Two‐dimensional crystallization of proteins on planar lipid films and structure determination by electron crystallography<sup>*</sup>

Brisson, Alain; Olofsson, Anders; Ringler, Philippe; Schmutz, Marc; Stoylova, Svetla

doi:10.1111/j.1768-322x.1994.tb00933.x

Cited by 33 publications

(32 citation statements)

References 32 publications

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“…[11][12][13][14] Within the 2D crystals, FVIII was present as a noncovalently linked heterodimer of HCs (domains A1-A2) and LCs (domains A3-C1-C2). 14 Very little material (of the order of micrograms) is needed to define a low-resolution 3D density map of a protein organized in 2D crystals by electron crystallography.…”

Section: Introductionmentioning

confidence: 99%

3-Dimensional structure of membrane-bound coagulation factor VIII: modeling of the factor VIII heterodimer within a 3-dimensional density map derived by electron crystallography

Stoilova‐McPhie

Villoutreix

Mertens

et al. 2002

Blood

151

175

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Despite recent studies, the organization of coagulation factor VIII (FVIII) on a phospholipid (PL) membrane is not known in detail. Thus, 2-dimensional (2D) crystals of human FVIII lacking the B domain were prepared for electron microscopy onto negatively charged PL monolayers. The 3-dimensional (3D) density map of the PL-bound FVIII protein was calculated at 1.5 nm. Existing atomic data and models for FVIII domains were fitted unambiguously within the 3D density map of the molecule. FVIII domains arrangement followed a compact spiral organization with the A3 domains in close association with the C1 and C2 domains near the PL surface. Viewed toward the membrane the A domains' heterotrimer is oriented side-on with the pseudo-3-fold axis almost parallel to the PL surface and A1 fully covering C1. The C2 domain is partially overlapped by the A2 domain of an adjacent molecule in the 2D crystal, favoring close packing. Viewed parallel to the membrane, C2 is slightly inclined to the PL surface covering an area of 12 nm 2 . Four C2 loops are embedded within the lipid monolayer at about 0.7 to 1.0 nm depth. C1 forms almost a right angle with C2, its long axis nearly parallel to the membrane. The proposed structure for membrane-bound FVIII results from modeling of the FVIII domains within a 3D density map obtained from electron crystallography and accords with the main biochemical and structural information known to date. A model is proposed for FVIIIa and factor IXa assembly within the membrane-bound factor X-activating complex. IntroductionFactor VIII (FVIII) is a plasma protein essential for blood coagulation and is deficient or defective in individuals with hemophilia A. FVIII is synthesized as a 300-kd precursor protein with domain structure A1-A2-B-A3-C1-C2. 1,2 In plasma it circulates as a series of noncovalently bound heterodimers, produced by proteolytic cleavage at the B-A3 junction or within the B domain. 3 Thus, FVIII in vivo consists of a heavy chain (HC) of variable molecular mass (from 90 kd, when only the A1-A2 domains are present, to 220 kd when the full-length B domain is present) and a light chain (A3-C1-C2; LC) of 76 kd molecular mass (for a review, see Lenting et al 4 ).In the process of blood coagulation, FVIII acts as a cofactor to the serine protease factor IXa (FIXa). Before the initiation of coagulation, FVIII circulates in a noncovalent complex with von Willebrand factor (VWF). Limited proteolysis near the N-terminus of LC by traces of thrombin removes a peptide containing a VWF-binding site, resulting in release of free FVIII. Further proteolysis between the A1 and A2 domains results in generation of FVIIIa, the active heterotrimeric cofactor. Binding of FVIIIa to negatively charged phospholipid (PL) and to factor IXa (FIXa) form the factor X-ase (FX-ase) complex. The product of the reaction, activated factor X (FXa), forms with activated factor V (cofactor FVa) a similar membrane-bound system, the prothrombinase complex that converts prothrombin to thrombin. 6 The serine proteases FIXa and F...

