Computational Investigation of the Prothrombinase Complex

COMPUTATIONAL INVESTIGATION OF THE PROTHROMBINASE COMPLEX

WILLIAM R. MARTIN

Bachelor of Science in Mathematics

University of Michigan - Dearborn

December 2004

Bachelor of Science in Chemistry

Cleveland State University

May 2013

Submitted in partial fulfillment of the requirements for the degree

DOCTOR OF PHILOSOPHY IN CLINICAL AND BIOANALYTICAL CHEMISTRY

CLEVELAND STATE UNIVERSITY

December 2018 We hereby approve this dissertation For William R. Martin Candidate for the Doctorate degree in Clinical and bioanalytical Chemistry For the Department of Chemistry And CLEVELAND STATE UNIVERSITY’S College of Graduate Studies by

Committee Chairperson: Dr. David Ball

Department and Date

Committee Member: Dr. Michael Kalafatis

Department and Date

Committee Member: Dr. Bin Su

Department and Date

Committee Member: Dr. Jacqueline Vitali

Department and Date

Committee Member: Dr. Anton Komar

Department and Date Student’s Date of Defense: December 7th, 2018 COMPUTATIONAL INVESTIGATION OF THE PROTHROMBINASE

COMPLEX

WILLIAM R. MARTIN

ABSTRACT

Prothrombinase is a central enzymatic complex in the coagulation cascade and is the primary activator for the conversion of prothrombin to thrombin. Composed of the enzymatic complex, activated factor X (fXa), and the regulatory subunit, activated factor

V (fVa), the complex in the presence of a phosphatidylserine-containing membrane surface is capable of activating thrombin at a catalytic efficiency 300,000-fold higher than the enzyme fXa alone. Here we use a combination of homology modeling and molecular dynamics simulations to construct a new model for the ternary complex with the incorporation of a phosphatidylserine-containing membrane surface. This new model will shed light on the importance of the membrane interaction in the complex-substrate interaction dynamic. Additionally, we investigate the mechanisms by which these mutations change the conformations of the ternary complex, inducing a potential change in the interaction between the prothrombinase complex, composed of activated factor V and activated factor X, and its substrate prothrombin.

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TABLE OF CONTENTS

Page

ABSTRACT ...... iii

LIST OF TABLES ...... x

LIST OF FIGURES ...... xi

CHAPTER

I INTRODUCTION

1.1 Hemostasis ...... 1

1.1.1 Primary Hemostasis ...... 2

1.1.2 Secondary Hemostasis ...... 3

1.2 Coagulation Factor V ...... 6

1.2.1 Activation of Factor V ...... 6

1.2.2 Models of fVa ...... 8

1.2.3 Inactivation of fVa ...... 8

1.3 Factor X ...... 9

1.3.1 Factor Xa inhibition/inactivation ...... 9

1.4 Prothrombin ...... 10

1.4.1 Prothrombin activation...... 10 iv

1.4.2 Thrombin...... 12

1.5 Prothrombinase ...... 13

1.6 Protein-Protein Interactions ...... 14

1.6.1 fVa-fXa interactions...... 14

1.6.2 Va-II interactions ...... 15

1.6.3 fXa-II interactions ...... 16

1.7 Protein Membrane Interactions ...... 16

1.7.1 Membrane-fVa interactions ...... 17

1.7.2 Membrane-Gla interactions ...... 17

II COMPUTATIONAL METHODS

2.1 Homology Modeling ...... 18

2.1.1 Fold assignment and target-template alignment ...... 19

2.1.2 Model building ...... 20

2.1.3 Model evaluation ...... 21

2.2 Molecular Dynamics ...... 21

2.2.1 Newtonian equation of motion...... 22

2.2.2 Potential energy function ...... 22

2.2.3 Periodic boundary conditions ...... 24

2.2.4 Particle-mesh Ewald ...... 24

2.3 Force Fields ...... 26

2.3.1 MARTINI force field ...... 27

2.3.2 CHARMM36m force field ...... 28

2.4 Software ...... 28

2.5 Hardware ...... 29

III COMPUTATIONAL INVESTIGATION OF PROTHROMBINASE AND ITS

COMPONENTS ON 10% PHOSPHATIDYLSERINE MEMBRANES

3.1 Introduction ...... 31

3.2 Methods...... 33

3.2.1 Homology model for factor Xa ...... 34

3.2.2 Homology model for prothrombin ...... 34

3.2.3 Homology model for factor Va ...... 34

3.2.4 Coarse-grained molecular dynamics (fVa only) ...... 35

3.2.5 Coarse-grained to all-atom conversion (fVa only) ...... 36

3.2.6 Molecular dynamics system construction ...... 37

3.2.7 Ternary prothrombinase construction ...... 38

3.2.8 Construction of systems for mutational analysis ...... 40

3.2.9 Molecular dynamics parameters ...... 40

3.3 Protein-Membrane Interactions for Single Proteins ...... 41

3.3.1 Factor Xa ...... 41

3.3.2 Prothrombin ...... 45

3.3.3 Factor Va ...... 49

3.4 Initial Construction of Ternary Prothrombinase Complex ...... 53

3.4.1 Membrane interaction in prothrombinase ...... 53

3.4.2 Protein-protein interactions ...... 54

3.5 Mutational Analysis on 10% PS Membrane ...... 56

3.5.1 No post-translational modifications ...... 57

3.5.2 695KFKF698 mutation ...... 60

3.5.3 695DFDF698 mutation ...... 62

3.5.4 Addition of sulfation on TYR 696 and TYR 698 ...... 64

3.5.5 700DE701 mutation ...... 67

3.5.6 Addition of all post-translational modifications ...... 68

3.5.7 334KF335 mutation ...... 71

3.5.8 Key Interactions/Future Directions ...... 73

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IV CONSTRUCTION OF A NEW MODEL FOR THE TERNARY

PROTHROMBINASE COMPLEX IN THE PRESENCE OF A

PHOSPHATIDYLSERINE MEMBRANE SURFACE

4.1 Introduction ...... 76

4.2 Methods...... 78

4.2.1 Homology model for factor Xa ...... 78

4.2.2 Homology model for prothrombin ...... 79

4.2.3 Homology model for factor Va ...... 79

4.2.4 Molecular dynamics system construction ...... 80

4.2.5 Ternary prothrombinase construction ...... 80

4.2.6 Molecular dynamics parameters ...... 81

4.3 Results ...... 82

4.3.1 fVa-membrane interaction ...... 82

4.3.2 Membrane interaction in GLA domain-containing proteins ...... 84

4.3.3 Interactions in the ternary prothrombinase complex ...... 86

V MUTATIONAL ANALYSIS OF THE INTERACTION ENERGIES BETWEEN

THE PROTHROMBINASE COMPLEX AND PROTHROMBIN

5.1 Introduction ...... 91

5.2 Methods...... 93 viii

5.2.1 System construction ...... 93

5.2.2 Molecular dynamics parameters ...... 94

5.2.3 Contact analysis parameters ...... 94

5.3 Results ...... 95

5.3.1 334KF335 mutation ...... 96

5.3.2 S478A, L480A, Q481A mutation in prothrombin ...... 99

5.3.3 700DE701 mutation in fVa ...... 100

5.3.4 695DFDF698 mutation ...... 101

5.3.5 695DYDY698 mutation ...... 103

5.3.6 695KFKF698 mutation ...... 104

5.3.7 Discussion ...... 105

REFERENCES ...... 109

LIST OF TABLES

Table Page

3.1 Initial contacting residues for fXa prior to simulation...... 42

3.2 Residues with heavy membrane contact over final 20 ns of trajectory...... 44

3.3 Initial contacting residues for II prior to simulation...... 46

3.4 Residues with heavy membrane contact over final 20 ns of trajectory...... 48

3.5 Spike regions in the C1 and C2 domains of fVa...... 50

3.6 Contacts from each mutant with membrane, by spike. C refers to an interaction with PC lipids, and S refers to interaction with PS lipids...... 51

4.1 Key regions in fVa for protein-membrane interaction...... 83

5.1 Non-bonded interaction energies between components of the prothrombinase complex and substrate. Values are in kcal/mol. WT is wild type, 334 is the 334KF335 mutant, 480 is the S478A, L480A, Q481A mutant of prothrombin, 700 is the 700DE701 mutant, and DFDF, DYDY, KFKF are the 695-698 region mutations of fVa...... 95

5.2 Regions of interest within fVa for substrate binding within the ternary prothrombinase complex...... 96

5.3 Summary of the interaction energies for each region...... 105

LIST OF FIGURES

Figure Page

1.1 Components of hemostasis upon vascular injury...... 2

1.2 Schematic of secondary hemostasis, including current therapeutics and their applications...... 5

1.3 Domain schematic of factor V. (A) Downward facing arrows indicate activation sites for thrombin. Upward facing arrows indicate inactivation sites for APC and plasmin. (B) Activated factor V. The A1 and A2 domains compose the heavy chain, which ass associates with the light chain via divalent cations. (C) Deactivation by APC results in the A2 domain dissociating as two separate fragments, A2N and A2C. Inactivation by thrombin occurs after cleavage at Arg 643...... 7

1.4 Domain schematic of prothrombin. Cleavage sites at Arg 271 and Arg 320 are indicated...... 10

1.5 Pathway of activation of prothrombin by factor Xa and prothrombinase. Pathway I occurs without the presence of the regulatory subunit fVa, while pathway II occurs in the prothrombinase complex...... 11

1.6 The central role of thrombin in hemostasis, as well as its involvement in proatherogenic cellular responses via PAR-1, PAR-3, and PAR-4 activation ...... 13

2.1 Steps in comparative protein structure modeling...... 19

2.2 Mapping of amino acids to interaction centers based on number of heavy atoms, increasing ratio for ring structures...... 27

3.1 Final position of fXa with respect to the membrane after 100 ns of molecular dynamics...... 43

3.2 Final frame interaction between fXa and membrane surface...... 45

3.3 Final position of II with respect to the membrane after 100 ns of molecular dynamics...... 47

3.4 Final frame interaction between II and membrane surface...... 49

3.5 Membrane contacts in fVa WT...... 52

3.6 Initial configuration of the ternary prothrombinase complex...... 53

3.7 Final position of the initial 100 ns simulation of the prothrombinase complex ...... 55

3.8 RMSD of each simulation over 100 nanoseconds...... 57

3.9 Final position of the prothrombinase complex after 100 ns with no post-translational modifications...... 59

3.10 Final position of the prothrombinase complex after 100 ns with the 685KFKF698 mutation...... 61

3.11 Final position of the prothrombinase complex after 100 ns with the 695DFDF698 mutation...... 63

3.12 Final position of the prothrombinase complex after 100 ns with the post-translational sulfation added at tyrosines 696 and 698...... 66

3.13. Final position of the prothrombinase complex after 100 ns with the 700DE701 mutation...... 68

3.14 Final position of the prothrombinase complex after 100 ns with the all post-translational modifications included...... 70

3.15 Final position of the prothrombinase complex after 100 ns with the 334KF335 mutation. All post-translational modifications are included...... 72

4.1 Interaction between the C1 and C2 domains in fVa and the PS- containing membrane surface. Light blue lines indicate hydrogen bonds...... 84

4.2 Interaction between the GLA domain of fXa and the PS-containing membrane surface. Calcium ions are indicated by green spheres...... 85

4.3 Protein-membrane interaction between the GLA domain of II and the PS-containing membrane surface...... 86

xii

4.4 Initial conformation of the ternary prothrombinase complex after MD simulations for all three proteins separately. Protein-protein interactions were selected to match crystal structures and accepted models as closely as possible without creating clashes...... 87

4.5 Final conformation of the ternary prothrombinase complex in the presence of a PS-containing membrane surface...... 89

5.1 Final pose of the prothrombinase complex with the 334KF335 mutation. fVa is pictured in green, II in blue, and fXa in purple...... 98

5.2 Final pose of the 695DFDF698 mutant...... 103

5.3 RMSD for all systems of alpha-carbons. All three proteins were considered for the RMSD...... 106

5.4 All-atom RMSF of each protein system...... 107

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CHAPTER I

INTRODUCTION

1.1 HEMOSTASIS

Hemostasis refers to the process by which bleeding at the site of injury is stopped while simultaneously maintaining blood flow throughout the rest of the vasculature1. An anticoagulant surface is maintained in blood vessels, protecting the blood from exposure to procoagulant components of the subendothelium2. But upon damage components of the subendothelial matrix are exposed to blood, specifically collagen, activating a cascade of processes such as the activation of platelets, initiating formation of a hemostatic plug3.

This process is tightly regulated; complications in the process could lead to excessive bleeding (hemorrhage) or excessive clotting (thrombosis).

There are three main mechanisms to hemostasis (Figure 1.1); vasoconstriction, platelet plug formation, and clot formation. Vasoconstriction and platelet plug formation are components of primary hemostasis, while clot formation is the major component of secondary hemostasis.

Figure 1.1 Components of hemostasis upon vascular injury. (www.thrombocyte.com)

1.1.1 Primary Hemostasis

Under normal circumstances, platelets are present in the blood at a concentration between 150 and 400 million per milliliter of blood4, and do not adhere to vessel surfaces or each other. Upon injury and the exposure of blood to the subendothelial matrix, platelets begin activation and adhesion at the injury site5. Attachment to the site of injury is facilitated by specific proteins on the surface of the platelet as well as the release of proteins from within platelets such as von Willebrand factor (vWf), factor V (fV), and fibrinogen6.

von Willebrand factor makes the surfaces of endothelial cells adhesive; in small blood vessels, this is enough to stop bleeding. In larger vessels, vWf acts as a bridge between endothelial collagen and platelets via surface receptors GpIb, promoting platelet adhesion.

Platelets adhere to the endothelial walls, releasing adenosine diphosphate (ADP), which helps with platelet aggregation, a powerful vasoconstrictor thromboxane A2 (TxA2), and fibrin stabilizing factor, factor XIII.

The receptors for these proteins are only activated upon injury to the blood vessel.

Upon activation, phosphatidylserine phospholipids are enzymatically flipped from the inner leaflet of the membrane surface to the outer, creating a negatively charged surface required for the assembly of numerous enzymatic complexes required for the coagulation cascade, otherwise known as secondary hemostasis7. The exposure of the membrane protein tissue factor (TF) as a result of the exposure of the subendothelial surface marks the start of secondary hemostasis.

1.1.2 Secondary Hemostasis

The classical view of the coagulation cascade (Figure 1.2) indicated two separate initiator pathways: the “intrinsic pathway”, in which the activation of factor XII, induced by contact with polyanions secreted by activated platelets, coincided with all clotting factors being present in the blood, and the “extrinsic pathway”, where TF was required in addition to circulating clotting factors8. However, current evidence indicates these pathways are not alternatives to each other, but the intrinsic pathway increases thrombin generation primarily initiated by the extrinsic pathway9.

The current view of secondary hemostasis separates coagulation into four phases8. An initiation phase (typically called the extrinsic pathway), where low amounts of active procoagulant factors are generated; an amplification phase, where the concentration of these factors is increased; a propagation phase, where coagulation factors bind phospholipids on

3 activated platelets; and a stabilization phase, where activation of factor XIII and of thrombin activatable fibrinolysis inhibitor (TAFI) gives the clot strength and stability.

