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Dimeric Organization of Blood Coagulation Factor VIII bound to Lipid Nanotubes.

Dalm D, Galaz-Montoya JG, Miller JL, Grushin K, Villalobos A, Koyfman AY, Schmid MF, Stoilova-McPhie S - Sci Rep (2015)

Bottom Line: Here we present the dimeric FVIII membrane-bound structure when bound to lipid nanotubes, as determined by cryo-electron microscopy.By combining the structural information obtained from helical reconstruction and single particle subtomogram averaging at intermediate resolution (15-20 Å), we show unambiguously that FVIII forms dimers on lipid nanotubes.We also demonstrate that the organization of the FVIII membrane-bound domains is consistently different from the crystal structure in solution.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, TX 77555, USA.

ABSTRACT
Membrane-bound Factor VIII (FVIII) has a critical function in blood coagulation as the pro-cofactor to the serine-protease Factor IXa (FIXa) in the FVIIIa-FIXa complex assembled on the activated platelet membrane. Defects or deficiency of FVIII cause Hemophilia A, a mild to severe bleeding disorder. Despite existing crystal structures for FVIII, its membrane-bound organization has not been resolved. Here we present the dimeric FVIII membrane-bound structure when bound to lipid nanotubes, as determined by cryo-electron microscopy. By combining the structural information obtained from helical reconstruction and single particle subtomogram averaging at intermediate resolution (15-20 Å), we show unambiguously that FVIII forms dimers on lipid nanotubes. We also demonstrate that the organization of the FVIII membrane-bound domains is consistently different from the crystal structure in solution. The presented results are a critical step towards understanding the mechanism of the FVIIIa-FIXa complex assembly on the activated platelet surface in the propagation phase of blood coagulation.

No MeSH data available.


Related in: MedlinePlus

Helical reconstruction of porcine Factor VIII bound to LNT.Orthogonal views along (top row) and perpendicular to (bottom row) the helical (z) axis of the final pFVIII-LNT helical reconstruction calculated from 10,430 helical segments and masked to 522 Å length (Supplements 1 and 2). The inner diameter of the helical tube is 200 Å. A. Density representation of the pFVIII-LNT helical reconstruction. The maximum density of the pFVIII-LNT helical reconstruction is shown with dark blue, 0.5 (50% of the maximum density level) is shown with medium blue and 0.2 (20% of the maximum density level) is shown with light blue. B. Surface representation of the pFVIII-LNT reconstruction drawn at 0.2 contour level (20% of the maximum density). The number of subunits viewed perpendicular to the helical axis is indicated, as well as the azimuthal angle (Δφ) between two adjacent subunits. A single helical strand from the 5 strand pFVIII-LNT helical structure shown on Fig. S3a is highlighted in dark blue. The volume corresponding to one asymmetric unit is indicated with a dashed oval line. C. Surface representation of the pFVIII-LNT cryo-EM map (light blue) superimposed with the segmented map corresponding to one asymmetric unit (dark blue). The cryo-EM map corresponding to a bare LNT (red) without attached pFVIII is superimposed with the pFVIII-LNT helical reconstruction to delineate the outer membrane surface of the LNT bilayer.
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f2: Helical reconstruction of porcine Factor VIII bound to LNT.Orthogonal views along (top row) and perpendicular to (bottom row) the helical (z) axis of the final pFVIII-LNT helical reconstruction calculated from 10,430 helical segments and masked to 522 Å length (Supplements 1 and 2). The inner diameter of the helical tube is 200 Å. A. Density representation of the pFVIII-LNT helical reconstruction. The maximum density of the pFVIII-LNT helical reconstruction is shown with dark blue, 0.5 (50% of the maximum density level) is shown with medium blue and 0.2 (20% of the maximum density level) is shown with light blue. B. Surface representation of the pFVIII-LNT reconstruction drawn at 0.2 contour level (20% of the maximum density). The number of subunits viewed perpendicular to the helical axis is indicated, as well as the azimuthal angle (Δφ) between two adjacent subunits. A single helical strand from the 5 strand pFVIII-LNT helical structure shown on Fig. S3a is highlighted in dark blue. The volume corresponding to one asymmetric unit is indicated with a dashed oval line. C. Surface representation of the pFVIII-LNT cryo-EM map (light blue) superimposed with the segmented map corresponding to one asymmetric unit (dark blue). The cryo-EM map corresponding to a bare LNT (red) without attached pFVIII is superimposed with the pFVIII-LNT helical reconstruction to delineate the outer membrane surface of the LNT bilayer.

