Limits...
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

Fitting of the FVIII-LNT structure within the EM map of the membrane-bound pFVIII-LNT dimer segmented from the helical and SPT reconstructions.A. Fitting of the FVIII-LNT structure from Fig. 1 to the cryo-EM map of the segmented dimer from the helical reconstruction, as shown on Fig. S5B. B. Fitting of the FVIII-LNT structure from Fig. 1 to the EM map of the segmented dimer from the SPT reconstruction as shown on Fig. S5A. The EM maps of the FVIII segmented dimer are shown as a grey surface. The FVIII A1 and A2 domains from the heavy chain are shown in pink and red, respectively. The FVIII A3, C1 and C2 domains from the light chain are shown in blue, light blue and cyan, respectively. The FVIII molecules are oriented in such a way as to interact with the LNT membrane with the identified residues from the apical loops of the C2 domain as shown on Fig. 1B and Fig. S5C.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4469981&req=5

f6: Fitting of the FVIII-LNT structure within the EM map of the membrane-bound pFVIII-LNT dimer segmented from the helical and SPT reconstructions.A. Fitting of the FVIII-LNT structure from Fig. 1 to the cryo-EM map of the segmented dimer from the helical reconstruction, as shown on Fig. S5B. B. Fitting of the FVIII-LNT structure from Fig. 1 to the EM map of the segmented dimer from the SPT reconstruction as shown on Fig. S5A. The EM maps of the FVIII segmented dimer are shown as a grey surface. The FVIII A1 and A2 domains from the heavy chain are shown in pink and red, respectively. The FVIII A3, C1 and C2 domains from the light chain are shown in blue, light blue and cyan, respectively. The FVIII molecules are oriented in such a way as to interact with the LNT membrane with the identified residues from the apical loops of the C2 domain as shown on Fig. 1B and Fig. S5C.

Mentions: To resolve the pFVIII molecular orientation within the membrane-bound dimer, the FVIII-LNT and FVIII crystal (FVIII-3D) structures from Fig. 1 were fitted within the pFVIII-LNT helical map with the rigid body docking algorithms implemented in the ‘fit in map’ option of the UCSF Chimera software6162 (Fig. 6A). To achieve this the helical pFVIII-LNT map corresponding to the membrane-bound FVIII dimer was further segmented to delineate the densities corresponding to each of the FVIII molecules (Fig. S5B). The FVIII-LNT molecules were then fitted with the rigid body algorithm implemented in the ‘fit to map’ option of UCSF Chimera, starting from different orientations of the FVIII-LNT structures, as detailed in the Materials and Methods section. The best FVIII-LNT structure fit within the segmented membrane-bound FVIII dimer map also allowed for the proper orientation of the C2 domains, such that their membrane-binding loops can interact with the negatively charged LNT membrane (Fig. 6A, Fig. S5B,C). The FVIII crystal (FVIII-3D) structure did not fit as well, as the juxtaposed configuration of the C domains could not be accommodated well in the pFVIII-LNT helical map (Fig. S5Da). The FVIII-LNT structure also fit well in the segmented dimer map from the pFVIII-LNT SPT reconstruction, which was further segmented to delineate the map/density corresponding to each FVIII molecule within the dimer (Fig. 6B, Fig. S5A, Fig. S4B). The lower resolution of the pFVIII-SPT structure resulted in a poorer fit of the FVIII-LNT structure. However, fitting of the FVIII crystal (FVIII-3D) structure within the pFVIII-SPT map left a considerable portion of the FVIII-HC outside of the molecular contour (Fig. S5Db). The orientation of the membrane-bound FVIII molecules obtained from the fitting of the FVIII-LNT structure was such that the main protein-protein interface within the membrane-bound dimer was at the level of the FVIII-heavy chains (HC:A1-A2 domains). This orientation of the FVIII monomers within the membrane-bound dimer does not obstruct the FVIIIa-FIXa interface encompassing the A2 and A3 domains, as shown by the almost identical activity measured for pFVIII in the presence and absence of LNT (Fig. 6, Supplement 6).