The extrinsic pathway is begun by exposure of subendothelial TF, which acts as a cofactor for factor VII (fVII), thereby promoting proteolysis and activation to fVIIa9. The complex activates trace amounts of factor IX (fIX) and factor X (fX) to fIXa and fXa via a

Ca2+ dependent mechanism. The resultant “extrinsic” fXa binds to the platelet surface, activating small amounts of prothrombin (II) to thrombin (IIa), as well as forming an intermediate in fIX activation, which is completed by the TF/fVIIa complex. This initiates a positive feedback loop whereby thrombin aids in further platelet activation as well as activation of factor V (fV) to fVa and factor VIII (fVIII) to fVIIIa. The fIXa and fVIIIa complex on the platelet surface, forming the “intrinsic” factor Xase, the main generator of fXa. Activated platelets provide the phosphatidylserine lipid surface for formation of the prothrombinase complex between the enzyme fXa and its regulatory subunit fVa.

Prothrombinase in the presence of a membrane surface is roughly 300,000-fold more active in activation of II to IIa than fXa alone10.

Figure 1.2 Schematic of secondary hemostasis, including current therapeutics and their

applications.

1.2 COAGULATION FACTOR V

Factor V is a 330,000 kDa protein composed of 2196 amino acids after pre-propeptide processing. 80% of venous factor V is located in the plasma and 20% in platelets, circulating at a concentration of roughly 20 nM. It is composed of three A domains, a B domain, and 2 C domains in an A1-A2-B-A3-C1-C2 configuration. The A domains share homology with both factor VIII and ceruloplastin11, while the C domains share homology with both factor VIII and discoidin, a slime mold protein12. Upon activation, the B domain is released, leaving a heavy chain (Mr 105,000) composed of the A1 and A2 domains of length 709 amino acids, and a light chain (Mr 74,000) composed of the A3, C1, and C2 domains, which associate via divalent metal ions.

1.2.1 Activation of Factor V

Factor V is activated by thrombin (Figure 1.3) via sequential clevages at Arg 709, Arg

1018, and Arg 1545. Initial cleavages at Arg 709 and Arg 1018 release the N-terminal portion of the B domain, resulting in a partially-active protein. The final cleavage at Arg

1545 is necessary for full activity of factor V as the regulatory subunit in prothrombinase.

Association with phosphatidylserine containing membrane surfaces is necessary for proper complex formation with fXa to become prothrombinase, which occurs through the C domains of the light chain. After association with the membrane surface, both the heavy and light chains of fVa interact with fXa, acting as the regulatory subunit within the prothrombinase complex. While not at a significant rate, factor V can also be activated by fXa and the prothrombin intermediate miezothrombin13.

Figure 1.3 Domain schematic of factor V. (A) Downward facing arrows indicate activation sites for thrombin. Upward facing arrows indicate inactivation sites for APC and plasmin. (B) Activated factor V. The A1 and A2 domains compose the heavy chain, which ass associates with the light chain via divalent cations. (C) Deactivation by APC

results in the A2 domain dissociating as two separate fragments, A2N and A2C.

Inactivation by thrombin occurs after cleavage at Arg 643. 7

1.2.2 Models of fVa

While no full crystal structure for factor Va exists, a number of partial structures, as well as homologous structures, have been solved. A crystal structure for the C2 domain has

14 15 been found , bovine fVai, inactivated by activated protein C , and most recently, a crystal structure for the prothrombinase complex from the venom of Pseudonaja textilis16. The

17 Pellequer model from 2000 was invalidated by the crystal structure of bovine fVai, which indicated the orientation of the C domains was such that the loops would be able to interact with the membrane. Two new models (Orban18 and Autin19) were generated based on this crystal structure, as well as the A domains of the prior theoretical model. The Orban model included the C-terminus of the A2 domain, while the Autin model included an interaction with factor Xa. Subsequent to these models, three models by the Pedersen20–22 group were generated; the first without the C-terminal loop, but with docked fXa, the second added prothrombin, and the third, most recent model, included the full ternary complex as well as the C-terminus.

1.2.3 Inactivation of fVa

Inactivation of fVa by activated protein C (APC) occurs via sequential cleavages at Arg

506, Arg 306, and Arg 67923. This inactivation is dependent on the presence of a membrane surface, as cleavage of fVa at Arg 306 is membrane dependent; however, cleavage at Arg

506 is not. It has been suggested that fV can act as an anticoagulant cofactor for APC in the inactivation of fVa24. Additionally, APC can inactivate fV in the presence of a membrane with sequential cleavage order Arg 306, Arg 506, Arg 679, and Lys 99425.

1.3 FACTOR X

Factor Xa is generated via cleavage of factor X by factor IXa with its cofactor factor

VIIIa, or by factor VIIa with its cofactor tissue factor, and is the enzyme in the prothrombinase complex. It is present in the blood as a glycoprotein with Mr 59,000. The heavy and light chains of factor Xa are connected by a single disulfide bond between residues 342 and 17226, and consist of 254 and 139 residues, respectively. The light chain is composed of an N-terminal GLA (γ-carboxyglutamic) domain, as well as two EGF

(epidermal growth factor) domains22. The heavy chain in the activated form is a single SP

(serine protease) domain, featuring the catalytic triad (His 236, Asp 282, and Ser 379).

Factor Xa alone has relatively low substrate specificity. It only reaches its full enzymatic activity when coupled with its regulatory subunit, factor Va, in the prothrombinase complex.

1.3.1 Factor Xa inhibition/inactivation

Due to its nature as a central enzyme in the coagulation cascade, fXa is a frequent target of therapeutics, with the goal of preventing thrombosis27. Rivaroxaban (Xarelto) and apixaban (Eliquis) are both competitive active site inhibitors of fXa with high bioavailability28. Fondaparinux (Arixtra) is a heparin-like drug which binds antithrombin, which inactivates fXa, thereby increasing inhibition29. Studies have been done using molecular docking to find inhibitors which interfere with the fXa-fVa interface in the prothrombinase complex30.

1.4 PROTHROMBIN

Prothrombin, the substrate for the prothrombinase complex, circulates in the blood at a

31 concentration of 1.4 µM with Mr 72,000 . Prothrombin is composed of 4 domains: an N- terminal GLA domain (residues 1-46), two kringle domains K1 ( residues 65-143) and K2

(residues 170-248), and a serine protease domain (residues 285-579, Figure 1.4) connected by three linker regions32. The linker region between the kringle domains lends to the conformational plasticity of the molecule33. Within the GLA domain are 10 γ- carboxyglutamic acid residues, which are post-translationally modified by carboxylase, for which vitamin K is a necessary cofactor, from glutamic acid residues34. This process is a frequent target of anticoagulants, such as warfarin, which competitively inhibits the vitamin K epoxide reductase complex 1 (VKORC1), a vitamin K activator35.

Figure 1.4 Domain schematic of prothrombin. Cleavage sites at Arg 271 and Arg 320

are indicated.

1.4.1 Prothrombin activation

Activation of prothrombin to thrombin by factor Xa is the result of two cleavages, one at Arg 271 and another at Arg 32036. The order of these cleavages determines the 10 intermediates formed; if cleaved at Arg 271 first, fragment 1·2 and prethrombin-2 are formed, while if the initial cleavage is at Arg 320, miezothrombin is formed (Figure 1.5).

Factor Xa is capable of converting prothrombin to thrombin via a separate pathway of that of the prothrombinase complex. However, this activation does not occur at a rate appropriate to maintain proper hemostasis. This is via the prethrombin-2 pathway, where

Arg 271 is cleaved first and Arg 320 is cleaved second.

Figure 1.5 Pathway of activation of prothrombin by factor Xa and prothrombinase.

Pathway I occurs without the presence of the regulatory subunit fVa, while pathway II

occurs in the prothrombinase complex.

1.4.2 Thrombin

Thrombin has a central, multifaceted role in hemostasis. Thrombin is the main activator of fibrinogen to fibrin, which upon polymerization, forms the clot once cross- linked by activated factor XIII, which is also activated by thrombin37. Thrombin also initiates positive feedback loops through activation of factor V, the regulatory subunit for the prothrombinase complex which is required for timely activation of thrombin, as well as factor XI, which activates the enzyme in tenase, the activator of factor X.

While the main function of thrombin is procoagulant in nature, it has other capabilities

(Figure 1.6): anticoagulant via complexation with thrombomodulin, which activates protein C, the main inhibitor of factor V; antifibrinolytic via activation of thrombin activatable fibrinolysis inhibitor (TAFI), which protects the fibrin clot against lysis38; proinflammatory via interaction with protease-activated receptors PAR-1, PAR-3, and

PAR-4; and anti-inflammatory through activation of APC.

Figure 1.6 The central role of thrombin in hemostasis, as well as its involvement in

proatherogenic cellular responses via PAR-1, PAR-3, and PAR-4 activation.

1.5 PROTHROMBINASE

Prothrombinase is the fVa/fXa complex which cleaves prothrombin to form thrombin.

In the absence of the regulatory subunit fVa, fXa is capable of this cleavage, but not at a rate capable of maintaining hemostasis10. The interaction of fVa with fXa in the presence

13 of calcium ions and a phosphatidylserine-containing membrane surface results in a

300,000-fold increase in the catalytic efficiency of fXa in generating thrombin10. This increase in catalytic efficiency is due to a 100-fold decrease in the Michaelis constant Km, as well as a 3000-fold increase in the catalytic constant kcat. In the absence of a membrane surface, the activation rate of prothrombin by prothrombinase is only 300 times that of factor Xa alone. The presence of the regulatory subunit stabilizes the complex, altering the pathway for which prothrombin activation occurs. It has been suggested that interaction between prothrombinase and its substrate is mediated by an interaction between anion binding exosite I (ABE-I) on prothrombin and the hirudin-like C-terminal loop of fVa39–42.

1.6 PROTEIN-PROTEIN INTERACTIONS

In an effort to uncover the mysteries of the prothrombinase complex, its construction, and how it interacts with the substrate, ongoing investigation attempts to determine which protein-protein interactions are key. Determination of key interaction sites could lead to novel targets for therapeutics, allowing non-active site drugs to be used. While direct inhibitors are used successfully to treat venous thromboembolism and stroke prevention in patients with atrial fibrilation43, the lack of specificity for the prothrombinase complex could lead to complications in processes not involving thrombin generation.

1.6.1 fVa-fXa interactions

Interaction between fVa and fXa, forming the prothrombinase complex, requires complete activation of fVa, and therefore removal of the B domain. The 1000-1008 region of factor V has been shown to inhibit binding to factor Xa44, indicating B domain removal 14 is required for proper prothrombinase assembly. Substitution of asparagine for Arg 347 in fXa has been shown to reduce regulatory subunit binding45. It was later determined that

Arg 347, Lys 351 and Lys 414 make up a binding epitope, while Glu 310 and Arg 306 form an extended region of this epitope46. Residues 311-325 of fVa have been suggested as a potential binding site for fXa, as a protein containing these residues was inhibitory for prothrombin generation in the presence of fVa, but not when the regulatory subunit was absent47. A similar study over the 323-331 stretch indicated direct inhibition of interaction between fXa and fVa48,49. Further studies have indicated residues 493-506, which contains an inactivation site for APC, may contribute to fVa-fXa binding as well50. Work on the same region indicated mutation of the 499-505 region was sufficient to greatly inhibit fVa- fXa interaction51. Later studies in the same region indicated a binding surface may exist centered on Arg 501, Arg 510, Ala 511, Asp 513, Asp 577, and Asp 578 of the factor Va

A2 domain52.

1.6.2 Va-II interactions

Interaction between fVa and the substrate are key to the timely activation of prothrombin. Site-directed mutagenesis studies have indicated the amino acid region 659-

15 cleavage, with substantial accumulation of meizothrombin56. ABE-I has been demonstrated to be key in fVa-accelerated prothrombin activation, as activation is inhibited in the presence of bothrojaracin, a snake venom protein which binds ABE-141.

1.6.3 fXa-II interactions

It has been reported that the interaction between prothrombin and factor Xa involves

Lys 276 on factor Xa57. It is also known that the catalytic triad of factor Xa interacts with

Arg 320 and Arg 271 of prothrombin, activating it to thrombin. A factor Va-dependent binding site for fXa on prothrombin has been found using synthetic peptide analysis, in the

473-487 region of prothrombin58. Further examination narrowed the region to the 478-482 stretch, with 478, 480, and 481 mutants inducing deficiency in clotting59.

1.7 PROTEIN MEMBRANE INTERACTIONS

Interactions between the proteins involved in the prothrombinase complex and a membrane surface are necessary for proper activation of thrombin60. While prothrombinase assembled on synthetic phospholipid vesicles proceed via the miezothrombin pathway, prothrombinase assembled on the surface of activated platelets has been shown to proceed via the pre2 pathway60 via a concerted mechanism61. It has also been suggested that prothrombinase assembled on synthetic vesicles differs from that assembled on platelets.

1.7.1 Membrane-fVa interactions

Mutations in both the C1 (Tyr 1956 and Leu 1957) as well as C2 (Trp 2063 and 2064) domains, as well as only the C1 mutation, impaired prothrombin activation in the presence of synthetic phospholipid vesicles when compared to wild type or the C2 mutation alone62,63. A previous study by the same group indicated the same C2 mutation inhibited membrane binding altogether64. Mutation of Arg 2023 and 2027 also showed decreased membrane binding in immobilized phosphatidylserine vesicles65.

1.7.2 Membrane-Gla interactions

Studies using deletion mutants of both fXa and II missing their gla domain have indicated their importance in membrane binding66. While investigation of the membrane binding action of the gla domains of fXa and II have not been researched extensively, it is still a long-used drug target. Warfarin is a competitive inhibitor of VKORC1, depleting functional vitamin K35. Vitamin K is an essential cofactor for glutamyl carboxylase, thereby preventing gamma carboxylation of key residues in factor X and II, resulting in impaired membrane binding67. Membrane binding through the gla domain is calcium (and possibly magnesium) ion dependent68. A recent study of the bovine gla domain using a highly-mobile membrane mimetic model indicated strong interaction between the gamma carboxyglutamic acid (CGU) and phosphatidylserine lipids through chelated calcium ions69.

CHAPTER II

COMPUTATIONAL METHODS

2.1 HOMOLOGY MODELING

Homology (or comparative) modeling predicts the 3D structure of a given protein sequence which is related to at least one known structure, based on its alignment to these proteins, also called templates.

There are four main steps to comparative modeling70. 1. Fold assignment, which identifies similarity between the target and templates. 2. Target-template alignment, which determines how well the target sequence matches up with the template sequence. 3. Model building, which uses the template in one of a number of ways (which will be described later) to obtain a possible model. This is followed by 4. Model evaluation, where the model is subjected to a number of tests to determine the potential “correctness” of the model.

Figure 2.1 gives an alternative, but equivalent, schematic for the steps for homology modeling.

Figure 2.1 Steps in comparative protein structure modeling.

