Factor XI Homodimer Formation Is Mediated by Electrostatic (Lys331 with Glu287) and Hydrophobic (Ile290 with Leu284 and Tyr329 with Tyr329) Interactions in the Apple 4 Domain and Is Essential for Normal Proteolytic Activation of Factor XI by Factor XIIa, Thrombin and Factor XIa.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2706-2706
Author(s):  
Wenman Wu ◽  
Dipali Sinha ◽  
James D. Lear ◽  
Paul C. Billings ◽  
Peter N. Walsh
Keyword(s):  
Disulfide Bond ◽  
Analytical Ultracentrifugation ◽  
Hydrophobic Interactions ◽  
The Other ◽  
Size Exclusion ◽  
Hek293 Cells ◽  
Salt Bridges ◽  
Factor Xi ◽  
Proteolytic Activation ◽  
Interchain Disulfide Bond

Abstract Factor XI (FXI), a coagulation protein essential for normal hemostasis, is a homodimer consisting of two identical subunits of 80 KDa linked by a disulfide bond formed by Cys321 within the Apple 4 (A4) domain of each subunit. Prekallikrein (PK), in spite of its high homology with FXI both in amino acid sequence and domain structure, is a monomer. Cys321 in PK forms an intrachain disulfide bond with Cys326. However FXI/C321S (in which interchain disulfide bond formation is precluded) is a noncovalent dimer. Thus, there are interacting residues between the two subunits of FXI that are responsible for mediating its unique homodimeric structure. Examination of the crystal structure of FXI (Papagrigoriou E, McEwan P, Walsh PN, Emsley J. Nature Structural & Molecular Biology. 2006;13:557–8) shows salt bridges between Lys331 of one subunit with Glu287 of the other subunit as well as hydrophobic interactions at the interface of the A4 domains involving Ile290, Leu284 and Tyr329. FXI/C321S, FXI/C321S/K331A, FXI/C321S/E287A, FXI/C321S/I290A, FXI/C321S/Y329A, FXI/C321S/L284A and FXI/C321S/K331R were expressed in HEK293 cells and characterized using size exclusion chromatography (SEC), analytical ultracentrifugation (AUC), and functional assays. Whereas FXI/C321S existed in a monomer/dimer equilibrium (Kd ∼40 nM) all other mutants were predominantly monomers by SEC with impaired dimer formation by AUC (Kd 3.4–38 μM). All the monomeric mutants when converted to the active enzyme, FXIa, were able to hydrolyze the small chromogenic substrate S-2366 with normal values of Km and Vmax and cleaved the macromolecular substrate FIX at both its scissile bonds at rates similar to those observed with wtFXIa strongly suggesting that all mutant proteins were properly folded. However all the monomeric mutants displayed impaired clotting activity in an APTT assay and displayed markedly decreased rates of activation by FXIIa or thrombin and autoactivation in the presence or absence of dextran sulfate. We conclude that salt bridges formed between Lys331 of one subunit and Glu287 of the other together with hydrophobic interactions of residues Ile290 with Leu284 and Tyr329 with Tyr329 and are essential for normal homodimer formation, which is essential for normal proteolytic activation of FXI by FXIIa, thrombin and FXIa either in solution or on an anionic surface.

scholarly journals Biochemical and biophysical characterization of purified native CD20 alone and in complex with rituximab and obinutuzumab

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Morgane Agez ◽  
Elodie Desuzinges Mandon ◽  
Thomas Iwema ◽  
Reto Gianotti ◽  
Florian Limani ◽  
...  
Keyword(s):  
B Cell ◽  
B Lymphocyte ◽  
Analytical Ultracentrifugation ◽  
B Cell Lymphoma ◽  
Integral Membrane Protein ◽  
Size Exclusion ◽  
Hek293 Cells ◽  
Extraction And Purification ◽  
Exclusion Chromatography ◽  
Specificity Of Binding

Abstract CD20 is a B-lymphocyte specific integral membrane protein, an activated-glycosylated phosphoprotein expressed on the surface of B-cells and a clinically validated target of monoclonal antibodies such as rituximab, ocrelizumab, ofatumumab and obinutuzumab in the treatment of all B cell lymphomas and leukemias as well as autoimmune diseases. Here, we report the extraction and purification of native CD20 from SUDHL4 and RAMOS cell lines. To improve the protein yield, we applied a calixarene-based detergent approach to solubilize, stabilize and purify native CD20 from HEK293 cells. Size Exclusion Chromatography (SEC) and Analytical Ultracentrifugation show that purified CD20 was non-aggregated and that CD20 oligomerization is concentration dependent. Negative stain electron microscopy and atomic force microscopy revealed homogenous populations of CD20. However, no defined structure could be observed. Interestingly, micellar solubilized and purified CD20 particles adopt uniformly confined nanodroplets which do not fuse and aggregate. Finally, purified CD20 could bind to rituximab and obinutuzumab as demonstrated by SEC, and Surface Plasmon Resonance (SPR). Specificity of binding was confirmed using CD20 antibody mutants to human B-cell lymphoma cells. The strategy described in this work will help investigate CD20 binding with newly developed antibodies and eventually help to optimize them. This approach may also be applicable to other challenging membrane proteins.