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)

Fitting of the FVIII-LNT structure within the EM map of the membrane-bound pFVIII-LNT dimer segmented from the helical and SPT reconstructions.A. Fitting of the FVIII-LNT structure from Fig. 1 to the cryo-EM map of the segmented dimer from the helical reconstruction, as shown on Fig. S5B. B. Fitting of the FVIII-LNT structure from Fig. 1 to the EM map of the segmented dimer from the SPT reconstruction as shown on Fig. S5A. The EM maps of the FVIII segmented dimer are shown as a grey surface. The FVIII A1 and A2 domains from the heavy chain are shown in pink and red, respectively. The FVIII A3, C1 and C2 domains from the light chain are shown in blue, light blue and cyan, respectively. The FVIII molecules are oriented in such a way as to interact with the LNT membrane with the identified residues from the apical loops of the C2 domain as shown on Fig. 1B and Fig. S5C.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Fitting of the FVIII-LNT structure within the EM map of the membrane-bound pFVIII-LNT dimer segmented from the helical and SPT reconstructions.A. Fitting of the FVIII-LNT structure from Fig. 1 to the cryo-EM map of the segmented dimer from the helical reconstruction, as shown on Fig. S5B. B. Fitting of the FVIII-LNT structure from Fig. 1 to the EM map of the segmented dimer from the SPT reconstruction as shown on Fig. S5A. The EM maps of the FVIII segmented dimer are shown as a grey surface. The FVIII A1 and A2 domains from the heavy chain are shown in pink and red, respectively. The FVIII A3, C1 and C2 domains from the light chain are shown in blue, light blue and cyan, respectively. The FVIII molecules are oriented in such a way as to interact with the LNT membrane with the identified residues from the apical loops of the C2 domain as shown on Fig. 1B and Fig. S5C.
Mentions: To resolve the pFVIII molecular orientation within the membrane-bound dimer, the FVIII-LNT and FVIII crystal (FVIII-3D) structures from Fig. 1 were fitted within the pFVIII-LNT helical map with the rigid body docking algorithms implemented in the ‘fit in map’ option of the UCSF Chimera software6162 (Fig. 6A). To achieve this the helical pFVIII-LNT map corresponding to the membrane-bound FVIII dimer was further segmented to delineate the densities corresponding to each of the FVIII molecules (Fig. S5B). The FVIII-LNT molecules were then fitted with the rigid body algorithm implemented in the ‘fit to map’ option of UCSF Chimera, starting from different orientations of the FVIII-LNT structures, as detailed in the Materials and Methods section. The best FVIII-LNT structure fit within the segmented membrane-bound FVIII dimer map also allowed for the proper orientation of the C2 domains, such that their membrane-binding loops can interact with the negatively charged LNT membrane (Fig. 6A, Fig. S5B,C). The FVIII crystal (FVIII-3D) structure did not fit as well, as the juxtaposed configuration of the C domains could not be accommodated well in the pFVIII-LNT helical map (Fig. S5Da). The FVIII-LNT structure also fit well in the segmented dimer map from the pFVIII-LNT SPT reconstruction, which was further segmented to delineate the map/density corresponding to each FVIII molecule within the dimer (Fig. 6B, Fig. S5A, Fig. S4B). The lower resolution of the pFVIII-SPT structure resulted in a poorer fit of the FVIII-LNT structure. However, fitting of the FVIII crystal (FVIII-3D) structure within the pFVIII-SPT map left a considerable portion of the FVIII-HC outside of the molecular contour (Fig. S5Db). The orientation of the membrane-bound FVIII molecules obtained from the fitting of the FVIII-LNT structure was such that the main protein-protein interface within the membrane-bound dimer was at the level of the FVIII-heavy chains (HC:A1-A2 domains). This orientation of the FVIII monomers within the membrane-bound dimer does not obstruct the FVIIIa-FIXa interface encompassing the A2 and A3 domains, as shown by the almost identical activity measured for pFVIII in the presence and absence of LNT (Fig. 6, Supplement 6).

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