scholarly journals SAXS analysis of the intrinsic tenase complex bound to lipid nanodisc highlights intermolecular contacts between factors VIIIa/IXa

2021 ◽  
Author(s):  
Kenneth C. Childers ◽  
Shaun C. Peters ◽  
Pete Lollar ◽  
H. Trent Spencer ◽  
Christopher B. Doering ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Lipid Membrane ◽  
Coagulation Cascade ◽  
Factor X ◽  
Protein Protein Interactions ◽  
Complex Assembly ◽  
Intermolecular Contacts ◽  
Membrane Bound ◽  
Saxs Analysis ◽  
Lipid Nanodiscs

The intrinsic tenase (Xase) complex, formed by factors (f)VIIIa and fIXa, forms on activated platelet surfaces and catalyzes the activation of factor X to Xa, stimulating thrombin production in the blood coagulation cascade. The structural organization of the membrane-bound Xase complex remains largely unknown, hindering our understanding of the structural underpinnings that guide Xase complex assembly. Here, we aimed to characterize the Xase complex bound to a lipid nanodisc with biolayer interferometry (BLI) and small angle X-ray scattering (SAXS). Using immobilized lipid nanodiscs, we measured binding rates and nanomolar affinities for fVIIIa, fIXa, and the Xase complex. An ab initio molecular envelope of the nanodisc-bound Xase complex allowed us to computationally model fVIIIa and fIXa docked onto a flexible lipid membrane and identify protein-protein interactions. Our results highlight multiple points of contact between fVIIIa and fIXa, including a novel interaction with fIXa at the fVIIIa A1-A3 domain interface. Lastly, we identified hemophilia A/B-related mutations with varying severities at the fVIIIa/fIXa interface that may regulate Xase complex assembly. Together, our results support the use of SAXS as an emergent tool to investigate the membrane-bound Xase complex and illustrate how mutations at the fVIIIa/fIXa dimer interface may disrupt or stabilize the activated enzyme complex.

scholarly journals SAXS Analysis of the Intrinsic Tenase Complex Bound to Lipid Nanodisc Highlights Intermolecular Contacts between Factors VIIIa/IXa

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2099-2099
Author(s):  
Kenneth C Childers ◽  
Shaun Peters ◽  
John S. Lollar ◽  
H. Trent Spencer ◽  
Christopher B Doering ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Lipid Membrane ◽  
Computational Modelling ◽  
Factor X ◽  
Protein Protein Interactions ◽  
Clotting Factors ◽  
Publicly Traded ◽  
Complex Assembly ◽  
The Past ◽  
Membrane Bound

Abstract The intrinsic tenase (Xase) complex is formed by activated clotting factors VIII (fVIIIa) and IX (fIXa) on activated platelet surfaces and catalyzes the activation of factor X to promote blood coagulation. The structural organization of the membrane-bound Xase complex remains largely unknown, hindering a mechanistic understanding of how mutations to fVIIIa or fIXa lead to thrombotic disorders. In the present study, we aimed to structurally characterize the Xase complex bound to a lipid nanodisc using biolayer interferometry and small-angle X-ray scattering. By immobilizing lipid nanodiscs, we measured binding rates and nanomolar affinities for fVIIIa, fIXa, and the Xase complex. An ab initio molecular envelope of the nanodisc-bound Xase complex allowed us to computationally model fVIIIa and fIXa docked onto a flexible lipid membrane and identify protein-protein interactions. Our results suggest that interactions between the Gla and catalytic domains of fIXa and the C2 and A2 domains of fVIIIa, respectively, mediate Xase complex assembly. Additionally, our computational modelling highlighted a novel interaction between fIXa residue K80 and an acidic patch formed at the fVIIIa A1/A3 domain interface. Our model of the Xase complex sheds light on how hemophilia A/B-related mutations with varying severities disrupt Xase complex assembly. We also speculate on how gain-of-function mutations to fIXa residue R338, such as the Padua (R338L) and Shanghai (R338Q) variants, bind to the fVIIIa A2 domain and stabilize the Xase complex, leading to excessive blood clotting. Together, our results support the use of SAXS as an emergent tool to investigate the membrane-bound Xase complex and illustrate how mutations at the fVIIIa/fIXa dimer interface may disrupt or stabilize the activated enzyme complex. Disclosures Lollar: Expression Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months, Patents & Royalties. Spencer: Expression Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months. Doering: Expression Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months.

scholarly journals Androgen signaling uses a writer and a reader of ADP-ribosylation to regulate protein complex assembly