Mentions: Previously, we have demonstrated that several FVIII forms organize helically when bound to negatively charged LNT3755 and that the asymmetric unit of the human and porcine FVIII helically organized on LNT consists of two FVIII molecules3856. 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. This was achieved by collecting more cryo-EM data of well ordered pFVIII-LNT helical tubes as previously described55 and applying a cosine mask to the helical segments to improve the efficiency of the 2D classification process (Supplement 1, Fig. S1). The final pFVIII-LNT helical structure was obtained from 10,430 helical segments (particles set) after 200 consecutive IHRSR refinement cycles (Supplement 2, Fig. S2). The helical parameters converged to a rise Δz = 36.0 Å and azimuthal angle Δϕ  = 35.5o, corresponding to a five strand helical structure (Fig. 2A,B Fig. S3). The resolution of the final pFVIII-LNT helical reconstruction was determined to be 15.5 Å at FSC = 0.143 according to the gold standard FSC criterion57 (Fig. S3C). The 3D volume of the pFVIII-LNT helical reconstruction was further segmented showing that the membrane-bound pFVIII molecules are organized as dimers within the asymmetric unit (Fig. S3A). To localize the exact position of the membrane bilayer we collected cryo-EM micrographs of LNT alone (with no FVIII attached) with the same lipid composition as the pFVIII-LNT and calculated a 3D structure following the same algorithms as for the pFVIII-LNT reconstruction (Fig. 2C). The volume corresponding to one asymmetric unit, as shown in Fig. 2C is equal to 1,029 × 103 Å3 and the surface area to 84.2 × 103 Å2. This volume can easily accommodate two FVIII molecules and part of the outer lipid layer of the LNT’s membrane, as the volume and surface area corresponding to the FVIII-3D structure (3CDZ) low-pass filtered to 25 Å are equal to 403.1 × 103 Å3 and 31.5 × 103 Å2, respectively and the volume and surface area corresponding to the FVIII-LNT structures in Fig. 1B, low-pass filtered to 25 Å are equal to 367.7 × 103 Å3 and 31.2 × 103 Å2, respectively. Therefore each asymmetric unit in the five strand pFVIII-LNT helical structure can accommodate two FVIII molecules organized as a membrane-bound dimer.


Dimeric Organization of Blood Coagulation Factor VIII bound to Lipid Nanotubes.

Dalm D, Galaz-Montoya JG, Miller JL, Grushin K, Villalobos A, Koyfman AY, Schmid MF, Stoilova-McPhie S - Sci Rep (2015)

Helical reconstruction of porcine Factor VIII bound to LNT.Orthogonal views along (top row) and perpendicular to (bottom row) the helical (z) axis of the final pFVIII-LNT helical reconstruction calculated from 10,430 helical segments and masked to 522 Å length (Supplements 1 and 2). The inner diameter of the helical tube is 200 Å. A. Density representation of the pFVIII-LNT helical reconstruction. The maximum density of the pFVIII-LNT helical reconstruction is shown with dark blue, 0.5 (50% of the maximum density level) is shown with medium blue and 0.2 (20% of the maximum density level) is shown with light blue. B. Surface representation of the pFVIII-LNT reconstruction drawn at 0.2 contour level (20% of the maximum density). The number of subunits viewed perpendicular to the helical axis is indicated, as well as the azimuthal angle (Δφ) between two adjacent subunits. A single helical strand from the 5 strand pFVIII-LNT helical structure shown on Fig. S3a is highlighted in dark blue. The volume corresponding to one asymmetric unit is indicated with a dashed oval line. C. Surface representation of the pFVIII-LNT cryo-EM map (light blue) superimposed with the segmented map corresponding to one asymmetric unit (dark blue). The cryo-EM map corresponding to a bare LNT (red) without attached pFVIII is superimposed with the pFVIII-LNT helical reconstruction to delineate the outer membrane surface of the LNT bilayer.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4469981&req=5

