Evaluation of the metal binding sites in a recombinant coagulation factor VIII identifies two sites with unique metal binding properties

Svensson, L. Anders; Thim, Lars; Olsen, Ole H.; Nicolaisen, Else Marie

doi:10.1515/hsz-2012-0298

Cited by 21 publications

(30 citation statements)

References 7 publications

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“…Calcium ions, one per complementary repeat, were inserted based on the structural alignments and adjusted slightly by hand to remove atomistic overlap. The resulting calcium-loaded LRP double complementary repeat 56 (LRP1 CR56) model was positioned relative to the light chain of FVIII (PDB: 4BDV, chain B) (43) to reconstruct the interaction motif of the LDLR-RAP complex in which CR5 interacts with Lys 2065 and CR6 interacts with Lys 2092 . The lysine residues of the FVIII light chain, Lys 2065 and Lys 2092 , were overlaid with the NZ atoms of the two interacting lysine residues (Lys 256 and Lys 270 ) of RAP.…”

Section: Methodsmentioning

confidence: 99%

Factor VIII Interacts with the Endocytic Receptor Low-density Lipoprotein Receptor-related Protein 1 via an Extended Surface Comprising “Hot-Spot” Lysine Residues

Biggelaar

Madsen

Faber

et al. 2015

Journal of Biological Chemistry

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Background: It is unclear how the LDL receptor family binds large protein ligands. Results: HDX and lysine scanning identified factor (F)VIII regions and specific lysine residues binding low-density lipoprotein receptor-related protein 1 (LRP1). Conclusion: FVIII-LRP1 interaction involves multiple "hot-spot" lysine residues in the A3C1 domains. Significance: Our study sheds light on interactions of complex ligands with the LDL receptor family.

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Section: Methodsmentioning

confidence: 99%

Factor VIII Interacts with the Endocytic Receptor Low-density Lipoprotein Receptor-related Protein 1 via an Extended Surface Comprising “Hot-Spot” Lysine Residues

Biggelaar

Madsen

Faber

et al. 2015

Journal of Biological Chemistry

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“…Because of limited proteolysis of the B domain, the molecular weight of FVIII in plasma ranges between 170 and 300 kDa . The crystal structures of FVIII show that the A domains are ordered in a triangular shape stacked on top of two C domains aligned in parallel …”

Section: Introductionmentioning

confidence: 99%

Unique surface‐exposed hydrophobic residues in the C1 domain of factor VIII contribute to cofactor function and von Willebrand factor binding

Przeradzka

Freato

Boon-Spijker

et al. 2020

Journal of Thrombosis and Haemostasis

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Background:The identity of the amino acid regions of factor VIII (FVIII) that contribute to factor IXa (FIXa) and von Willebrand factor (VWF) binding has not been fully resolved. Previously, we observed that replacing the FVIII C1 domain for the one of factor V (FV) markedly reduces VWF binding and cofactor function. Compared to the FV C1 domain, this implies that the FVIII C1 domain comprises unique surfaceexposed elements involved in VWF and FIXa interaction.Objective: The aim of this study is to identify residues in the FVIII C1 domain that contribute to VWF and FIXa binding. Methods:Structures and primary sequences of FVIII and FV were compared to identify surface-exposed residues unique to the FVIII C1 domain. The identified residues were replaced with alanine residues to identify their role in FIXa and VWF interaction. This role was assessed employing surface plasmon resonance analysis studies and enzyme kinetic assays.Results: Five surface-exposed hydrophobic residues unique to the FVIII C1 domain, ie, F2035, F2068, F2127, V2130, I2139 were identified. Functional analysis indicated that residues F2068, V2130, and especially F2127 contribute to VWF and/or FIXa interaction. Substitution into alanine of the also surface-exposed V2125, which is spatially next to F2127, affected only VWF binding. Conclusion:The surface-exposed hydrophobic residues in C1 domain contribute to cofactor function and VWF binding. These findings provide novel information on the fundamental role of the C1 domain in FVIII life cycle. K E Y W O R D Sfactor VIII, factor IXa, kinetics, surface plasmon resonance, Von Willebrand factor | 365 PRZERADZKA Et Al.

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“…[39][40][41][45][46][47][48] The structure of human recombinant FVIII lacking the B domain as organized in 3D crystals in solution has been resolved at 4 Å by X-ray crystallography. 43,45,49 The structure of plasma derived human FVIII organized in membranebound 2D crystals was calculated at 15 Å by electron crystallography. Further fitting of the X-ray coordinates (3CDZ) 45 in the EM map resolved the FVIII membrane-bound domain organization in the 2D crystals (3J2Q).…”

Section: Introductionmentioning

confidence: 99%

Lipid nanotechnologies for structural studies of membrane-associated proteins

et al. 2014

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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.

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Evaluation of the metal binding sites in a recombinant coagulation factor VIII identifies two sites with unique metal binding properties

Cited by 21 publications

References 7 publications

Factor VIII Interacts with the Endocytic Receptor Low-density Lipoprotein Receptor-related Protein 1 via an Extended Surface Comprising “Hot-Spot” Lysine Residues

Factor VIII Interacts with the Endocytic Receptor Low-density Lipoprotein Receptor-related Protein 1 via an Extended Surface Comprising “Hot-Spot” Lysine Residues

Unique surface‐exposed hydrophobic residues in the C1 domain of factor VIII contribute to cofactor function and von Willebrand factor binding

Lipid nanotechnologies for structural studies of membrane-associated proteins

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