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Chun-Song Yang ◽  
Kasey Jividen ◽  
Teddy Kamata ◽  
Natalia Dworak ◽  
Luke Oostdyk ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Interactions ◽  
Prostate Cancer Cells ◽  
Protein Protein Interactions ◽  
Post Translational Modification ◽  
Complex Assembly ◽  
Adp Ribosylation ◽  
Signaling Axis ◽  
Regulate Protein ◽  
Protein Complex Assembly

AbstractAndrogen signaling through the androgen receptor (AR) directs gene expression in both normal and prostate cancer cells. Androgen regulates multiple aspects of the AR life cycle, including its localization and post-translational modification, but understanding how modifications are read and integrated with AR activity has been difficult. Here, we show that ADP-ribosylation regulates AR through a nuclear pathway mediated by Parp7. We show that Parp7 mono-ADP-ribosylates agonist-bound AR, and that ADP-ribosyl-cysteines within the N-terminal domain mediate recruitment of the E3 ligase Dtx3L/Parp9. Molecular recognition of ADP-ribosyl-cysteine is provided by tandem macrodomains in Parp9, and Dtx3L/Parp9 modulates expression of a subset of AR-regulated genes. Parp7, ADP-ribosylation of AR, and AR-Dtx3L/Parp9 complex assembly are inhibited by Olaparib, a compound used clinically to inhibit poly-ADP-ribosyltransferases Parp1/2. Our study reveals the components of an androgen signaling axis that uses a writer and reader of ADP-ribosylation to regulate protein-protein interactions and AR activity.

scholarly journals De novo design of a reversible phosphorylation-dependent switch for membrane targeting

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Leon Harrington ◽  
Jordan M. Fletcher ◽  
Tamara Heermann ◽  
Derek N. Woolfson ◽  
Petra Schwille
Keyword(s):  
Protein Interactions ◽  
Lipid Membrane ◽  
De Novo ◽  
Protein Localization ◽  
Protein Structures ◽  
Spatiotemporal Pattern ◽  
Membrane Targeting ◽  
Protein Protein Interactions ◽  
Reversible Phosphorylation ◽  
Potential Applications

AbstractModules that switch protein-protein interactions on and off are essential to develop synthetic biology; for example, to construct orthogonal signaling pathways, to control artificial protein structures dynamically, and for protein localization in cells or protocells. In nature, the E. coli MinCDE system couples nucleotide-dependent switching of MinD dimerization to membrane targeting to trigger spatiotemporal pattern formation. Here we present a de novo peptide-based molecular switch that toggles reversibly between monomer and dimer in response to phosphorylation and dephosphorylation. In combination with other modules, we construct fusion proteins that couple switching to lipid-membrane targeting by: (i) tethering a ‘cargo’ molecule reversibly to a permanent membrane ‘anchor’; and (ii) creating a ‘membrane-avidity switch’ that mimics the MinD system but operates by reversible phosphorylation. These minimal, de novo molecular switches have potential applications for introducing dynamic processes into designed and engineered proteins to augment functions in living cells and add functionality to protocells.

SPR Analysis of Protein-Protein Interactions Involving Cytochromes P450 and Cytochrome b5 Integrated into Lipid Membrane

2020 ◽  
Vol 14 (2) ◽  
pp. 168-173
Author(s):  
L. A. Kaluzhskiy ◽  
P. V. Ershov ◽  
K. S. Kurpedinov ◽  
D. S. Sonina ◽  
E. O. Yablokov ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Lipid Membrane ◽  
Cytochromes P450 ◽  
Cytochrome B5 ◽  
Protein Protein Interactions

scholarly journals Proximity Labeling to Map Host-Pathogen Interactions at the Membrane of a Bacteria Containing Vacuole inChlamydia trachomatisInfected Human Cells

2019 ◽  
Author(s):  
Macy G. Olson ◽  
Ray E. Widner ◽  
Lisa M. Jorgenson ◽  
Alyssa Lawrence ◽  
Dragana Lagundzin ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Host Pathogen Interactions ◽  
Inclusion Membrane ◽  
Eukaryotic Proteins ◽  
Host Pathogen ◽  
Membrane Bound ◽  
Labeling System ◽  
Chlamydial Inclusion