2.1.1 Fold assignment and target-template alignment

Typically, the fold assignment step is paired with target-template alignment. The identification of suitable templates is usually determined by searching some sort of database for protein structures, such as the RCSB protein data bank (PDB)71. This can be easily done using the target sequence using BLAST72. BLAST uses a pairwise sequence alignment method, looking for high sequence identity. Current methods simultaneously determine target-template alignment along with fold assignment. Without a model to be used as a template, one with high enough similarity, we cannot proceed and assume we will obtain useful results. These methods are capable of finding homologous structures

19 with high sequence identity, but fail when the sequence identity falls into the “twilight zone”, or a range of roughly 20-35% identity with template. One way to overcome this is to use profile-sequence alignment methods, which are derived from the multiple sequence alignment using a position specific scoring matrix (PSSM). This “profile” is used to search against the database of template sequences. These methods are more sensitive to finding potential templates in the “twilight zone”. For profile-sequence alignment, PSI-BLAST73 is the most used algorithm.

In the second step, the restraints from the first step, along with the CHARMM74 force field, are combined into an objective function, similar to those used in molecular dynamics programs. The function depends on the Cartesian coordinates of the atoms that form the modeled molecules. The function is made up of a quadratic function, harmonic lower/upper bounds, a weighted sum of a few Gaussian functions, Coulomb’s law, cubic splines, and the Lennard-Jones potential function. This function is optimized in Cartesian space, yielding the final model. Any restraints derived from experimental data can easily be implemented by adding them to the first step, the homology-derived constraints.

2.1.2 Model building

Model building is the third step in the process. In some cases, especially when identity between the target and template is in the 30-50% range, there can be some variance in loops, even while core regions are typically well conserved. These loops can play important roles in protein function and structure, as has been noted for prothrombin in our case. The most recent crystal structure, which will be used in our complex model, is missing the linker region. This will have to be modelled using the loop modeling module

20 within MODELLER. Loop modeling is a mini protein-folding problem, in that a somewhat ab initio approach must be taken. The structure will be calculated based almost solely on the sequence itself. Loop modeling in MODELLER uses an optimization-based approach, relying on conjugate gradients and molecular dynamics with simulated annealing.

2.1.3 Model evaluation

The last step of homology modeling is evaluation of the model. There are numerous programs which can be used here. There are algorithms to determine whether the correct template was used or not; these use statistical potentials to assess compatibility. When the sequence identity is low, it is possible to generate a model with many errors. Programs such as DOPE75 and VERIFY3D76 can be used to verify compatibility. In addition, the models should be evaluated for self-consistency, ensuring the restraints used to find it were met. Programs such as WHAT-IF77 and PROCHECK78 can determine if the model has any stereochemical errors or have residues in disallowed regions via Ramachandran plot79, a way to visualize energetically allowed regions for backbone dihedral angles.

2.2 MOLECULAR DYNAMICS

Molecular dynamics is solving the classical equations for motion for a large collection of atoms. In order to do this, we need a few pre-determined parameters for the calculation.

First, we must have some way to describe the interactions between atoms in the system.

From this, given atomic positions, we can calculate potential energy, forces, and if

21 necessary, forces on the container walls. We use some sort of force field to describe these interactions, and in the event the force field is insufficient, we can use electronic structure calculations to supplement our chosen force field. Second, we need a way to numerically integrate the equations of motion for the atoms in the molecule. Third, we need some initial conditions, such as positions and velocities, in order to solve the equations of motion.

2.2.1 Newtonian equation of motion

The Newtonian equation of motion (2.1) is

̈ 휕 푚훼푟⃗훼 = − 푈푡표푡푎푙(푟⃗훼, 푟⃗2, . . , 푟⃗푁), 훼 = 1,2, . . , 푁 (2.1) 휕푟⃗훼

where mα is the mass of the atom, 푟⃑⃑⃑훼 is its position, and Utotal is the total potential energy, which is dependent on all atomic positions and therefore couples the motion of atoms. This potential energy, which is represented via the force field, is the most important part of the simulation, since it must not only represent all atomic interactions, but it must also be in a form which can be calculated quickly.

2.2.2 Potential energy function

The potential energy function (2.2) has the following form:

푈푡표푡푎푙 = 푈푏표푛푑 + 푈푎푛푔푙푒 + 푈푑푖ℎ푒푑푟푎푙 + 푈푣푎푛 푑푒푟 푊푎푎푙푠 + 푈퐶표푢푙표푚푏 (2.2)

The first three terms describe bonded interactions from stretching (2.3), bending (2.4), and torsional (2.5) movement:

푏표푛푑 2 푈푏표푛푑 = ∑ 푘푖 (푟푖 − 푟0푖) (2.3) 푏표푛푑푠푖 22

푎푛푔푙푒 2 푈푎푛푔푙푒 = ∑ 푘푖 (휃푖 − 휃0푖) (2.4) 푎푛푔푙푒푖

푑푖ℎ푒푑푟푎푙 푈푑푖ℎ푒푑푟푎푙 = ∑ 푘푖 [1 + cos(푛푖휙푖 − 훾푖)] (2.5) 푑푖ℎ푒푑푟푎푙푖

The above equations count every covalent bond in the system, the angles between pairs of covalent bonds sharing a common atom between them (at the vertex), and atom pairs separated by three covalent bonds, with the central bond subject to the torsional angle.

Bond stretching is governed by r, bond angles by θ, and ϕ the “improper” dihedral. The fourth and fifth terms in the potential energy function describe non-bonding interactions:

12 6 휎푖푗 휎푖푗 푈푣푑푊 = ∑ ∑ 4휀푖푗 (( ) − ( ) ) (2.6) 푟푖푗 푟푖푗 푖 푗>푖

푞푖푞푗 푈퐶표푢푙표푚푏 = ∑ ∑ (2.7) 4휋휀0푟푖푗 푖 푗>푖

These correspond to the van der Waals forces (2.6), approximated using a Lennard-

Jones 6-12 potential, and electrostatic interactions (2.7).

The parameters for the interactions given have been determined using a combination of empirical techniques and quantum mechanical calculations and are included in the force field parameter files.

2.2.3 Periodic boundary conditions

To avoid issues with the boundaries of the system, periodic boundary conditions are typically used in molecular dynamics simulations. The particles are enclosed in a “cell” which is replicated to infinity around this central cell; as such, when a particle leaves the cell on one side, it is replaced on the other side by a copy. Each particle is influenced by all particles in the system, as well as the particles imaged in the surrounding cells. Because all cells are exact copies of each other, they all move together and only need to be represented once in the simulation. However, the interactions exist between every nonbonded pair of atoms in the system. Because of this, the van der Waals interaction is truncated at some user defined distance. The periodicity of the system is used to calculate the full electrostatic interaction using the particle-mesh Ewald method (PME).

2.2.4 Particle-mesh Ewald

The long-range electrostatic interactions with periodic boundary conditions are described using Ewald summation. This is more reliable than using a cutoff scheme, though the artificial periodicity can lead to bias in free energy. This bias can lead to artificial stability in a protein which should have unfolded quickly. This sum (2.8) involves the following terms (dropping ¼πε0):

퐸퐸푤푎푙푑 = 퐸푑푖푟 + 퐸푟푒푐 + 퐸푠푒푙푓 + 퐸푠푢푟푓푎푐푒 (2.8)

The terms are defined as follows (2.9-2.12):

−휋2|푚⃑⃑⃑⃗|2 푁 2 1 푒 훽2 퐸 = ∑ |∑ 푞 푒2휋i푚⃑⃑⃑⃗∙푟⃗푖| (2.10) 푟푒푐 2휋푉 |푚⃑⃑⃗|2 푖 푚⃑⃑⃑⃗≠0 푖=1

푁 훽 2 퐸푠푒푙푓 = − ∑ 푞푖 (2.11) √휋 푖=1

푁 2 2휋 퐸푠푢푟푓푎푐푒 = |∑ 푞푖푟⃗푖| (2.12) (2휀푠 + 1)푉 푖=1

Here, qi and 푟⃗푖 are the charge and position for atom i, and 푛⃑⃗푟 is the lattice vector. We define

푛⃑⃗푟 = 푛1푎⃗1 + 푛2푎⃗2 + 푛3푎⃗3 (2.13)

with 푎⃗1, ⃑⃑⃑푎⃗2, 푎⃗3 independent base vectors for an arbitrary simulation box, and

푛 , 푛 , 푛 ∈ ℤ. ∑′ … denotes a summation excluding the 푛⃑⃗ = 0 term in the case where 1 2 3 푛⃑⃗푟 푟 i = j. The “excluded” summation is the set of atom pairs which should have their electrostatic interactions excluded. The lattice vector for the (i, j) pair that minimizes |푟⃗푖 −

푟⃗푗 + 푣⃗푖푗| is denoted by 푣⃗푖푗. β adjusts the workload distribution for direct and reciprocal 25 sums, while εs is the dielectric constant of the surroundings for the simulation box, which in our case, as in most cases, will be water. V is the volume of the box, and 푚⃑⃑⃗ is the reciprocal vector, defined as

⃑⃗ ⃑⃗ ⃑⃗ 푚⃑⃑⃗ = 푚1푏1 + 푚2푏2 + 푚3푏3 (2.14)

⃑⃗ ⃑⃑⃗ ⃑⃗ where 푚1, 푚2, 푚3 ∈ ℤ, and 푏1, 푏2, 푏3 are the reciprocal base vectors defined such that

⃑⃗ 푎⃗훼 ∙ 푏훽 = 훿훼훽, 훼, 훽 ∈ {1,2,3}. The function erfc(x) is the complementary error function:

∞ 2 2 푒푟푓푐(푥) = ∫ 푒−푡 푑푡 (2.15) √휋 푥

The Ewald sum has terms for direct sum, reciprocal sum, self-energy, and surface energy, respectively.

2.3 FORCE FIELDS

The force field is a mathematical description of the dependence of the energy of the particles in a system on their coordinates80. These parameters are found using a combination of experimental data and ab initio calculations. The force field replaces the true potential functions with a simplified model according to equations 2.2 through 2.7 above. Herein the MARTINI81–84 force field will be implemented in an effort to induce membrane interaction taking a coarse-grained approach; for all-atom simulations the

CHARMM36m85 force field will be used.

2.3.1 MARTINI force field

The MARTINI force field, on average, uses a 4 to 1 ratio; that is, every 4 heavy atoms are represented by one single interaction center. Only four types of interaction centers are considered: polar, apolar, nonpolar, and charged (Figure 2.2). Nonbonded interactions follow the same Lennard-Jones 12-6 potential as in equation 2.6. However, coulombic interactions are shifted slightly, dividing equation 2.7 by an additional dielectric constant, which is set to 15 for explicit screening. Bonded interactions follow similar forms as equations 2.3 through 2.5. For ring molecules, the 4:1 ratio used does not preserve geometry well. The number of interaction centers is increased to the minimum required to maintain ring geometry, leading to 2 or 3 to 1 ratios.

Figure 2.2 Mapping of amino acids to interaction centers based on number of heavy

atoms, increasing ratio for ring structures.

2.3.2 CHARMM36m force field

The CHARMM force field uses a similar implementation of the potential energy function as above, with the addition of an Urey-Bradley term, which is a harmonic term in the distance between atoms 1 and 3 of some angle terms:

2 ∑ 퐾푈퐵(푆 − 푆0) (2.16) 푈퐵

Intermolecular parameters for the CHARMM force field are based on ab initio interaction calculations; the values then for these parameters were determined using structural and vibrational data. For the intramolecular terms, initial geometries were obtained using gas phase calculations. Force constants associated with bond length and angles, dihedrals, and improper torsions were adjusted using vibrational data. Vibrational calculations were done using a scaled HF/6-31G(d), with the scaling factor determined via comparison to experimental data where available.

2.4 SOFTWARE

A number of web services and software packages were used in this study. Homology modeling will use MODELLER70 within the Chimera software suite86. Homologous structures were found using the BLAST module73 within Chimera, with selection of a model coming from a mix of DOPE score75 and visual inspection. Systems for molecular dynamics simulation were constructed using CHARMM-GUI87. For systems using the

MARTINI force field, the MARTINI maker within CHARMM-GUI84 were used to construct the system, as well as for the MARTINI to all-atom conversion. Systems using only the CHARMM36m force field were constructed using the membrane builder in

CHARMM-GUI87–91. Addition of post-translational modifications were accomplished using SwissSidechain92 rotamer library within Chimera, with parameterization coming from SwissParam93. Modification of parameters to properly work with the CHARMM force field was done by hand.

Molecular dynamics simulations were completed using the GROMACS software suite94,95. Software versions used include 5.1.2, 2016.1, and 2018.2. GPU acceleration was achieved using the CUDA programing architecture96. Graphics were generated using

Chimera or VMD97. Plots were generated using standard GROMACS tools, and plotted using XMGRACE in Ubuntu 17.10. MDTraj98 was implemented for multi-trajectory analysis. Contact analysis data is generated using PyContact99 and CONAN100.

Electrostatic interaction energies will be calculated using gRINN101.

2.5 HARDWARE

Molecular dynamics simulations were run at the Ohio Supercomputer Center102 on the

Owens cluster. The Owens cluster is an 824-node Dell-built supercomputer. Each node uses a Dell PowerEdge C6320 mainboard with two 14 core Intel Xenon E5-2680 v4

Broadwell processors, each node being equipped with 128 GB of memory. GPU acceleration is achieved using NVIDIA Tesla P100 (Pascal architecture) GPUs.

Minimization and equilibration for some systems were carried out on desktop PCs built specifically for the task on the campus of Cleveland State University. Three PCs using

Intel i5-6600K processors with 16 GB of RAM and GTX 1070 GPUs, as well as three PCs with Intel i5-4590 processors, were used for some simulations. In addition to these

29 desktops, two PCs, one with an Intel i7-4770 processor and the other with an i7-3770, and

32 GB of RAM, were used for computationally intensive analysis.

CHAPTER III

COMPUTATIONAL INVESTIGATION OF PROTHROMBINASE AND ITS

COMPONENTS ON 10% PHOSPHATIDYLSERINE MEMBRANES

3.1 INTRODUCTION

Prothrombinase is the enzymatic complex which cleaves prothrombin to form thrombin. In the absence of the regulatory subunit factor Va (fVa), the enzymatic subunit factor Xa (fXa) is capable of this cleavage, but not at a rate which is conducive to proper hemostasis. The interaction of fVa with fXa in the presence of calcium ions and a phosphatidylserine-containing membrane surface results in a 300,000-fold increase in the catalytic efficiency of fXa in generating thrombin10, also resulting in a reversal of the order of cleavages of prothrombin (II) from the prothrombin-2 pathway (Arg 271 followed by

Arg 320) to the meizothrombin pathway (Arg 320 followed by Arg 271)36. This increase in catalytic efficiency is due to a 100-fold decrease in the Michaelis constant Km, as well as a 3000-fold increase in the catalytic constant kcat. In the absence of a membrane surface, the activation rate of prothrombin by prothrombinase is only 300 times that of factor Xa alone. 31

Factor Va is generated by cleavage of factor V, a plasma protein with concentration of

10 roughly 20 µM and Mr of 330,000 . Factor V is composed of three A domains, a B domain, and two C domains. Upon activation via cleavage, the B domain is released as two heavily glycosylated fragments, with Mr of 150,000 and 71,000. The remaining two subunits (heavy and light chains) bind to each other through hydrophobic interactions, which are exposed following interaction with calcium ions. While a complete crystal structure for factor Va does not yet exist, a number of partial structures, as well as homologous structures, have been solved. A crystal structure for the C2 domain has been

14 15 found , bovine fVai, inactivated by activated protein C , and most recently, a crystal structure for the prothrombinase complex from the venom of Pseudonaja textilis16. Factor

Va is known to interact with phosphatidylserine-containing membrane surfaces via its C- domains63,64.