f2: Helical reconstruction of porcine Factor VIII bound to LNT.Orthogonal views along (top row) and perpendicular to (bottom row) the helical (z) axis of the final pFVIII-LNT helical reconstruction calculated from 10,430 helical segments and masked to 522 Å length (Supplements 1 and 2). The inner diameter of the helical tube is 200 Å. A. Density representation of the pFVIII-LNT helical reconstruction. The maximum density of the pFVIII-LNT helical reconstruction is shown with dark blue, 0.5 (50% of the maximum density level) is shown with medium blue and 0.2 (20% of the maximum density level) is shown with light blue. B. Surface representation of the pFVIII-LNT reconstruction drawn at 0.2 contour level (20% of the maximum density). The number of subunits viewed perpendicular to the helical axis is indicated, as well as the azimuthal angle (Δφ) between two adjacent subunits. A single helical strand from the 5 strand pFVIII-LNT helical structure shown on Fig. S3a is highlighted in dark blue. The volume corresponding to one asymmetric unit is indicated with a dashed oval line. C. Surface representation of the pFVIII-LNT cryo-EM map (light blue) superimposed with the segmented map corresponding to one asymmetric unit (dark blue). The cryo-EM map corresponding to a bare LNT (red) without attached pFVIII is superimposed with the pFVIII-LNT helical reconstruction to delineate the outer membrane surface of the LNT bilayer.
Mentions: Previously, we have demonstrated that several FVIII forms organize helically when bound to negatively charged LNT3755 and that the asymmetric unit of the human and porcine FVIII helically organized on LNT consists of two FVIII molecules3856. 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. This was achieved by collecting more cryo-EM data of well ordered pFVIII-LNT helical tubes as previously described55 and applying a cosine mask to the helical segments to improve the efficiency of the 2D classification process (Supplement 1, Fig. S1). The final pFVIII-LNT helical structure was obtained from 10,430 helical segments (particles set) after 200 consecutive IHRSR refinement cycles (Supplement 2, Fig. S2). The helical parameters converged to a rise Δz = 36.0 Å and azimuthal angle Δϕ  = 35.5o, corresponding to a five strand helical structure (Fig. 2A,B Fig. S3). The resolution of the final pFVIII-LNT helical reconstruction was determined to be 15.5 Å at FSC = 0.143 according to the gold standard FSC criterion57 (Fig. S3C). The 3D volume of the pFVIII-LNT helical reconstruction was further segmented showing that the membrane-bound pFVIII molecules are organized as dimers within the asymmetric unit (Fig. S3A). To localize the exact position of the membrane bilayer we collected cryo-EM micrographs of LNT alone (with no FVIII attached) with the same lipid composition as the pFVIII-LNT and calculated a 3D structure following the same algorithms as for the pFVIII-LNT reconstruction (Fig. 2C). The volume corresponding to one asymmetric unit, as shown in Fig. 2C is equal to 1,029 × 103 Å3 and the surface area to 84.2 × 103 Å2. This volume can easily accommodate two FVIII molecules and part of the outer lipid layer of the LNT’s membrane, as the volume and surface area corresponding to the FVIII-3D structure (3CDZ) low-pass filtered to 25 Å are equal to 403.1 × 103 Å3 and 31.5 × 103 Å2, respectively and the volume and surface area corresponding to the FVIII-LNT structures in Fig. 1B, low-pass filtered to 25 Å are equal to 367.7 × 103 Å3 and 31.2 × 103 Å2, respectively. Therefore each asymmetric unit in the five strand pFVIII-LNT helical structure can accommodate two FVIII molecules organized as a membrane-bound dimer.

Bottom Line: Here we present the dimeric FVIII membrane-bound structure when bound to lipid nanotubes, as determined by cryo-electron microscopy.By combining the structural information obtained from helical reconstruction and single particle subtomogram averaging at intermediate resolution (15-20 Å), we show unambiguously that FVIII forms dimers on lipid nanotubes.We also demonstrate that the organization of the FVIII membrane-bound domains is consistently different from the crystal structure in solution.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, TX 77555, USA.

ABSTRACT
Membrane-bound Factor VIII (FVIII) has a critical function in blood coagulation as the pro-cofactor to the serine-protease Factor IXa (FIXa) in the FVIIIa-FIXa complex assembled on the activated platelet membrane. Defects or deficiency of FVIII cause Hemophilia A, a mild to severe bleeding disorder. Despite existing crystal structures for FVIII, its membrane-bound organization has not been resolved. Here we present the dimeric FVIII membrane-bound structure when bound to lipid nanotubes, as determined by cryo-electron microscopy. By combining the structural information obtained from helical reconstruction and single particle subtomogram averaging at intermediate resolution (15-20 Å), we show unambiguously that FVIII forms dimers on lipid nanotubes. We also demonstrate that the organization of the FVIII membrane-bound domains is consistently different from the crystal structure in solution. The presented results are a critical step towards understanding the mechanism of the FVIIIa-FIXa complex assembly on the activated platelet surface in the propagation phase of blood coagulation.

No MeSH data available.


Related in: MedlinePlus