AbstractAs an obligate intracellular pathogenic bacterium,C. trachomatisdevelops within a membrane-bound vacuole, termed the inclusion. The inclusion membrane is modified by chlamydial inclusion membrane proteins (Incs), which act as the mediators of host-pathogen interactions. Anin vivounderstanding of Inc-Inc and Inc-eukaryotic protein interactions and how these contribute to overall host-chlamydial interactions at this unique membrane is lacking. Previous bacterial two-hybrid studies established that certain Incs have the propensity to bind other Incs while others have limited Inc-Inc interactions. We hypothesize some Incs organize the inclusion membrane whereas other Incs bind eukaryotic proteins to promote chlamydial-host interactions. To test this hypothesis, we used the ascorbate peroxidase proximity labeling system (APEX2), which labels proximal proteins with biotinin vivo, and chose to analyze Inc proteins with varying Inc-binding propensities. We inducibly expressed these Incs fused to APEX2 inChlamydia trachomatisL2, verified their localization and labeling activities by transmission electron microscopy, and used affinity purification-mass spectrometry to identify biotinylated proteins. To analyze our mass spectrometry results for statistical significance, we used Significance Analysis of INTeractome (SAINT), which demonstrated that our Inc-APEX2 constructs labeled Inc proteins as well as known and previously unreported eukaryotic proteins that localize to the inclusion. Our results broadly support two types of Inc interactions: Inc-Inc versus Inc-host. One eukaryotic protein, LRRFIP1 (LRRF1) was found in all of our Inc-APEX2 datasets, which is consistent with previously published AP-MS datasets. For the first time, we demonstrate by confocal and super-resolution microscopy that endogenous LRRF1 localizes to the chlamydial inclusion. We also used bacterial two-hybrid studies and pulldown assays to determine if LRRF1 was identified as a true interacting protein or was proximal to our Inc-APEX2 constructs. Combined, our data highlight the utility of APEX2 to capture the complexin vivoprotein-protein interactions at the chlamydial inclusion.Author summaryMany intracellular bacteria, including the obligate intracellular pathogenChlamydia trachomatis, grow within a membrane-bound “bacteria containing vacuole” (BCV) that, in most cases, prevents association with the lysosome. Secreted cytosolic effectors modulate host activity, but an understanding of the host-pathogen interactions that occur at the BCV membrane is limited by the difficulty in purifying membrane fractions from infected host cells. Here, we used the ascorbate peroxidase proximity labeling system (APEX2), which labels proximal proteins with biotinin vivo, to study the interactions that occur at the chlamydial vacuolar, or inclusion, membrane. The inclusion membrane is modified by chlamydial type III secreted inclusion membrane proteins (Incs), which act as the mediators of host-pathogen interactions. Our results broadly support two types of Inc interactions: Inc-Inc versus Inc-host. Our data highlight the utility of APEX2 to capture the complex protein-protein interactions at a membrane sitein vivoin the context of infection.

scholarly journals Surface-dependent reactions of the vitamin K-dependent enzyme complexes

Blood ◽  
1990 ◽  
Vol 76 (1) ◽  
pp. 1-16 ◽  
Author(s):  
KG Mann ◽  
ME Nesheim ◽  
WR Church ◽  
P Haley ◽  
S Krishnaswamy
Keyword(s):  
Tissue Factor ◽  
Vitamin K ◽  
Blood Clotting ◽  
Catalytic Efficiency ◽  
Coagulation Cascade ◽  
Factor Xii ◽  
Factor X ◽  
Membrane Bound ◽  
Factor X Activation ◽  
Enzyme Complexes

Abstract During the past 20 years contributions from many laboratories have led to the development of isolation procedures, delineation of primary structures, and more recently, to the expression of recombinant proteins associated with the coagulation cascade. In general, studies of coagulation proteins under defined conditions have demonstrated the prescience of Davie and Ratnoff and MacFarlane in their proposals of the coagulation cascade. The more recent discovery of thrombomodulin by Esmon et al has led to the identification and characterization of components of the vitamin K-dependent anticoagulant pathway. In this review we have attempted to analyze and compare the functional properties of each of the vitamin K-dependent enzyme complexes associated with the procoagulant and anticoagulant phases of blood clotting. Although dissimilarities exist, the vitamin K-dependent complexes have analogous requirements and appear to function with a common general mode of organization. Membrane-bound cofactors serve as anchoring sites for the appropriate membrane-binding enzymes. This process localizes the complex on the membrane surface and increases the catalytic efficiency for substrate utilization. Complex formation provides extraordinary improvements in the catalytic efficiency for the complexes as compared with their soluble enzyme components. Membrane- bound complexes provide a mechanism that can be regulated at a site by membrane presentation, zymogen activation, and cofactor activation or presentation. The kinetic constants obtained for the various coagulation reactions determined in vitro provide some insights into how these pathways may function in vivo. The catalytic efficiency (kcat/Km) for factor X activation by factor VIIIa/factor IXa is far in excess of the catalytic efficiency of activation of factor X by tissue factor/factor VIIa (Table 3). This may provide a rational interpretation for the observation that patients with hemophilia A and B bleed even though they appear to have an alternative pathway to factor X activation. In addition, tissue factor is not ordinarily presented by the vascular tissue that has direct access to blood. However, it appears that extravascular constitutive tissue factor is available once the blood vessel becomes disrupted. The efforts to identify the initiating reactions of the blood coagulation process have not been unambiguously successful. We conclude that factor VII is most likely a zymogen, just as are the other proenzymes of the blood clotting process. In addition, it is difficult to rationalize the importance of the intrinsic pathway of coagulation involving factor XII, prekallikrein, and high molecular weight kininogen since the congenital absence of any one of these factors does not result in abnormal bleeding.