Factor Xa is generated via cleavage of factor X by factor IXa with its cofactor factor

VIIIa, or by factor VIIa with its cofactor tissue factor, and is the enzyme in the prothrombinase complex. It is present in the blood as a glycoprotein with Mr 59,000. The heavy and light chains of factor Xa are connected by a single disulfide bond between residues 172 and 34226, and consist of 254 and 139 residues, respectively. The light chain is composed of an N-terminal GLA (γ-carboxyglutamic) domain, as well as two EGF

(epidermal growth factor) domains22. These post-translational modifications are essential to membrane interaction103, which occurs via the GLA domain. The heavy chain in the activated form is a single SP (serine protease) domain, featuring the catalytic triad (His

236, Asp 282, and Ser 379). Factor Xa alone has relatively low substrate specificity, and

32 only reaches its full enzymatic activity when coupled with its regulatory subunit, factor Va, in the prothrombinase complex.

Prothrombin is the substrate for the prothrombinase complex. Activation of prothrombin to thrombin by factor Xa is the result of two cleavages, one at Arg271 and another at Arg 32036. The order of these cleavages determines the intermediates formed; if cleaved at Arg 271 first, fragment 1·2 and prethrombin-2 are formed, while if the initial cleavage is at Arg 320, miezothrombin is formed. Prothrombin is composed of 4 domains:

An N-terminal GLA domain, two kringle domains K1 and K2, and a serine protease domain. The linker region between the kringle domains lends to the conformational plasticity of the molecule33. Factor Xa is capable of converting prothrombin to thrombin via a separate pathway of that of the prothrombinase complex. However, this conversion does not occur at a rate appropriate for proper hemostasis. This is via the prethrombin-2 pathway, where Arg 271 is cleaved first and Arg 320 is cleaved second.

3.2 METHODS

All models built with MODELLER70 version 9.15 using UCSF Chimera 1.11.286 as a graphical user interface. All crystal structures used were obtained from the Protein Data

Bank71. For all models, 20 poses were generated without hydrogens using thorough optimization. Final models were selected based on DOPE75 score as well as visual inspection. Similarity to currently published models17,18,22 was taken into consideration as well. All sequences were taken from Uniprot26. Sequence alignment and percent homology were determined using the BLOSUM62104 algorithm in BLAST73. Post- translational modifications were added using SwissSidechain92 within Chimera.

3.2.1 Homology model for factor Xa

To construct the model for fXa, crystal structures of the partial heavy chain with partial light chain (1XKA)105 and the gamma-carboxyglutamic acid containing (GLA) domain

(1IOD)106 were used. The GLA domain from 1IOD was placed below the crystal structure for the heavy chain and remainder of the light chain from 1XKA, and a series of loop optimizations were carried out to obtain the final model. Calcium ions were chelated to the beta-hydroxyaspartic (BHD) and gamma-carboxyglutamic acid (CGU) residues.

3.2.2 Homology model for prothrombin

The homology model for prothrombin (II) was constructed using the crystal structure for the 154-167 deletion mutant (5EDM)33. The Pedersen model22 was used to inform a proper distance for the reconstruction of the deleted residues, with the 5EDM crystal structure being used as the template for both the SP/K2 side of the linker region, as well as the K1/GLA side.

3.2.3 Homology model for factor Va

14 15 Crystal structures of the C2 domain (1CZT) and bovine fVai (1SDD) were oriented using the crystal structure of prothrombinase from the venom of P. textilis (4BXS)16. Due to the higher homology in bovine fVa with respect to that of the brown snake venom prothrombinase, the C2 domain orientation from bovine fVa was selected preferentially, as there is a marked rotation away from the C1 domain in the structure from the venom prothrombinase. The C-terminal loop was initially chosen to resemble that of the Pedersen model22. However, in the absence of prothrombin in the initial studies, the C-terminal loop

34 is unlikely to contribute to membrane binding due to its structure. Post-translational modifications of fVa were not included in the initial studies where the protein was not complexed with fXa.

3.2.4 Coarse-grained molecular dynamics (fVa only)

The model built by homology above was converted to a coarse-grained (CG) representation using CHARMM-GUI87, using the bilayer builder within the MARTINI maker84 input generator. Location with respect to the membrane was chosen based on currently available experimental data, indicating the C1 and C2 domains are key for proper membrane interaction63,107–109. The MARTINI 2.2 force field81–83 was used for the protein, using non-polarizable water and an elastic network for the protein. The CG simulation was conducted in GROMACS 5.1.294. A lipid membrane composed of 297 palmitoyl oleoyl- phosphatidylcholine (PC) molecules and 33 palmitoyl-oleoyl-phosphatidylserine (PS) molecules per side of the bilayer with the C-domains of fVa situated at the membrane surface. A 10% PS membrane was chosen as optimal activity for the prothrombinase complex is observed on bilayer surfaces containing 10-15 mol% phosphatidylserine (PS).

29527 CG water particles, without polarizability, were added, along with 375 sodium ions and 297 chloride ions, neutralizing the system to a 0.15 molar NaCl environment. The initial box size for the simulation was 151.5×151.5×216 Å, containing a total of 41268 CG particles, which can be seen in Figure 3 without solvent and ions. Prior to production, 6864 steps of steepest descent soft-core-minimization (negating the need for double precision) were carried out, at which point the algorithm converged to machine precision. This was followed by another 15001 steps of steepest descent, again reaching machine precision.

Five equilibration steps followed, with reduced positional restraints and increased timestep between steps, with a final timestep of 20 fs. Temperature was kept constant at 310 K using the velocity rescaling thermostat110. A Berendsen barostat111 was used to apply semi- isotropic coupling with a reference pressure of 1 bar, a compressibility of 3.0×10-4 bar-1, and a coupling constant of 5.0 ps. Van der Waals interactions were smoothly shifted to zero between 9 and 12 Å. The production simulation time was 4 µs.

3.2.5 Coarse-grained to all-atom conversion (fVa only)

A frame from the trajectory roughly 1 µs into the simulation was selected for conversion to the all-atom case. Conversion was done using the MARTINI to All-atom

Converter84. After conversion, the number of PC, PS, and ion molecules remained the same. As the MARTINI force field converts 4 water molecules into one water bead, the number of water molecules quadrupled to 118108. The complete system consisted of

464685 atoms in a 145×145×218 Å simulation box. All steps of the simulation were completed in GROMACS 5.1.2 using the CHARMM36 force field112. The converted system was minimized using steepest descent for 10589 steps, at which point the maximum force was reduced below 1000 kJ mol-1 nm-1. Minimization was followed by six equilibration steps, each reducing positional restraints on heavy atoms (4000, 2000, 1000,

500, 200, and 50 kcal mol-1 Å-2 for backbone, and 2000, 1000, 500, 200, 50, and 0 kcal mol-1 Å-2 for side chain, respectively). The timestep was changed from 1 fs in steps 1, 2, and 3 to 2 fs for all subsequent steps. After equilibration, 10 ns of production dynamics were completed using a 2 fs timestep. The temperature was maintained at 310.15 K using the Nosé-Hoover thermostat113 with time constant of 1.0 ps and the membrane, protein,

36 and solvent coupled separately. A Parrinello-Rahman barostat114 was used to apply semi- isotropic coupling with a reference pressure of 1 bar, a compressibility of 4.5×10-4 bar-1, and coupling constant of 5.0 ps. The van der Waals interactions were smoothly switched to zero between 10 and 12 Å.

3.2.6 Molecular dynamics system construction

For both fXa and II, the selected model was placed on a membrane surface in a water box with calcium and chloride ions using CHARMM-GUI87. In addition, CHARMM-GUI was used to add hydrogens to the system. The protein, water, ions, and membrane were parameterized using the CHARMM36 force field112, while both CGU and BHD residues were parameterized using SwissParam93. A square membrane patch roughly 152 angstroms per side composed of 90% PC and 10% PS was added, with the GLA domain of factor Xa placed at the surface. Ions were added to neutralize the system at a concentration of 0.15 molar, and water molecules added, with the final dimension of the box being roughly 177 angstroms for fXa and 222 angstroms for II. In total, 612 PC molecules and 68 PS molecules were used in the membrane patch. For fXa, 96068 water molecules, 302 calcium ions, and 537 chloride ions were added. In addition, 10 calcium ions were chelated to the CGU and BHD residues. For II, 128907 water molecules, 403 calcium ions, and 710 chloride ions were used. As opposed to using steered molecular dynamics, the protein was placed with the GLA domain in contact with the membrane surface directly.

For fVa, the final frame from the coarse-grained to all-atom conversion was moved to a new membrane with mutations to the C-terminus of the heavy chain added:

695DFDF698 and 695KFKF698. This region has been shown to be important in binding with prothrombin42; however, in the current study the mutations are only for testing of mutation techniques and have no impact on membrane interaction. However, an interaction between the C-terminus and a portion of the light chain was observed to have different indications based on these mutations, so the mutations are noted. The new membrane measured roughly 200 angstroms per side, leading to systems with over 700,000 atoms each. The system was again neutralized with sodium chloride to a roughly 0.15 molar concentration, with water molecules filling the rest of the box, with Z-dimension of

192 angstroms. Two calcium ions and one copper ion were placed as per the crystal structure of the brown snake prothrombinase16. The protein was placed slightly further out of the membrane with the intent to show the interaction was not based on initial placement only.

3.2.7 Ternary prothrombinase construction

To construct the ternary prothrombinase complex, frames from the single-protein simulations for fVa, fXa, and II were chosen such that contacts between proteins could be achieved while maintaining membrane contact. The protein-protein contacts were chosen to be similar to those of existing homology models for the ternary prothrombinase complex22, as well as those in the venom prothrombinase16. The initial position was chosen to sit fVa deeper in the membrane than has been seen in single protein simulations to ensure all three proteins have proper contact with the membrane. It was determined during the single protein simulations that the depth of membrane penetration fluctuated significantly throughout the trajectory. Due to the flexibility of both fXa and II, the system was placed

38 such that both were in contact with the membrane surface, which meant the penetration from fVa was higher than in the single protein case. Simulations on the C-domains of fVa

(not shown) indicated the initial depth in the membrane had no influence on the final interactions with the membrane, so long as contact was present initially.

In the fVa simulation, the C-terminal loop of the heavy chain interacted strongly with the N-terminus of the light chain. This was moved to interact with prothrombin. No post- translational modifications were added to fVa for this initial study, though gamma- carboxyglutamic acid and beta-hydroxyaspartic acid residues were retained in prothrombin and fXa, as proteins lacking the gamma-carboxyglutamic acid post-translational modifications have been shown to have significantly reduced membrane affinity.

The completed model was placed on a membrane surface in a water box with calcium and chloride ions using CHARMM-GUI87. In addition, CHARMM-GUI was used to add hydrogens to the system. The protein, water, ions, and membrane were parameterized using the CHARMM36112 force field, while both CGU and BHD residues were parameterized using SwissParam93. A square membrane patch roughly 200 angstroms per side composed of 90% PC and 10% PS was added, with the complex situated such that all three proteins were in contact with the membrane. Ions were added to neutralize the system at a concentration of 0.15 molar, and water molecules added, with the final dimension of the box being roughly 212 angstroms. In total, 1050 PC molecules and 117 PS molecules were used in the membrane patch. 206064 water molecules, 663 calcium ions, and 1195 chloride ions were added, neutralizing the system. In addition, 23 calcium ions were chelated to the CGU and BHD residues, and an additional 2 calcium ions, one copper ion,

39 and one sodium ion were placed as in the crystal structure for the brown snake prothrombinase16 and the heavy chain for fXa105.

3.2.8 Construction of systems for mutational analysis

The final frame of molecular dynamics simulation of the above construction was used as the initial structure for mutational analysis. Sulfotyrosines (TYS), gamma- carboxyglutamic acids (CGU), and beta-hydroxyaspartic acid (BHD) were added using swisssidechain92 and parameterized using swissparam93. Phosphothreonine (TPO2) was added using CHARMM-GUI87 at the time of system creation. In addition, all mutations were made using CHARMM-GUI. Each system was created as described previously. In short, the protein was moved to a new membrane system at the same depth from the last frame of the original simulation. The size of the membrane initially used was 250 angstroms per side, but was reduced to 200 angstroms per side to save on computation time once it was determined the extra surface had no impact on the protein movement. Calcium and chloride ions were added, and the box filled with water molecules. See table 1 for final molecule counts for each mutant.

3.2.9 Molecular dynamics parameters

An initial energy minimization was carried out until the maximum force was smaller than 1000.0 kJ mol-1 nm-1. Following minimization, a series of 6 equilibration steps were carried out, each step gradually reduced restraints. Temperature coupling for the protein, membrane, and solvent/ions were done separately using a Berendsen thermostat111 at

310.15 K with a 1 ps coupling constant. Linear center of mass motion removal was done every 100 steps, with the entire system as the only group. Hydrogen bond constraints used 40 the LINCS algorithm115. A pair list for cutoff was constructed using the Verlet scheme116, with an update frequency of every 20 evaluations and cutoff distance of 1.2 nm. PME electrostatics were chosen, with a cutoff distance of 1.2 nm. van Der Waals forces were smoothly switched to zero between 1.0 and 1.2 nm using a force-switch modifier. The first three steps of equilibration used a 1 fs timestep over 25000 steps. Step 3 added semi- isotropic pressure coupling using a Berendsen barostat111 with coupling constant of 5.0 ps and compressibility of 4.5e-05 bar-1 in both the x-y direction and z direction, with reference pressure of 1 bar. For steps 4, 5, and 6, a timestep of 2 fs was used and the simulation time was increased to 50000 steps. The production step was run for 100 ns with a Nose-

Hoover113 temperature coupling and Parrinello-Rahman114 barostat.

3.3 PROTEIN-MEMBRANE INTERACTIONS FOR SINGLE PROTEINS

3.3.1 Factor Xa

The initial conformation placed the gla domain of fXa at the surface of the membrane.

As opposed to taking a steered approach, the protein was placed to begin with interaction with the membrane. Residues contacting the membrane in the initial conformation are outlined in Table 3.1.

fXa Residue Contacting Lipid Types ALA 1 PC ASN 2 PC SER 3 PC PHE 4 PC LEU 5 PC CGU 6 PC MET 8 PC LYS 9 PC HIS 12 PC, PS CGU 14 PC, PS MET 18 PC, PS CGU 19 PC, PS CGU 20 PC, PS THR 21 PC

Table 3.1 Initial contacting residues for fXa prior to simulation.