scholarly journals A comprehensive analysis of novel disulfide bond introduction site into the constant domain of human Fab

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hitomi Nakamura ◽  
Moeka Yoshikawa ◽  
Naoko Oda-Ueda ◽  
Tadashi Ueda ◽  
Takatoshi Ohkuri
Keyword(s):  
Thermal Stability ◽  
Pichia Pastoris ◽  
Protein Stability ◽  
Disulfide Bond ◽  
Binding Activity ◽  
Comprehensive Analysis ◽  
The Other ◽  
Constant Domain ◽  
Intermolecular Disulfide Bond ◽  
Sds Page

AbstractGenerally, intermolecular disulfide bond contribute to the conformational protein stability. To identify sites where intermolecular disulfide bond can be introduced into the Fab’s constant domain of the therapeutic IgG, Fab mutants were predicted using the MOE software, a molecular simulator, and expressed in Pichia pastoris. SDS-PAGE analysis of the prepared Fab mutants from P. pastoris indicated that among the nine analyzed Fab mutants, the F130C(H):Q124C(L), F174C(H):S176C(L), V177C(H):Q160C(L), F174C(H):S162C(L), F130C(H):S121C(L), and A145C(H):F116C(L) mutants mostly formed intermolecular disulfide bond. All these mutants showed increased thermal stability compared to that of Fab without intermolecular disulfide bond. In the other mutants, the intermolecular disulfide bond could not be completely formed, and the L132C(H):F118C(L) mutant showed only a slight decrease in binding activity and β-helix content, owing to the exertion of adverse intermolecular disulfide bond effects. Thus, our comprehensive analysis reveals that the introduction of intermolecular disulfide bond in the Fab’s constant domain is possible at various locations. These findings provide important insights for accomplishing human Fab stabilization.

scholarly journals P granules extend the nuclear pore complex environment in the C. elegans germ line

2011 ◽  
Vol 192 (6) ◽  
pp. 939-948 ◽  
Author(s):  
Dustin L. Updike ◽  
Stephanie J. Hachey ◽  
Jeremy Kreher ◽  
Susan Strome
Keyword(s):  
Nuclear Pore Complex ◽  
Germ Plasm ◽  
Germ Line ◽  
Hydrophobic Interactions ◽  
Nuclear Periphery ◽  
Size Exclusion ◽  
Nuclear Pore ◽  
Complex Environment ◽  
P Granules ◽  
C Elegans

The immortal and totipotent properties of the germ line depend on determinants within the germ plasm. A common characteristic of germ plasm across phyla is the presence of germ granules, including P granules in Caenorhabditis elegans, which are typically associated with the nuclear periphery. In C. elegans, nuclear pore complex (NPC)–like FG repeat domains are found in the VASA-related P-granule proteins GLH-1, GLH-2, and GLH-4 and other P-granule components. We demonstrate that P granules, like NPCs, are held together by weak hydrophobic interactions and establish a size-exclusion barrier. Our analysis of intestine-expressed proteins revealed that GLH-1 and its FG domain are not sufficient to form granules, but require factors like PGL-1 to nucleate the localized concentration of GLH proteins. GLH-1 is necessary but not sufficient for the perinuclear location of granules in the intestine. Our results suggest that P granules extend the NPC environment in the germ line and provide insights into the roles of the PGL and GLH family proteins.

scholarly journals Effects of an Interchain Disulfide Bond on Tropomyosin Structure: A Molecular Dynamics Study

2018 ◽  
Vol 19 (11) ◽  
pp. 3376 ◽  
Author(s):  
Natalia A. Koubassova ◽  
Sergey Y. Bershitsky ◽  
Andrey K. Tsaturyan
Keyword(s):  
Heart Failure ◽  
Molecular Dynamics ◽  
Disulfide Bond ◽  
Coiled Coil ◽  
Middle Part ◽  
Actin Binding ◽  
Cross Linking ◽  
Interchain Disulfide Bond ◽  
Structural And Functional Properties ◽  
The Cross