scholarly journals Membrane Binding Promotes Annexin A2 Oligomerization

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1169 ◽  
Author(s):  
Anna Lívia Linard Matos ◽  
Sergej Kudruk ◽  
Johanna Moratz ◽  
Milena Heflik ◽  
David Grill ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Biological Membrane ◽  
Membrane Binding ◽  
Annexin A2 ◽  
Protein Protein Interactions ◽  
Mutant Proteins ◽  
Lipid Domain ◽  
Protein Protein Interaction ◽  
Membrane Bound ◽  
Charged Membranes

Annexin A2 (AnxA2) is a cytosolic Ca2+ regulated membrane binding protein that can induce lipid domain formation and plays a role in exocytosis and endocytosis. To better understand the mode of annexin-membrane interaction, we analyzed membrane-bound AnxA2 assemblies by employing a novel 3-armed chemical crosslinker and specific AnxA2 mutant proteins. Our data show that AnxA2 forms crosslinkable oligomers upon binding to membranes containing negatively charged phospholipids. AnxA2 mutants with amino acid substitutions in residues predicted to be involved in lateral protein–protein interaction show compromised oligomer formation, albeit still being capable of binding to negatively charged membranes in the presence of Ca2+. These results suggest that lateral protein–protein interactions are involved in the formation of AnxA2 clusters on a biological membrane.

scholarly journals Kinetics of Factor X activation by the membrane-bound complex of Factor IXa and Factor VIIIa

2004 ◽  
Vol 381 (3) ◽  
pp. 779-794 ◽  
Author(s):  
Mikhail A. PANTELEEV ◽  
Evgueni L. SAENKO ◽  
Natalya M. ANANYEVA ◽  
Fazoil I. ATAULLAKHANOV
Keyword(s):  
Experimental Studies ◽  
Coagulation Cascade ◽  
Factor X ◽  
Complex Assembly ◽  
High Affinity ◽  
Factor Ixa ◽  
Activated Platelets ◽  
Factor Viiia ◽  
High Affinity Site ◽  
Factor X Activation

Intrinsic tenase consists of activated Factors IX (IXa) and VIII (VIIIa) assembled on a negatively charged phospholipid surface. In vivo, this surface is mainly provided by activated platelets. In vitro, phosphatidylcholine/phosphatidylserine vesicles are often used to mimic natural pro-coagulant membranes. In the present study, we developed a quantitative mathematical model of Factor X activation by intrinsic tenase. We considered two situations, when complex assembly occurs on either the membrane of phospholipid vesicles or the surface of activated platelets. On the basis of existing experimental evidence, the following mechanism for the complex assembly on activated platelets was suggested: (i) Factors IXa, VIIIa and X bind to their specific platelet receptors; (ii) bound factors form complexes on the membrane: platelet-bound Factor VIIIa provides a high-affinity site for Factor X and platelet-bound Factor IXa provides a high-affinity site for Factor VIIIa; (iii) the enzyme–cofactor–substrate complex is assembled. This mechanism allowed the explanation of co-operative effects in the binding of Factors IXa, VIIIa and X to platelets. The model was reduced to obtain a single equation for the Factor X activation rate as a function of concentrations of Factors IXa, VIIIa, X and phospholipids (or platelets). The equation had a Michaelis–Menten form, where apparent Vmax and Km were functions of the factors’ concentrations and the internal kinetic constants of the system. The equation obtained can be used in both experimental studies of intrinsic tenase and mathematical modelling of the coagulation cascade. The approach of the present study can be applied to research of other membrane-dependent enzymic reactions.

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