII are successfully organized helically on lipid nanotubes, which is the most critical step. The next critical step is to preserve the sample in thin amorphous ice by flash freezing
.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
JEM2100 with LaB6	JEOL Ltd.	JEM-2100	operated at 200 kV
with TEMCON software	JEOL Ltd.
Gatan626 Cryo-holder	Gatan, Inc.	626.DH	cooled to -175 °C
with temperature controler unit	Gatan, Inc.
Gatan 4K x 4K CCD camera	Gatan, Inc.	US4000	4,096 x 4,096 pixel at 15 μm/pixel physical resolution
Solarus Model 950 plasma cleaner	Gatan, Inc.
Vitrobot Mark IV	FEI
Carbon coated 300-mesh 3 mm copper grid	Ted Pella	01821	plasma cleaned for 10 sec on high power
Quantifoil R2/2 300 mesh	Electron Microscopy Sciences	Q225-CR2	Carbon coated 300-mesh Cu grids with 2 mm in diameters holes
Uranyl acetate dihydrate	Ted Pella	19481	1% solution, filtered
Galactosyl ceramide	Avanti Polar Lipids Inc.	860546
Dioleoyl-sn-glycero-phospho-L-serine	Avanti Polar Lipids Inc.	840035
EM software Digital Micrograph	Gatan, Inc.		http://www.gatan.com/DM/
EM software EMAN	free download		http://blake.bcm.edu/emanwiki/EMAN/
EM software Spider	free download		http://spider.wadsworth.org/spider_doc/spider/docs/spider.html
EM software IHRSR	free download		Programs available from Edward H. Egelman http://people.virginia.edu/~ehe2n/
EM software (IMOD)	free download		http://bio3d.colorado.edu/imod/
EM software (SerialEM)	free download		ftp://bio3d.colorado.edu/pub/SerialEM/
UCSF-Chimera	free download		http://www.cgl.ucsf.edu/chimera/download.html