The final conformation (Figure 3.1) lends to the flexibility of the C-terminal loop in the light chain, allowing significant deviation from the more vertical starting position. The

RMSD for the system rose slowly for the first 40 nanoseconds, leveling off and oscillating between roughly 1 and 1.5 nanometers from the starting structure. Residue-by-residue fluctuations are as to be expected, with the highest fluctuations occurring at the ends of both the heavy and light chains, and in loops surrounding the catalytic triad in the heavy chain.

Figure 3.1 Final position of fXa with respect to the membrane after 100 ns of molecular

dynamics.

Membrane contacts over the trajectory shifted slightly as the GLA domain settled into place, though the contacting residues did not significantly change. While ALA 1 maintained weak contact early in the trajectory, it lost contact by the last 20 ns. HIS 12,

CGU 14, and MET 18 began the trajectory with some membrane contact, but did not stay in range over a significant portion of the simulation. MET 8 has very little contact at the beginning of the trajectory, but over the last 30 ns has significant membrane contact, while

CGU 7, LYS 10, and ARG 15 all began the simulation outside of contact range, but maintained greater than 25% contact over the last 20 ns (or in the case of ARG 15, 43 significant contact with both PC and PS, Table 3.2). Contact area between fXa and the membrane didn't fluctuate significantly, though it stabilized after 80 ns. A final snapshot of membrane binding can be seen in Figure 3.2. The wireframe atoms represent the membrane surface, and protein residues with lower than 25% binding over the last 20 ns are not pictured. Due to the lack of lipid mobility afforded by an all-atom membrane simulation (10-8 cm2 s-1), it is difficult to study specific binding sites for PS versus PC using a biologically relevant membrane composition. However, membrane binding was not prevented by this, and interactions between CGU residues and PS residues (via Ca2+ ions) are still evident. Even so, interaction between CGU residues and PS lipids is known to be required for proper membrane binding; the overall lack of proper CGU-PS interactions could inform the overall lack of depth of interaction. fXa Residue Contacting Lipid Types ASN 2 PC SER 3 PC PHE 4 PC LEU 5 PC CGU 6 PC CGU 7 PC MET 8 PC LYS 9 PC ARG 15 PC, PS CGU 19 PC, PS CGU 20 PC, PS

Table 3.2 Residues with heavy membrane contact over final 20 ns of trajectory.

Figure 3.2 Final frame interaction between fXa and membrane surface.

3.3.2 Prothrombin

For II, the choice was made to not chelate the CGU residues with calcium ions as was done for fXa, as there was significant interchange between the initially chelated calcium ions and the solute ions. This decision had no impact on membrane binding or maintaining

45 interaction between CGU residues and calcium ions throughout the simulation. As with fXa, the gla domain was placed at the surface of the membrane, with initial contacts as in

Table 3.3.

II Residue Contacting Lipid Types ASN 2 PS THR 3 PC, PS PHE 4 PC, PS LEU 5 PC, PS CGU 6 PC CGU 7 PC VAL 8 PC, PS TYR 24 PC CGU 25 PC, PS PHE 28 PC CGU 29 PC

Table 3.3 Initial contacting residues for II prior to simulation.

The long linker region between the kringle domains gives significant flexibility, allowing large movements in the serine protease domain in solution relative to the membrane-interacting GLA domain (Figure 3.3). Not surprisingly, this conformational plasticity is evident in the RMSD plot, which shows spikes up to 2.5 nanometers of deviation from starting position. The root-mean squared fluctuation (RMSF) paints a better picture of the stability of the model, indicating extensive fluctuations only occurring in areas of interaction with the membrane and on exterior loops in the serine protease domain, as well as the linker region, where relatively heavy fluctuation did occur. The final position

(Figure III.3) after 100 ns indicates a shift toward a closed conformation after beginning in a fully opened configuration.

Figure 3.3 Final position of II with respect to the membrane after 100 ns of molecular

dynamics.

The contact area between the protein and the membrane surface fluctuated significantly over roughly the first 60 ns of simulation but stabilized after 70 ns. Many of the contacts within the kringle domain were not maintained for more than 10% of the entire trajectory,

47 with the exception of THR 102 and 103, as well as PRO 105. These contacts disappeared by the end of the trajectory (Table 3.4), once again with significant interaction between

CGU residues and PS lipids (Figure 3.4). While fXa had only slight change in protein- membrane interaction between the initial conformation and the final frame, prothrombin had significantly reduced membrane interaction. It is likely that this loss of interaction is due to reduced PS relative to what would be found in vivo. That is, PS lipids are likely to aggregate, creating binding sites as opposed to having a random distribution.

II Residue Contacting Lipid Types PHE 4 PC ARG 9 PC, PS LYS 10 PS CGU 25 PS PHE 28 PC CGU 29 PC CGU 32 PC, PS

Table 3.4 Residues with heavy membrane contact over final 20 ns of trajectory.

Figure 3.4 Final frame interaction between II and membrane surface.

3.3.3 Factor Va

The RMSD for all three simulations had a similar profile, all tending to level off after the halfway point of the simulation. The value of the RMSD for each settled in somewhere

49 between 7 and 9 angstroms for all three simulations, which would be considered high for a typical globular protein. Considering the size of the structure and potential for movement in the loop regions, it was not determined to be abnormal. It is clear from the RMSF that the C-terminal loop of the heavy chain, as well as the loop in the light chain from ALA

1749 to GLU 1776, have significant fluctuation when compared to the rest of the protein.

The 1749-1776 loop appeared to be stabilized some by the interaction from the C-terminal loop in the unmodified and 695KFKF698 mutant roughly 30 residues upstream from the loops. In the 695DFDF698 mutant, the C-terminal loop moves away from the bulk of the protein, lending to the higher RMSF values in the C-terminus, and likely the increased fluctuations in the 1749-1776 loop. The mutation to positively charged lysine residues in the 695KFKF698 mutant allowed the formation of salt bridges with ASP 1625 and GLU

1686 throughout the trajectory, while in the unmodified fVa TYR 698 made hydrogen bonds with ASN 1550 and ARG 1552. The removal of the ability to hydrogen bond from the TYR to PHE mutation allowed the 695DFDF698 mutants tail to move away from the bulk of the protein.

fVa Spike 1 Spike 2 Spike 3 Spike 4 C1 G1902-Y1903 N1913-Y1917 Y1956-C1960 A2020-N2022 C2 W2063-W2064 Q2078-N2082 L2116-S2117 W2180-Q2182

Table 3.5 Spike regions in the C1 and C2 domains of fVa.

There was significant difference in the final structures between the models, even though they all started with identical starting location and their only differences were in regions not related to membrane binding. Based on the final frame, it appears the C2 domain may have lower binding affinity to the membrane than C1. Over the entire trajectory the DFDF mutants spike 1 (Table 3.5) has very low contact with the membrane in TRP 2063 but contact throughout with TRP 2064. Conversely, both residues contact the membrane consistently in both the KFKF and unmodified simulations, though the latter more so than the former. In all simulations, spike 2 maintains consistent contact with the membrane, while spike 3 has low contact in the DFDF mutant and unmodified fVa (but high contact in the KFKF mutant), and spike 4 has virtually no membrane contact throughout. In the

C1 domain, spikes 1, 2, and 3 all make strong contacts with the membrane throughout each simulation, while spike 4 has spottier but still relatively consistent contact (Figure 3.5). A summary of the final contacts can be found in Table 3.6.

C1 C2 Spike # 1 2 3 4 1 2 3 4 WT C C C C C C,S C KFKF C C C C,S C C S DFDF C C,S S,C C,S C C,S

Table 3.6 Contacts from each mutant with membrane, by spike. C refers to an

interaction with PC lipids, and S refers to interaction with PS lipids.

Figure 3.5 Membrane contacts in fVa WT.

3.4 INITIAL CONSTRUCTION OF TERNARY PROTHROMBINASE COMPLEX

Figure 3.6 Initial configuration of the ternary prothrombinase complex.

3.4.1 Membrane interaction in prothrombinase

The initial construction of the system was as mentioned in the methods section (Figure

3.6). There was significant shifting in the prothrombinase complex within the timeframe of the simulation. Much of this occurred in the C domains of fVa, as was to be expected since the initial placement was deeper in the membrane than indicated in the single protein

53 studies. Many of the same interactions present in the single protein cases remained throughout the trajectory for the complex. In addition, spike 4 in the C1 domain maintained strong contact via ASN 2022 throughout, while spike 1 in the C2 domain also had strong, consistent interaction. Between these spikes lies spottier contacts, most of which occurred at the beginning of the trajectory; these contacts were not expected to remain as the proteins settled into place on the membrane.

In contrast to that of fVa, fXa maintained its increased contacts throughout the trajectory. Strong interactions within the first 20 residues of the light chain remained, with additional interactions in CGU 25, CGU 29, VAL 30, and CGU 32 maintained throughout.

Contacts between II and the membrane were similar to those in the substrate-only simulation. Strong contacts in CGU 29 and 32 were once again present throughout the trajectory, other contacts were spotty and fluctuated in distance throughout the trajectory.

Only CGU 7 had strong interaction throughout the trajectory.

3.4.2 Protein-protein interactions

When compared to the solution model published by the Pedersen lab, there are a number of similarities in key residues with significant protein-protein interaction, though not always between the same pairs. It is evident that the region from ASP 504 through

ARG 506 in the heavy chain of fVa is very important in binding to II. In addition, interaction in the region between HIS 318 and LYS 320 came into play after 30 nanoseconds of simulation, indicating a significant shift in the proteins. The most interesting interaction, or lack thereof, is in the C-terminal loop of the heavy chain. Though the system was constructed such that it would be in range of prothrombin at the start of the 54 trajectory, interaction is not maintained (Figure 3.7). It would seem evident that sulfation of the tyrosines at 696 and 698 are important to binding with prothrombin; further investigation of this interaction would be necessary.

Figure 3.7 Final position of the initial 100 ns simulation of the prothrombinase complex.

3.5 MUTATIONAL ANALYSIS ON 10% PS MEMBRANE

The final pose from the previous simulation (Figure 3.7) was used as the starting point for all subsequent simulations. The C-terminal loop of fVa was placed back in the exosite of prothrombin to confirm the hypothesis that the interaction loss in the first 100 nanoseconds was due to lack of post-translational modifications in TYR 696 and TYR 698.

Each system was placed on a new membrane system as noted above, with modifications added and mutations implemented as noted in the section headings. Unless otherwise specified, no post-translational modifications were included initially. Figure 3.8 shows the root-mean squared deviation (RMSD) of each simulation, with each modification listed, and WT indicative of all post-translational modifications being included.

Figure 3.8 RMSD of each simulation over 100 nanoseconds.

3.5.1 No post-translational modifications

The RMSD plot indicates a relatively stable structure, leveling off after roughly the first 20 nanoseconds of the simulation. While the overall RMSD is higher than that of the other simulations, it is evident from the RMSF that much of the deviation lies in the C- terminal tail of the heavy chain of fVa. While fluctuation exists in the residues contacting the membrane surface, the majority of the movement is within the C-terminal tail of the fVa heavy chain.

Based on the contact data, it is evident residues 503 through 512 in the fVa heavy chain are important in either binding to II or fXa. Residues 665-680 show strong interaction with II, with the 668-671 stretch showing interaction with fXa as well. Residues 215 and

216 in fXa have strong interaction with the 270-273 region of II. The same region in II also has strong interaction with the 439-443 region in the heavy chain of fXa.

Interestingly, the 681-709 region of fVa did not maintain contact with II. Numerous studies have been carried out showing the 695-698 region is key to the fVa-II interaction42,117–119. The lack of interaction would indicate the importance of sulfation of the tyrosine residues at positions 696 and 698 (Figure 3.9).

Figure 3.9 Final position of the prothrombinase complex after 100 ns with no post-

translational modifications.

Protein membrane interactions are significantly stronger in fXa and II than in the single protein case. While in the single protein case, weak interactions in CGU 7 was present, in the ternary complex, a strong interaction with a phosphatidylserine lipid exists. While no interactions beyond THR 21 remained throughout the trajectory in the single protein case, strong interactions with PS residues persisted for CGU 25, 29, and 32. Similarly, while no

59 additional interactions between the membrane and II appeared versus the single protein case, the interactions between CGU residues and the membrane were significantly stronger.

The fVa-membrane interaction maintained similar contacts to those in the single protein case. It appears based on the final position after simulation, the poor interaction between fVa and II could have caused a poor interaction between spike 1 in the C2 domain in fVa and the membrane.

3.5.2 695KFKF698 mutation

The protein with no post-translational modifications was used as a template for the mutation, and therefore no other post-translational modifications were added to fVa. Not surprisingly, the RMSD and RMSF for this mutant are similar to that of the unmodified protein. The residues with highest fluctuation again lie in the C-terminal tail of the fVa heavy chain, though the highest fluctuation begins closer to the C-terminus than in the unmodified protein.

Protein-protein interactions in this mutant are very similar to those in the unmodified protein (Figure 3.10). While interaction between fVa and II extends further toward the C- terminus in the mutant than in the unmodified protein, much of the interaction profile is the same. The bulk of the C-terminal tail does not maintain contact with prothrombin, and residues in the 503-506 and 665-673 region have significant interaction with residues in II. fXa-II and fVa-fXa interactions are nearly identical to those in the unmodified protein.

Protein-membrane interactions are also similar; it is likely the differences lie in the random distribution of the PS lipids in the membrane causing small differences in membrane binding, and the speed at which lipids are able to move through the membrane are outside

60 the timescale of these simulations. The final pose indicates less movement of the C- terminus of the fVa heavy chain away from II, and along with it a better interaction with the membrane in spike 1 of the C2 domain.

Figure 3.10 Final position of the prothrombinase complex after 100 ns with the

685KFKF698 mutation.

3.5.3 695DFDF698 mutation

The 695DFDF698 mutation, surprisingly, did not have a similar binding profile to those of the previous two simulations. The RMSD was markedly lower, with stability closer to

0.5 nanometers over most of the trajectory, while the 695KFKF698 and unmodified proteins had RMSD values settling in closer to 1 nanometer. The key difference is evident in the RMSF, which indicates much lower fluctuation in the C-terminal tail of the fVa heavy chain. While the protein-protein interactions indicate a similar interaction profile between fVa-fXa and fXa-II, the C-terminal tail of the fVa heavy chain maintains contact with II through a hydrophobic interaction between PHE 698 and ILE 398.

The most interesting result from this simulation is the interaction between fVa and the membrane (Figure 3.11). Over the final 20 nanoseconds of the trajectory, contacts between fVa and the membrane are conspicuously absent. The final pose indicates a contortion of fXa and II away from fVa, removing it from its initial seat in the membrane. Upon further inspection, though, it is clear that a lack of interaction between the C1 and C2 domains in fVa with PS-type lipids in the membrane weakened the protein-membrane interaction, allowing fVa to freely detach. While this is an unfortunate consequence of the lack of lipid interchange possible in this type of membrane system, it does show the importance of interaction with the charged PS-type lipids in the membrane for proper protein-membrane interaction.

Figure 3.11 Final position of the prothrombinase complex after 100 ns with the

695DFDF698 mutation.