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Section: Introductionmentioning

confidence: 99%

3-Dimensional structure of membrane-bound coagulation factor VIII: modeling of the factor VIII heterodimer within a 3-dimensional density map derived by electron crystallography

Stoilova‐McPhie

Villoutreix

Mertens

et al. 2002

Blood

151

175

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“…This technology has been highly successful for annexin V, due to its property of binding specifically to negatively charged phospholipids such as phosphatidylserine in the presence of Ca 2+ ions. 6,7 Annexin V molecules are monomeric in solution but, after binding to a membrane, spontaneously form tightly bound trimers. The process of trimerization is extremely rapid, and electron crystallography evidence suggests that there is no significant change in the molecular conformation upon binding to the membrane.…”

Section: Introductionmentioning

confidence: 99%

Modeling the Phase Behavior of the Membrane Binding Protein Annexin V

et al. 2002

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The bulk thermodynamic properties of proteins originate from a varied and complex combination of interactions. We propose a simple model for the formation of ordered two-dimensional aggregates based on the interactions between pairs of annexin V molecules. Simulations of this model are shown to reproduce the experimental observations of a honeycomb (p6) and a triangular (p3) crystalline phase. The simulations indicate that the transition between these two phases is first order. While this model is extremely simple in that it relies only on hard body and short-range directional interactions, it nevertheless captures the essential physics of the interactions between the protein molecules and reproduces the phase behavior observed in electron microscopy and atomic force microscopy experiments.

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“…The mechanism of binding involves an extremely rapid first stage in which three proteins cluster together in an almost irreversible manner to form a trimer. 9,10 The strongly bound trimer structure behaves as a single species once it is absorbed onto the surface of the lipid mono or bilayer. The association of the trimer to a planar substrate seems to be sufficient for triggering the self-assembly into 2D crystals and it has long been known that annexin V can crystallize in two crystal forms.…”

Section: Methodsmentioning

confidence: 99%

Computer simulation of the phase behavior of a model membrane protein: Annexin V

Bates

Noro

Frenkel

2002

The Journal of Chemical Physics

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The bulk thermodynamic properties of membrane proteins originate from a complex combination of molecular interactions. We propose a simple model based on the pair interactions between a model membrane protein, annexin V. The experimental observations of a honeycomb ͑p6͒ and a triangular ͑p3͒ phase are successfully reproduced with Monte Carlo computer simulations. Grand canonical simulations and a newly developed ''strip''-move constant pressure technique reveal the stability of a dilute fluid phase and a dense solid phase, not observed with the current experimental technology. While this model is extremely simple in that it relies only on hard-body and short-range directional interactions, it nevertheless captures the essential physics of the interactions between the protein molecules and reproduces the phase behavior observed in experiments.

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Two‐dimensional crystallization of proteins on planar lipid films and structure determination by electron crystallography^*

Cited by 33 publications

References 32 publications

3-Dimensional structure of membrane-bound coagulation factor VIII: modeling of the factor VIII heterodimer within a 3-dimensional density map derived by electron crystallography

3-Dimensional structure of membrane-bound coagulation factor VIII: modeling of the factor VIII heterodimer within a 3-dimensional density map derived by electron crystallography

Modeling the Phase Behavior of the Membrane Binding Protein Annexin V

Computer simulation of the phase behavior of a model membrane protein: Annexin V

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Two‐dimensional crystallization of proteins on planar lipid films and structure determination by electron crystallography*

Cited by 33 publications

References 32 publications

3-Dimensional structure of membrane-bound coagulation factor VIII: modeling of the factor VIII heterodimer within a 3-dimensional density map derived by electron crystallography

3-Dimensional structure of membrane-bound coagulation factor VIII: modeling of the factor VIII heterodimer within a 3-dimensional density map derived by electron crystallography

Modeling the Phase Behavior of the Membrane Binding Protein Annexin V

Computer simulation of the phase behavior of a model membrane protein: Annexin V

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Two‐dimensional crystallization of proteins on planar lipid films and structure determination by electron crystallography^*