Tropomyosin (Tpm) is a coiled-coil actin-binding dimer protein that participates in the regulation of muscle contraction. Both Tpm chains contain Cys190 residues which are normally in the reduced state, but form an interchain disulfide bond in failing heart. Changes in structural and functional properties of Tpm and its complexes with actin upon disulfide cross-linking were studied using various experimental methods. To understand the molecular mechanism underlying these changes and to reveal the possible mechanism of the involvement of the cross-linking in heart failure, molecular dynamics (MD) simulations of the middle part of Tpm were performed in cross-linked and reduced states. The cross-linking increased bending stiffness of Tpm assessed from MD trajectories at 27 °C in agreement with previous experimental observations. However, at 40 °C, the cross-linking caused a decrease in Tpm stiffness and a significant reduction in the number of main chain hydrogen bonds in the vicinity of residues 133 and 134. These data are in line with observations showing enhanced thermal unfolding of the least stable part of Tpm at 30–40 °C and accelerated trypsin cleavage at residue 133 at 40 °C (but not at 27 °C) upon cross-linking. These results allow us to speculate about the possible mechanism of involvement of Tpm cross-linking to heart failure pathogenesis.

scholarly journals A Dimerization Site at SCR-17/18 in Factor H Clarifies a New Mechanism for Complement Regulatory Control

2021 ◽  
Vol 11 ◽  
Author(s):  
Orla M. Dunne ◽  
Xin Gao ◽  
Ruodan Nan ◽  
Jayesh Gor ◽  
Penelope J. Adamson ◽  
...  
Keyword(s):  
Host Cell ◽  
Analytical Ultracentrifugation ◽  
Dissociation Constants ◽  
Alternative Pathway ◽  
Factor H ◽  
Regulatory Control ◽  
Size Exclusion ◽  
Complement Factor ◽  
Dimer Formation ◽  
Self Association

Complement Factor H (CFH), with 20 short complement regulator (SCR) domains, regulates the alternative pathway of complement in part through the interaction of its C-terminal SCR-19 and SCR-20 domains with host cell-bound C3b and anionic oligosaccharides. In solution, CFH forms small amounts of oligomers, with one of its self-association sites being in the SCR-16/20 domains. In order to correlate CFH function with dimer formation and the occurrence of rare disease-associated variants in SCR-16/20, we identified the dimerization site in SCR-16/20. For this, we expressed, in Pichia pastoris, the five domains in SCR-16/20 and six fragments of this with one-three domains (SCR-19/20, SCR-18/20, SCR-17/18, SCR-16/18, SCR-17 and SCR-18). Size-exclusion chromatography suggested that SCR dimer formation occurred in several fragments. Dimer formation was clarified using analytical ultracentrifugation, where quantitative c(s) size distribution analyses showed that SCR-19/20 was monomeric, SCR-18/20 was slightly dimeric, SCR-16/20, SCR-16/18 and SCR-18 showed more dimer formation, and SCR-17 and SCR-17/18 were primarily dimeric with dissociation constants of ~5 µM. The combination of these results located the SCR-16/20 dimerization site at SCR-17 and SCR-18. X-ray solution scattering experiments and molecular modelling fits confirmed the dimer site to be at SCR-17/18, this dimer being a side-by-side association of the two domains. We propose that the self-association of CFH at SCR-17/18 enables higher concentrations of CFH to be achieved when SCR-19/20 are bound to host cell surfaces in order to protect these better during inflammation. Dimer formation at SCR-17/18 clarified the association of genetic variants throughout SCR-16/20 with renal disease.

scholarly journals A Relaxed Specificity in Interchain Disulfide Bond Formation Characterizes the Assembly of a Low-Molecular-Weight Glutenin Subunit in the Endoplasmic Reticulum

2008 ◽  
Vol 149 (1) ◽  
pp. 412-423 ◽  
Author(s):  
Alessio Lombardi ◽  
Alessandra Barbante ◽  
Pietro Della Cristina ◽  
Daniele Rosiello ◽  
Chiara Lara Castellazzi ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Endoplasmic Reticulum ◽  
Disulfide Bond ◽  
Bond Formation ◽  
Glutenin Subunit ◽  
Low Molecular Weight ◽  
Disulfide Bond Formation ◽  
Interchain Disulfide Bond

Structure of β-casein

1979 ◽  
Vol 46 (2) ◽  
pp. 281-282 ◽  
Author(s):  
Shunrokuro Arima ◽  
Ryoya Niki ◽  
Kenji Takase
Keyword(s):  
Electron Microscopy ◽  
Phosphate Buffer ◽  
Analytical Ultracentrifugation ◽  
Hydrophobic Interactions ◽  
Gel Filtration ◽  
Random Coil ◽  
Free Energies ◽  
Gel Filtration Chromatography ◽  
Increasing Temperature

SUMMARYThe temperature and concentration dependent association of β-casein was studied by means of viscometry, gel filtration chromatography, electron microscopy, analytical ultracentrifugation and UV difference spectrophotometry. Degrees of polymerization of 12, 22 and 49 and free energies of association of –21, –23 and – 25 kJ/mole monomer were found at temperatures of 10, 15 and 20 °C respectively in 0·2 M Na phosphate buffer pH 6·7.Monomeric β-casein was not a completely random coil but became more compact with increasing temperature, due to hydrophobic interactions.

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