3.5.4 Addition of sulfation on TYR 696 and TYR 698

The addition of post-translational sulfation on the tyrosines at positions 696 and 698 of the fVa heavy chain result in a markedly different interaction profile over all proteins. The

RMSD showed the lowest deviation in all of the proteins simulated, and a look at the RMSF indicates a low fluctuation overall throughout the protein. While the simulations without sulfation had high fluctuation in the C-terminus of the fVa heavy chain, movement here only existed in the final few residues. While interactions in the 318-320 region and the

372-375 region of fVa with II exist in the other simulations, they either did not have both sets interacting, or the strength of interaction was lower.

These additional strong interactions appear to have caused a small shift in the fVa-fXa and fXa-II interactions. While many of the same interactions exist, such as residues in the

503-505 region, there are fewer consistently strong residue-residue interactions than in the non-sulfated cases. ARG 506, which had multiple strong interactions in the other runs, doesn’t maintain interaction with II with sulfations added. This may be due to the tighter interaction with II, as all interactions between ARG 506 and II are sidechain-backbone interactions while in the non-sulfated runs, some of the interactions were sidechain- sidechain. The fXa-II interaction, overall, appears to be much weaker than in the non- sulfated cases. Additionally, a number of contacts between the light chain of fXa and the

GLA domain of II appear, which are not present in the non-sulfated runs.

Interestingly, while there are very few interactions between the protein complex and

PS lipids, there is consistent interaction with the membrane throughout the trajectory. This is contradictory to the result for the 695DFDF698 mutant, where the lack of fVa-PS

64 interaction resulted in the protein moving away from the membrane surface. It is possible that the interaction between LYS 2116 in spike 3 of the C2 domain and PS residues was enough to keep the protein seated, as this is the only strong interaction throughout the simulation. The final pose (Figure 3.12) indicates the sulfation at 696 and 698 are key to proper fVa-II interaction.

Figure 3.12 Final position of the prothrombinase complex after 100 ns with the post-

translational sulfation added at tyrosines 696 and 698.

3.5.5 700DE701 mutation

The 700DE701 mutant was created with the sulfation on 696 and 698, as in the previous section, intact. Experimental data suggests an accumulation of meizothrombin in prothrombinase made with a regulatory subunit having this mutation56. While the RMSD of this protein is similar to that of the other sets, the RMSF indicates significant movement in the C domains of fVa. Movement in the C-terminus of the fVa heavy chain is limited even further than in the non-mutated version of this protein.

The fVa-II interaction is similar to that of the system lacking the 700DE701 mutation, with the greatest differences being in the light chain. However, a consequence of the addition of negatively charged residues is a hairpinning in the C-terminus; for instance, interaction between the mutated GLU 701 and the terminal ARG 709 causes a rigidification of this region, which was still quite flexible in the non-mutated case. Interaction between fVa and fXa is nearly unchanged from that of the system lacking the 700DE701 mutation.

While the non-mutated system had several interactions between the light chain of fXa and the GLA domain of II, these are not as present in the mutated system (Figure 30). This is likely due to the C2 domain moving away from the membrane surface, causing a contortion of the full complex (Figure 3.13).

Figure 3.13. Final position of the prothrombinase complex after 100 ns with the

700DE701 mutation.

3.5.6 Addition of all post-translational modifications

Upon adding the final post-translational modifications (phosphorylation at THR 612 and sulfation at TYR 665, or WT), additional stabilization appears to have occurred in the 68 complex. The C-terminal tail of the fVa heavy chain had significantly lower fluctuation than in the other systems, though a higher fluctuation did exist in the 340-359 loop on fVa heavy chain.

The lack of movement in the C-terminus is evident from the contact data, which shows residues 705-709 of the fVa heavy chain strongly interacting with II. This interaction was not seen in any of the other systems, and likely stems from the stabilization of the proteins caused by the phosphorylation at THR 612 and sulfation of TYR 665. The most similar system (lacking these two modifications) lacks interaction between II and TYR 665, as well as the contacts in the 571-575 region. Systems lacking phosphorylation on THR 612 have interaction between the threonine residue and the light chain of fVa, whereas phosphorylation diminishes this interaction. It is possible this lack of interaction allows for greater ability to interact with II.

While the interactions between fVa and fXa are very similar to the interactions in the other systems, the fXa-II interactions are quite different. While the same interactions have been seen in one system or another, the contact combination here is markedly different than the next closest system. Interactions between the light chain of fXa and the GLA domain of II are far less present, though some prevalent interactions in other systems have shown to be prevalent here.

The protein-membrane interactions once again display the importance of PS lipids in membrane binding. While fVa has strong membrane interaction in both the C1 and C2 domains, II has significantly diminished membrane interaction when compared to the other systems (Figure 3.14). However, the entire system has good membrane contact overall; a

69 better distribution (or more altogether) of PS lipids could lead to a slightly different interaction.

Figure 3.14 Final position of the prothrombinase complex after 100 ns with the all post-

translational modifications included.

3.5.7 334KF335 mutation

The 334KF335 mutant was constructed with all post-translational modifications present. As may be expected, the RMSD is similar to that of the previous system; however, the RMSF tells a different story. The C-terminus has significant fluctuation, especially when compared to that of the non-mutated system with the same post-translational modifications. In fact, the C-terminus of fVa does not stay in contact with II throughout the trajectory. The contact profile for the fVa-fXa interaction differs only slightly from that of the wild-type, though the fXa-II interaction is quite different, having more long- held contacts than wild-type. However, in none of the protein-protein interactions are residues 334 or 335 involved.

The intraprotein interactions of the 334KFKF335 mutant and wild-type indicate a loss of interaction with TYR 564 and VAL 565 from the mutated region in the mutant which are maintained in wild-type prothrombinase, and a much weaker interaction with GLU 525 when compared to wild-type. The loss of interaction with TYR 564 and VAL 565 likely had implications upstream, as the wild type has significant contact in the region from 571-

575, while the 334KF335 mutant lacks this interaction.

Though a few spikes in the C1 domain did not have contact with PS lipids, overall this system had high protein-membrane contact. PS lipids were more present around both fXa and II, leading to better interaction, specifically with CGU type residues. A large fluctuation in the membrane surface (Figure III.15) appears to have cause the C domains

71 to come closer to each other than in the other modeled systems, as well as a pulling of the light chain of fXa down well below the plane of the C domains of fVa.

Figure 3.15 Final position of the prothrombinase complex after 100 ns with the

334KF335 mutation. All post-translational modifications are included.

3.5.8 Key Interactions/Future Directions

The results here indicate the importance of post-translational modifications to enzymatic complex-substrate binding. It is evident that not only is the 695-698 stretch of the heavy chain of fVa is important for interaction with II, as indicated by experimental data42,117–119, but the sulfation of the tyrosine residues in the region is necessary for proper fVa-II interaction. All systems lacking this post-translational modification had lowered interaction between II and the C-terminal tail of the fVa heavy chain. Addition of phosphorylation at THR 612 and sulfation at TYR 665 improved the overall fVa-II interaction as well. The results of the 334KF335 mutant indicate that, while these residues do not directly interact with either fXa or II, their intraprotein interactions can regulate complex-substrate interactions via conformational changes stemming from lost residue- residue interactions. A number of interactions persisted through each of the systems as well. For the most part, all fVa-fXa interactions were present in each of the systems; even those interactions not displayed in the figures were present, though not as prominent. The

503-506 region of fVa made a number of contacts not only with fXa, but with II as well.

Further study of this region with the Leiden mutation (R506Q) may give insight into the possibility of different protein-protein interactions caused by this mutant.

It also became clear through the analysis of protein-membrane interactions that PS lipids have great importance in maintaining contact. While plenty of experimental data64,120,121 backs up the notion that PS lipids are necessary for proper membrane interaction in all three proteins, computational data confirming this, as well as indicating consequences of lack thereof, only strengthen the hypothesis that PS is required.

Upon final analysis of the results, it is clear the choice of a full membrane system with

90% of lipids being of PC type and 10% PS led to some issues with protein-membrane interaction. Due to the random placement of PS lipids throughout the membrane, not all pertinent protein-PS interactions were present at any given time. The timescale for interchange of lipids within the membrane is longer than allowed by the simulation times.

There are a few ways to remedy this; the first of which would involve increasing the percentage of PS lipids beyond the amount typically found in platelets, to overcome the issue with lipid-type interchange. The other option would be using a highly-mobile membrane mimetic (HMMM)122 system for initial equilibration of the protein-membrane interactions, then converting to a full-length lipid system.

The HMMM system would also potentially allow the investigation of complex- substrate binding in the presence of the membrane system without docking the substrate a priori. While the systems herein were built such that II had already interfaced with the complex, increasing computational power along with a membrane mimetic that allows fast diffusion might allow a more “organic” complex-substrate interaction by placing only the

C-terminus of the fVa heavy chain on the exosite of prothrombin, and allowing the interaction to evolve as it would in vivo. Such a system would require a much larger membrane surface than used in the current study, and as such may require much more computing power than is available at this time.

Preliminary investigation of HMMM systems using the final pose of the wild-type system above verified the belief that a full-PS system would be more appropriate. A significant increase in protein-membrane interactions occurred, with many strengthening as the system was run. However, membrane penetration was not impacted; the depth in 74 the membrane did not significantly change. Furthermore, previous simulations using a full-tailed lipid system indicated rapid equilibration of the protein in the membrane when additional depth was added initially. Full lipid systems with 100% PS will need to be investigated to determine if proper protein-protein and protein-membrane interactions are inhibited significantly by the choice of a 10% PS system. It is expected that this type of system, while unrealistic overall, will give a more appropriate binding system for the proteins, where aggregation of PS lipids on the surface of would be likely to occur.

CHAPTER IV

CONSTRUCTION OF A NEW MODEL FOR THE TERNARY

PROTHROMBINASE COMPLEX IN THE PRESENCE OF A

PHOSPHATIDYLSERINE MEMBRANE SURFACE

4.1 INTRODUCTION

Prothrombinase is the activated factor V (fVa)/activated factor X (fXa) complex which cleaves prothrombin to form thrombin. In the absence of the regulatory subunit fVa, fXa is capable of this cleavage, but not at a rate capable of maintaining hemostasis10. The interaction of fVa with fXa in the presence of calcium ions and a phosphatidylserine- containing membrane surface results in a 300,000-fold increase in the catalytic efficiency of fXa in generating thrombin10. This increase in catalytic efficiency is due to a 100-fold decrease in the Michaelis constant Km, as well as a 3000-fold increase in the catalytic constant kcat. In the absence of a membrane surface, the activation rate of prothrombin by prothrombinase is only 300 times that of fXa alone. The presence of the regulatory subunit stabilizes the complex, altering the pathway for which prothrombin activation occurs. It 76

has been suggested that interaction between prothrombinase and its substrate is mediated by an interaction between anion binding exosite I (ABE-I) on prothrombin and the hirudin-like C-terminal loop of fVa39–42.

Prothrombin, the substrate for the prothrombinase complex, circulates in the blood at a

31 concentration of 1.4 µM with Mr of 72,000 . Prothrombin is composed of 4 domains: an

N-terminal GLA domain (residues 1-46), two kringle domains K1 (residues 65-143) and

K2 (residues 170-248), and a serine protease domain (residues 285-579) connected by three linker regions32. The linker region between the kringle domains lends to the conformational plasticity of the molecule33. Within the GLA domain are 10 γ- carboxyglutamic acid residues, which are post-translationally modified by carboxylase, for which vitamin K is a necessary cofactor, from glutamic acid residues34. This process is a frequent target of anticoagulants, such as warfarin, which competitively inhibits the vitamin K epoxide reductase complex 1 (VKORC1), a vitamin K activator35.

While no full crystal structure for factor Va exists, a number of partial structures, as well as homologous structures, have been solved. A crystal structure for the C2 domain

14 15 has been found , bovine fVai, inactivated by activated protein C , and most recently, a crystal structure for the prothrombinase complex from the venom of Pseudonaja textilis16.

17 The Pellequer model from 2000 was invalidated by the crystal structure of bovine fVai, which indicated the orientation of the C domains was such that the loops would be able to interact with the membrane. Two new models (Orban18 and Autin19) were generated based on this crystal structure, as well as the A domains of the prior theoretical model. The Orban model included the C-terminus of the A2 domain, while the Autin model included an interaction with factor Xa. Subsequent to these models, three models by the Pedersen20–22 77 group were generated; the first without the C-terminal loop, but with docked fXa, the second added prothrombin, and the third, most recent model, included the full ternary complex as well as the C-terminus. Here we present a new model constructed from membrane-equilibrated structures for fVa, fXa, and II. The complete ternary prothrombinase complex constructed from these systems has also been equilibrated on a phosphatidylserine (PS) membrane surface.

4.2 METHODS

All models built with MODELLER70 version 9.15 using UCSF Chimera 1.11.286 as a graphical user interface. All crystal structures used were obtained from the Protein Data

Supercomputer Center102.

4.2.1 Homology model for factor Xa

To construct the model for fXa, crystal structures of the partial heavy chain with partial light chain (RCSB ID: 1XKA)105 and the gamma-carboxyglutamic acid containing (GLA) domain (1IOD)106 were used. The GLA domain from 1IOD was placed below the crystal structure for the heavy chain and remainder of the light chain from 1XKA, and a series of 78 loop optimizations were carried out to obtain the final model. Calcium ions were chelated to the beta-hydroxyaspartic acid (BHD) and gamma-carboxyglutamic acid (CGU) residues.

4.2.2 Homology model for prothrombin

4.2.3 Homology model for factor Va

14 15 Crystal structures of the C2 domain (1CZT) and bovine fVai (1SDD) were oriented using the crystal structure of prothrombinase from the venom of P. textilis (4BXS)16. Due to the higher homology in bovine fVa with respect to that of the brown snake venom prothrombinase, the C2 domain orientation from bovine fVa was selected preferentially, as there is a marked rotation away from the C1 domain in the structure from the venom prothrombinase. The C-terminal loop was initially chosen to resemble that of the Pedersen model22. Post-translational modifications of fVa were added using SwissSidechain for the sulfotyrosine residues, while the phosphothreonine was added using CHARMM-GUI90 at the time of system generation.

4.2.4 Molecular dynamics system construction

All systems were placed at the surface of an individual membrane patch, each consisting of only palmitoyl-oleoyl-phosphatidylserine (POPS) type lipids. Initial equilibration attempts using non-zero concentrations of palmitoyl oleoyl- phosphatidylcholine (POPC) type lipids yielded poor results for membrane interaction.

Initial dynamics on a 10% POPS, 90% POPC membrane for 300 ns yielded membrane binding, but lacked interactions with phosphatidylserine (PS) type lipids as would be expected in vivo. These systems were moved to full POPS lipid patches for further membrane binding analysis. For fXa and II, the membranes measured 150 angstroms per side, while the membrane patch for fVa measured 200 angstroms per side. Each system was constructed using the membrane builder within CHARMM-GUI89. Each system was neutralized to a 0.15 molar concentration of calcium chloride to account for the calcium- dependent nature of the membrane binding in the GLA domains of fXa and II.

4.2.5 Ternary prothrombinase construction

The completed model was placed on a membrane surface in a water box with calcium and chloride ions using CHARMM-GUI87. In addition, CHARMM-GUI was used to add

80 hydrogens to the system. The protein, water, ions, and membrane were parameterized using the CHARMM36m112 force field, while both CGU and BHD residues were parameterized using SwissParam93. A square membrane patch roughly 200 angstroms per side of POPS lipids was added, with the complex situated such that all three proteins were in contact with the membrane using positions from the single protein simulations for initial depth placement. Ions were added to neutralize the system at a concentration of 0.15 molar, and water molecules added, with the final dimension of the box being roughly 212 angstroms. In total, the entire solvated system with protein, membrane, and solvent totaled

742,221 atoms.

4.2.6 Molecular dynamics parameters

310.15 K with a 1 ps coupling constant. Linear center of mass motion removal was done every 100 steps, with the entire system as the only group. Hydrogen bond constraints used the LINCS algorithm115. A pair list for cutoff was constructed using the Verlet scheme116, with an update frequency of every 20 evaluations and cutoff distance of 1.2 nm. PME electrostatics were chosen, with a cutoff distance of 1.2 nm. van der Waals forces were smoothly switched to zero between 1.0 and 1.2 nm using a force-switch modifier. The first three steps of equilibration used a 1 fs timestep over 25,000 steps. Step 3 added semi- isotropic pressure coupling using a Berendsen barostat111 with coupling constant of 5.0 ps

81 and compressibility of 4.5e-05 bar-1 in both the x-y direction and z direction, with reference pressure of 1 bar. For steps 4, 5, and 6, a timestep of 2 fs was used and the simulation time was increased to 50,000 steps. A total of 300 ns of full molecular dynamics were used for the single proteins, while 200 ns of dynamics for the ternary complex were completed with a Nose-Hoover113 temperature coupling and Parrinello-Rahman114 barostat. Following these initial simulations, an additional cycle of minimization, equilibration, and 100 ns of dynamics were carried out for each system in triplicate, with the only change from the above parameters being an increase to 500,000 steps for the final equilibration step. These final systems were used for analysis of protein-membrane and protein-protein interactions.

Contact data was generated using pycontact99 using default settings.

4.3 RESULTS

4.3.1 fVa-membrane interaction

Key residues in the C1 and C2 domain for membrane binding are given in Table 4.1123.

In the C1 domain, spikes 1 and 2 make multiple hydrogen bonds between the both the side chains and the backbones of the protein and the head groups of the lipid surface (Figure

4.1). In spike 1, G1902 and Y1903 maintain hydrogen bonds throughout all three trajectories, while in spike 2 G1915 and S1916 do the same, with the glycine residues making H-bonds through backbone residues. Spike 3 maintains contact throughout the trajectory, but only makes hydrogen bonds through K1954 and K1958. The tyrosine and leucine residues between the two lysines maintain contact, but do not maintain hydrogen bonds throughout the entire trajectory. Spike 4 has similar membrane interaction, where residues Y2021-R2023 all make strong hydrogen bonds throughout.

The C2 domain is where the impact of a high PS concentration in the membrane was most evident. In previous studies of a membrane system containing 10% PS, only very weak interaction was observed in spike 3, and no interaction in spike 4 at all. With a full

PS membrane, spikes 1, 2, and 3 all had very strong hydrogen bonding, while spike 4 also made contact with the membrane in all three trajectories, though not to the same extent as the other three spikes. Spike 1 assumed significant depth, with residues 2060-2068 all maintaining hydrogen bonds throughout the trajectories. Similarly, all residues in spike 2 hydrogen bonded with the lipid heads, either through their backbones or the sidechains.

Spike 3 again had hydrogen bonding from C2113 through E2119, which is significant compared to previous studies as mentioned above. Spike 4 maintained hydrogen bonds with lipids through Q2182. These contacts will be maintained upon construction of the ternary complex, and will be used to validate the final pose.

fVa Spike 1 Spike 2 Spike 3 Spike 4 C1 G1902-Y1903 N1913-Y1917 Y1956-C1960 A2020-N2022 C2 W2063-W2064 Q2078-N2082 L2116-S2117 W2180-Q2182

Table 4.1 Key regions in fVa for protein-membrane interaction.

Figure 4.1 Interaction between the C1 and C2 domains in fVa and the PS-containing

membrane surface. Light blue lines indicate hydrogen bonds.

4.3.2 Membrane interaction in GLA domain-containing proteins

For both fXa (Figure 4.2) and II (Figure 4.3), much of the contribution was through calcium-dependent interaction between CGU residues and the PS lipid heads of the membrane surface. In fXa, the first 10 residues all had some level of membrane interaction, which includes CGU residues at positions 6 and 7. Additionally, CGU residues 19, 20, 25,

29, and 32 all contributed significantly to membrane binding.

II had a similar binding profile to that of fXa. The first 10 residues all contributed (to varying extents) to membrane binding, with CGU 6 and 7 maintaining strong interaction via chelated calcium ions as in fXa. CGU residues at 16, 25, 26, 29, and 32 rounded out

84 the contacting gamma-carboxyglutamic acid residues, which made up the majority of the consistent contacts.

Figure 4.2 Interaction between the GLA domain of fXa and the PS-containing membrane

surface. Calcium ions are indicated by green spheres.

Figure 4.3 Protein-membrane interaction between the GLA domain of II and the PS-

containing membrane surface.

4.3.3 Interactions in the ternary prothrombinase complex

As was expected when using a homogenous membrane system, the protein-membrane interactions did not change when compared to the non-complexed systems. Residues 1 through 5 of II did have a tendency to be in contact with the membrane with lower frequency than in the single protein cases, though the membrane-CGU interactions from the single protein cases did not change in the complex. For fVa and fXa, interactions were as described previously. Figure 4.4 depicts the starting structure for the complex, composed of the MD-equilibrated protein structures.

Figure 4.4 Initial conformation of the ternary prothrombinase complex after MD simulations for all three proteins separately. Protein-protein interactions were selected

to match crystal structures and accepted models as closely as possible without creating

clashes.

A number of regions within fVa appear to have significant importance in proper binding of the substrate (Figure 4.5). In the fVa heavy chain, the E372-E374 region formed salt bridges with some combination of R498, R500, E414 and E549 of II in all studied

87 trajectories. The short stretch from L503-R506, which also contains a cleavage site for

APC, appears to serve a dual role in the construction of the complex. Interaction with prothrombin in the L260-E262 region as well as E254 and E255 was prevalent, with multiple hydrogen bonds formed over the simulation. This region of fVa appears to be just as important for fVa-fXa interaction, with the S354-T359 and T275-Y279 regions of fXa again forming a number of hydrogen bonds. Not surprisingly, fXa-II interactions also occur in these regions, where N272-E277 in the fXa heavy chain interact strongly with

E249-E254 in prothrombin. T570-C575 interacted strongly with the N162-Q169 region of prothrombin, and specifically with L167 which formed hydrogen bonds with G573 and

C575 for more than 90% of the simulations.

Closer to the C-terminus of fVa there were a large number of weak interactions with prothrombin. Nearly every residue in the stretch between E662 and E691 interacted weakly with prothrombin, with a few lasting hydrogen bonds appearing within the region.

While the sulfotyrosines at 696 and 698 do interact with prothrombin, the interaction is not strong implying these interactions are easily reversible when releasing thrombin after activation. L706-R709 additionally make significant contact with prothrombin.

Interaction between prothrombin and the light chain of fVa could be key as well. The

T149-S160 stretch of prothrombin formed multiple interactions with the light chain of fVa, with many being hydrogen bonds which held for large portions of the trajectory. While the interaction between the light chain of fVa and prothrombin is rarely studied, the number of interactions present should not be ignored.

Aside from those mentioned above, a few other regions appear to be key in the construction of the ternary complex. The short stretch of T626-D628 in fVa interacts with

R347 and N348 of fXa, while the stretch between S664 and P671 make contact with the region between R424 and A434. In the light chain, R1687 forms salt bridges and hydrogen bonds with fXa in the D346-S349 stretch. The remainder of the fXa-II interaction is composed of an interaction between E216 in fXa and E269 in prothrombin, and a pair of interacting stretches: N272-K276 in fXa with C248-E254 and K435-T443 of fXa with

E444 in prothrombin.

Figure 4.5 Final conformation of the ternary prothrombinase complex in the presence of

a PS-containing membrane surface.

It should be noted that, although it is true that the protein-membrane interactions are

PS-dependent, there is a difference between a prothrombinase complex bound to PS- containing vesicles and prothrombinase bound to a platelet surface. It has been demonstrated previously61 that activation of prothrombin on an activated platelet follows the pre2-pathway via a concerted mechanism, while the meizothrombin pathway is dominant in prothrombinase in the presence of PS-containing vesicles. In our model, R271 is significantly closer to the active site of fXa than R320, though significantly longer simulation would be required for a significant shift to occur from the initial distances.

Further investigation on the composition of the binding sites of the full complex on the platelet surface could potentially uncover the mystery in this pathway difference.

CHAPTER V

MUTATIONAL ANALYSIS OF THE INTERACTION ENERGIES BETWEEN

THE PROTHROMBINASE COMPLEX AND PROTHROMBIN

5.1 INTRODUCTION

Prothrombinase is a protein complex composed of the enzymatic subunit factor Xa (fXa) and its regulatory subunit factor Va (fVa), for which its substrate is prothrombin (II). The conversion of II to thrombin (IIa) occurs on a phosphatidylserine membrane surface in the presence of calcium ions10, and is a central process in the coagulation cascade. Both fVa and the membrane surface are required for proper clotting, and result in a 300,000-fold increase in catalytic efficiency over fXa alone without the presence of a membrane surface.

To this end, a number of studies have looked into the impact of certain amino acids on protein-protein interactions in the ternary prothrombinase complex. Interaction between fVa and fXa, forming the prothrombinase complex, requires complete activation of fVa,

91 and therefore removal of the B domain. The 1000-1008 region of factor V has been shown to inhibit binding to factor Xa44, indicating B domain removal is required for proper prothrombinase assembly. Substitution of asparagine for Arg 347 in fXa has been shown to reduce regulatory subunit binding45. It was later determined that Arg 347, Lys 351 and

Lys 414 make up a binding epitope, while Glu 310 and Arg 306 form an extended region of this epitope46. Residues 311-325 of fVa have been suggested as a potential binding site for fXa, as a protein containing these residues was inhibitory for prothrombin generation in the presence of fVa, but not when the regulatory subunit was absent47. A similar study over the 323-331 stretch indicated direct inhibition of interaction between fXa and fVa48,49.

Further studies have indicated residues 493-506, which contains an inactivation site for

APC, may contribute to fVa-fXa binding as well50. Work on the same region indicated mutation of the 499-505 region was sufficient to greatly inhibit fVa-fXa interaction51.

Later studies in the same region indicated a binding surface may exist centered on Arg 501,

Arg 510, Ala 511, Asp 513, Asp 577, and Asp 578 of the factor Va A2 domain52.

Interaction between fVa and the substrate are key to the timely activation of prothrombin. Site-directed mutagenesis studies have indicated the amino acid region 659-

663 in the fVa heavy chain contributes to cofactor function53. Mutation of 695DYDY698 in the fVa heavy chain to 695KFKF698 resulted in reduced cofactor activity within prothrombinase42. Further charge inversion studies on the 334-335 region of the fVa heavy chain indicate this region may be required for enzyme and substrate rearrangement, though affinity for fXa was not reduced when compared to wild type54,55. Another charge inversion mutation 700NR701 to 700DE701 resulted in significant delay in prothrombin cleavage, with substantial accumulation of meizothrombin56. ABE-I has been 92 demonstrated to be key in fVa-accelerated prothrombin activation, as activation is inhibited in the presence of bothrojaracin, a snake venom protein which binds ABE-141.

It has been reported that the interaction between prothrombin and factor Xa involves

Lys 276 on factor Xa57. It is also known that the catalytic triad of factor Xa interacts with

Arg 320 and Arg 271 of prothrombin, activating it to thrombin. A factor Va-dependent binding site for fXa on prothrombin has been found using synthetic peptide analysis, in the

473-487 region of prothrombin58. Further examination narrowed the region to the 478-482 stretch, with 478, 480, and 481 mutants inducing deficiency in clotting59.

In the present study, molecular dynamics simulations are used to determine the interactions within these protein complexes, and the impact a number of mutations may have.

5.2 METHODS

5.2.1 System construction

The initial model was taken from the initial construction of the prothrombinase complex on the membrane surface after various equilibration and dynamics steps, summing to a total of 200 nanoseconds of molecular dynamics. Sulfotyrosines (TYS), gamma- carboxyglutamic acids (CGU), and beta-hydroxyaspartic acid (BHD) were added using

SwissSidechain92 and parameterized using SwissParam93. Phosphothreonine was added using CHARMM-GUI87 at the time of system creation. In addition, all mutations were made using CHARMM-GUI. Each system was created as described previously. In short, each mutated protein was constructed on a full phosphatidylserine membrane surface with the same initial conditions of that of the wild type system. Membrane size remained at 200

93 angstroms per side, with a 0.15 molar calcium chloride concentration and TIP3 water molecules to fill the remainder of the simulation box.

5.2.2 Molecular dynamics parameters

The parameters used for the molecular dynamics simulation were as described previously. In short, the CHARMM36m112 force field was used to parameterize the protein, and all simulations were done using GROMACS 2018.294. A brief minimization was followed by 6 equilibration steps, each with reduced restraints and increased time with respect to the previous step, switching from a 1 fs timestep to 2 fs for the 4th step and all subsequent steps. Production over 100 ns followed equilibration. Each mutation was simulated in triplicate using different initial velocity seeds. The velocity seeds were kept consistent between mutations.

5.2.3 Contact analysis parameters

Contact data was generated using PyContact99 using default parameters. For the present study, 6 mutant variations of the wild type have been investigated. In fVa, mutants are 334KF335, 695DYDY698 (no post-translational modification), 695DFDF698,

695KFKF698, and 700DE701. In prothrombin, residues 478, 480, and 481 were investigated with alanine mutations. Protein-protein and residue-by-residue interaction energies were generated in gRINN101 using standard parameters. Interaction energies were taken from the final 20 ns of the trajectory.

5.3 RESULTS

Table 5.1 contains a summary of the interaction energies between the prothrombinase

complex and the substrate. The interaction energy is defined as the sum of the electrostatic

and van der Waals terms, summed over all interactions between each protein, informing

the magnitude of the values.

Seed WT 334 480 700 DFDF DYDY KFKF

S1 fVaHC:II -440.54 165.37 311.19 -68.83 -312.14 -139.08 -38.28

fVaLC:II -211.16 -117.34 -231.46 -265.00 -198.50 -239.40 -72.72

fXa:II -41.21 -109.08 -128.32 -18.33 -76.36 -111.08 -99.27

fVa/fXa:II -692.91 -61.05 -48.58 -352.16 -587.00 -489.56 -210.27

S2 fVaHC:II -240.34 -46.32 58.27 -14.39 -75.24 46.33 -115.85

fVaLC:II -245.89 -343.22 -203.57 -161.58 -186.05 -473.84 -248.10

fXa:II 36.92 -70.65 -175.75 -84.62 -119.46 -16.36 -365.86

fVa/fXa:II -449.31 -460.19 -321.04 -260.58 -380.75 -443.87 -729.81

S3 fVaHC:II -6.49 -172.46 34.37 96.84 1.29 -425.56 -40.61

fVaLC:II -289.57 -180.90 -253.81 -170.90 -135.07 -209.13 -245.38

fXa:II 8.73 -62.41 55.14 -61.47 -225.03 -85.00 62.67

fVa/fXa:II -287.33 -415.78 -164.30 -135.53 -358.82 -719.69 -223.32

Table 5.1 Non-bonded interaction energies between components of the prothrombinase

complex and substrate. Values are in kcal/mol. WT is wild type, 334 is the 334KF335

mutant, 480 is the S478A, L480A, Q481A mutant of prothrombin, 700 is the 700DE701

mutant, and DFDF, DYDY, KFKF are the 695-698 region mutations of fVa.

Key interacting regions have been identified previously for the wild type system, summarized in Table 5.2. These regions will be investigated for changes in binding energy.

Any additional regions of interest in mutated systems will also be considered. Seed 1 will be used for investigation of differential interactions.

Region Label fVa region Region 1 K320-E323 Region 2 E372-K378 Region 3 S502-I514 Region 4 T570-V580 Region 5 K650-K680 Region 6 M681-R709

Table 5.2 Regions of interest within fVa for substrate binding within the ternary

prothrombinase complex.

5.3.1 334KF335 mutation

While the mutated region does not have a direct influence on the protein-protein interactions within the complex, there is a significant impact on the interprotein interactions within fVa. In wild type fVa, a strong interaction between D334 and K361 results, with an average interaction energy of roughly –61.6 kcal/mol. However, the charge inversion on the aspartic acid to a lysine results in a repulsion (10.5 kcal/mol) and therefore differential interaction further along. The repulsive interaction with K361 in the mutant results in this

96 residue interacting with D353 very strongly (-68.6 kcal/mol) in an interaction not present at all in wild type. The impact of this interaction is a loss of interaction between D353 and

K646, which is moderately strong in the wild type (-15.8 kcal/mol) but has nearly no presence in the mutant, resulting in overall conformational differences. The mutation of residue 335 from tyrosine to phenylalanine was not as impactful; while interaction with

E525 from this position is reduced (-16 kcal/mol to –2.7), no impact is seen in further interactions.

Due to these conformational changes, per-residue interactions were significantly impacted. Charged interactions in region 1 between K320 and D495 as well as between

E323 and R498 had their interaction energy significantly reduced in the mutant. Within the region 2, a moderate interaction between E372 and R500 was barely present in the mutant. While the interaction between E374 in fVa and R498 in II is very strong in the wild type (-63 kcal/mol), it is barely present in the mutant (-3 kcal/mol), dominated by the repulsion from E414 and E549 in II (roughly 12 and 23 kcal/mol in WT, 36 and 44 kcal/mol in the mutant, respectively). An interaction between E374 and R553 in II is not present in

WT, but is relatively strong (-26 kcal/mol) in the mutant. There is a shift in the interprotein interactions within II in this region leading to this new interaction. In the wild type, E549 interacts strongly with R498, while in the mutant this interaction is absent, replaced with

R553. The small conformational differences caused by the 334 charge inversion in fVa appear to have caused a conformational shift in II due to the protein-protein interactions present. Regions 4 and 5 did not have significant changes in their interaction energies when compared to wild type.

These conformational changes had significant impact on the interaction between the C- terminus of fVa and II. In the wild type, R709 in fVa makes attractive interactions with both K372 and E466 of II, with a repulsion interaction with R388 between of lower magnitude between. However, these interactions are nonexistent in the mutant, with the only strong interaction between R709 and II being with E345, which is a sidechain- backbone interaction and therefore strongly repulsive. The final frame of the mutant

(Figure 5.1) would seem to indicate the C-terminus is losing contact with II altogether, and the interaction data would support this, with most interactions having a positive, and therefore repelling, interaction energy.

Figure 5.1 Final pose of the prothrombinase complex with the 334KF335 mutation. fVa

is pictured in green, II in blue, and fXa in purple.

5.3.2 S478A, L480A, Q481A mutation in prothrombin

The difference in the interaction energies between II and the heavy chain of fVa here are significant; a change of nearly 750 kcal/mol. Practically every key interaction is reduced in this mutation. In region 1 of the wild type, a –67 kcal/mol interaction between

K320 and D495 is significantly diminished (-9 kcal/mol in the mutant), and an interaction between E323 and R498 is absent. This region contributes roughly -87 kcal/mol to the interaction energy in the wild type, but only -2.65 kcal/mol in the mutant. The conformational differences are evident in region 2, where a repulsion of roughly 18 kcal/mol in the wild type is over 110 kcal/mol in the mutant. The key difference is the increased repulsion from E414 in prothrombin, which contributes roughly 56 kcal/mol to repulsion in the wild type and over 100 kcal/mol in the mutant.

Closer to the C-terminus, the repulsive interactions still overwhelm the attractive forces. While regions 3 and 4 do not have significant changes in interaction energy versus wild type, region 5 increases the repulsion by just over 230 kcal/mol, while the same region in wild type increases the attraction slightly, roughly -30 kcal/mol. The overall contribution to the interaction of the remaining 30 residues (region 6) of the C-terminus in the mutant is negligible, while in the wild type, the interaction is strengthened by roughly

-240 kcal/mol.

The rationale behind the change in the interaction energies is not terribly clear. The alanine mutations do not seem to contribute as significantly to conformational changes as the previous mutation in any specific interaction, though the interprotein interactions did shift. While the S478A mutation had nearly zero impact on the interprotein interaction

99 energies, the L480A and Q481A mutations did, with the former going from roughly -61 kcal/mol to -52 kcal/mol, and the latter from -72 to -49 kcal/mol. These changes in the intramolecular interaction energies could lead to significant enough conformational changes to impact the interaction with the enzymatic complex overall.

5.3.3 700DE701 mutation in fVa

The interaction between the heavy chain of fVa and the substrate is very similar in this mutation as to the prothrombin mutation through the first 375 residues of fVa, with a slightly positive interaction energy, though the repulsive contribution of E414 in prothrombin are significantly diminished in this mutation. This repulsion interaction is replaced by D552, which contributes around 78 kcal/mol to the repulsion, compared to under 13 kcal/mol for E414. This is mostly due to a strong attraction with K378 of -53 kcal/mol, which while present in the previous mutant (and the wild type), is not as significant. However, the interaction energy of this mutation still is roughly 46 kcal/mol in the first 500 residues of fVa combined, while in the wild type it is roughly -84 kcal/mol.

Region 3, where interactions between fVa and both fXa and II occur, had an interaction energy around -50 kcal/mol in both the mutant and wild type.

Region 4 again has slightly lower interaction energy in the mutant than in the wild type, mostly due to a stronger repulsion by around 16 kcal/mol in the fVa-D578 to II-E249 contact. As in the previous mutant, region 5 contributed significantly to repulsion of the substrate, with an increase in repulsion by over 90 kcal/mol. The C-terminal loop, however, does improve the attractive interaction, by just over -120 kcal/mol. This region does include the two mutated residues, N700D and R701E. Residue 700 had nearly no

100 impact on the interaction with II in either the wild type or in the mutant. However, the charge inversion at position 701 resulted in the interaction changing from an attractive interaction with E345 (-62 kcal/mol) in the wild type to a repelling interaction with D371

(48 kcal/mol) in the mutant. This interaction change alone accounts for nearly all of the difference in the interaction energies between these regions.

5.3.4 695DFDF698 mutation

As with the other mutations, the attraction between the substrate and region 1 is diminished, though only slightly in this case. The overall difference is a decrease in attractive energy by roughly -30 kcal/mol, lower than in the previous mutations. Region

2, on the other hand, had a very similar interaction energy, contributing roughly -20 kcal/mol in the wild type and -24 kcal/mol in the mutant. An interesting interaction between regions 2 and 3 not present in the wild type to the same extent is a strong attraction between R400 in fVa and D495 in II (-10 kcal/mol in wild type, -50 kcal/mol in the mutant), which would contribute significantly if not for a counteracting interaction with D401 (30 kcal/mol), an interaction that is present in the wild type system (37 kcal/mol). Region 3 contributes to substrate binding more strongly in the mutant (-91 kcal/mol) than in the wild type (-50 kcal/mol) due to a significantly weaker repulsion in D504 (96 kcal/mol in wild type, 1.5 kcal/mol in mutant). Stronger interactions in R506 and R510 in the wild type system counter roughly half of the difference.

Region 4 did not have a significant contribution to attractive forces in the mutant; a change of less than -10 kcal/mol was observed, while in the wild type the energy changed by roughly -37 kcal/mol. A repulsion between D577 in fVa and E168 in II, while present

101 in wild type, was much stronger in the mutant. Small differences in other interactions comprise the remainder of the difference. On the N-terminal side of region 5, the binding energy of the mutant is actually lower than that of the wild type by roughly 36 kcal/mol.

However, E168 in II strongly repels both D660 and E662 in fVa. In the wild type, the overall contribution is 76 kcal/mol, while in the mutant the contribution to the repelling energy is 164 kcal/mol. K680 in the mutant makes an extremely strong (-90 kcal/mol) interaction with the C-terminus of II, resulting in the difference in interaction energy through region 5 to remain roughly the same.

The mutation in region 6 illuminates the importance of interprotein interactions.

Through A694, the interaction energy for the remainder of the protein is nearly 100 kcal/mol lower in the mutant than in the wild type, indicating a much stronger interaction.

However, while D695 and D697 made slightly stronger interactions with II in the mutant, the difference in the intramolecular interactions in F696 and F698 in the mutant significantly impacted the interaction with the substrate. Why the sulfotyrosines in these positions do not make large contributions to the binding energy with the substrate (though

TYS698 does contribute -35 kcal/mol to the interaction), they largely repel the surrounding residues, allowing them to interact only with the substrate. In contrast, the intramolecular interaction energy in F696 and F698 are -44 and -49 kcal/mol, respectively. The impact of this is evident in the remaining interaction with the substrate. While the N699-R709 stretch in the wild type contributes -235 kcal/mol to the interaction energy, the same region in the mutant contributes -19 kcal/mol. Figure 5.2 shows the differential in binding between the

C-terminus in the mutant versus the wild type, which detached from prothrombin roughly

40 nanoseconds into the trajectory. 102

Figure 5.2 Final pose of the 695DFDF698 mutant.

5.3.5 695DYDY698 mutation

In an effort to determine the importance of the sulfation on the tyrosines at positions

696 and 698 of fVa, a non-sulfated mutant was created. Interaction in region 1 was significantly diminished, with an overall energy from this region of roughly -21 kcal/mol.

Region 2 resulted in a repulsion of around 38 kcal/mol, with the culprit once again being

E414. R400 and D401 make no interaction with the substrate as they do in the wild type, as well as in other mutants. Region 3 only makes an attractive contribution of -24 kcal/mol, significantly lower than that of the previous mutant and the wild type. As in the previous

103 mutant, region 4 makes nearly no contribution to either attraction of repulsion of the substrate.

Within region 5, D173 and D207 in prothrombin repel fVa significantly, resulting in an overall repulsion in the interaction energy of roughly 48 kcal/mol for the region. The interaction through region 5 in the previous mutant was just over -300 kcal/mol stronger than in the non-sulfated mutant. A strong (-73 kcal/mol) interaction between R684 in region 6 and the C-terminus of II makes the overall interaction attractive from the N- terminus to this point again. Once again, the tyrosines at 696 and 698, like the phenylalanines in the previous mutant, interact with the surrounding residues, with intramolecular interaction energies of -41 and -38 kcal/mol, respectively. These intramolecular interactions were not quite as inhibitory as they were in the previous mutant, but the contribution of the N699-R709 stretch was roughly -70 kcal/mol.

5.3.6 695KFKF698 mutation

The final mutation had very similar binding to the 478-480-481 mutant above in regions

1 and 2, and therefore very similar interaction energy. Region 3 was similar to that of the

DYDY mutant, as was region 4, though the D578-E249 repulsion was especially strong in the KFKF mutant. Region 5 had a strong repulsion between both D660 and E662 with

E168, which largely counteracted any interactions contributing to attractive interactions, similar to that of the DFDF mutation. As in the DFDF mutant, K680 in fVa makes a strong interaction with the C-terminus of II, though not to quite the same magnitude as in the

DFDF mutant (-66 kcal/mol).

104

Once again, in region 6 the intramolecular interactions dominate the conformation.

While the intramolecular interactions in F696 and F698 are similar (-46 and -39 kcal/mol, respectively) the interaction energies in K695 and K697 with respect to the rest of the heavy chain of fVa are increased (-25 to -59 kcal/mol for residue 695 and -36 to -48 kcal/mol for residue 697). However, these interactions do not diminish the interaction in this region to the extent they do in the DFDF mutation, with the interaction being more similar to that of the DYDY mutation.

5.3.7 Discussion

WT 334 480 7001 DFDF DYDY KFKF

R1 -88.79 -20.31 -2.58 2.99 -50.71 -21.42 3.53

R2 -19.32 24.60 116.03 37.11 -24.18 37.65 117.92

R3 -49.46 -12.21 -37.30 -59.85 -93.02 -24.02 -21.99

R4 -37.07 -35.09 -2.36 -24.86 -8.94 5.48 16.71

R5 -30.37 82.92 233.46 94.56 -31.22 47.86 65.31

R6 -240.58 133.96 0.21 -121.97 -74.32 -203.81 -213.38

Table 5.3 Summary of the interaction energies for each region.

The interaction energies are summarized in Table 5.3. The most interesting result here is the conformational change induced in the substrate by the type of interaction it has with the C-terminal tail of fVa. Even small changes, such as removal of sulfation, resulted in significant changes in the interactions. The root-mean-squared deviation (RMSD) plot

(Figure 5.3) indicates relatively stable systems considering their size, with all mutations

105 remaining below 6 angstroms, and the only system with significant movement being the wild type.

Figure 5.3 RMSD for all systems of alpha-carbons. All three proteins were considered

for the RMSD.

The root-mean-squared fluctuation (RMSF, Figure 5.4) plot shows similar movement in all residues, with the exception of the C-terminus of fVa in the DFDF mutant. This region lost contact with the substrate, and therefore had extra fluctuation.

106

Figure 5.4 All-atom RMSF of each protein system.

There are further experiments which could be done on this system to better understand the interaction mechanisms between the enzymatic complex and the substrate prothrombin, though at the current time they may not be computationally feasible. Through use of a highly mobile membrane mimetic, it could be possible to set up a system where the C- terminus has been placed to interact with prothrombin, but where prothrombin is not in full contact with the enzymatic complex. However, there are still issues with both the timescale that might be needed (likely microseconds of simulation for the proper lateral movement), as well as the system size. The systems constructed here use a 200 angstrom long by 200 107 angstrom wide membrane patch; the authors would estimate a membrane patch of at least

300 angstroms would be required, increasing the possible system size close to 1.7 million atoms. The combination of system size and timescale required may put such a simulation out of reach currently, but with GPU scalability and ever increasing computational power, such a system could be possible within the next few years!

108

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