scholarly journals Engineering the Fab fragment of the anti-IgE omalizumab to prevent Fab crystallization and permit IgE-Fc complex crystallization

2020 ◽  
Vol 76 (3) ◽  
pp. 116-129
Author(s):  
Alkistis N. Mitropoulou ◽  
Tom Ceska ◽  
James T. Heads ◽  
Andrew J. Beavil ◽  
Alistair J. Henry ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Immunoglobulin E ◽  
Structural Basis ◽  
Cross Linking ◽  
Fab Fragment ◽  
Crystal Forms ◽  
High Affinity ◽  
Affinity Interaction ◽  
Packing Interactions

Immunoglobulin E (IgE) plays a central role in the allergic response, in which cross-linking of allergen by Fc∊RI-bound IgE triggers mast cell and basophil degranulation and the release of inflammatory mediators. The high-affinity interaction between IgE and Fc∊RI is a long-standing target for therapeutic intervention in allergic disease. Omalizumab is a clinically approved anti-IgE monoclonal antibody that binds to free IgE, also with high affinity, preventing its interaction with Fc∊RI. All attempts to crystallize the pre-formed complex between the omalizumab Fab and the Fc region of IgE (IgE-Fc), to understand the structural basis for its mechanism of action, surprisingly failed. Instead, the Fab alone selectively crystallized in different crystal forms, but their structures revealed intermolecular Fab/Fab interactions that were clearly strong enough to disrupt the Fab/IgE-Fc complexes. Some of these interactions were common to other Fab crystal structures. Mutations were therefore designed to disrupt two recurring packing interactions observed in the omalizumab Fab crystal structures without interfering with the ability of the omalizumab Fab to recognize IgE-Fc; this led to the successful crystallization and subsequent structure determination of the Fab/IgE-Fc complex. The mutagenesis strategy adopted to achieve this result is applicable to other intractable Fab/antigen complexes or systems in which Fabs are used as crystallization chaperones.

scholarly journals Structure of lipoprotein lipase in complex with GPIHBP1

2019 ◽  
Vol 116 (21) ◽  
pp. 10360-10365 ◽  
Author(s):  
Rishi Arora ◽  
Amitabh V. Nimonkar ◽  
Daniel Baird ◽  
Chunhua Wang ◽  
Chun-Hao Chiu ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Lipoprotein Lipase ◽  
Structural Information ◽  
Density Lipoprotein ◽  
Lipid Binding ◽  
Structural Basis ◽  
Maturation Factor ◽  
Lipase Maturation Factor 1 ◽  
Hydrolysis Of

Lipoprotein lipase (LPL) plays a central role in triglyceride (TG) metabolism. By catalyzing the hydrolysis of TGs present in TG-rich lipoproteins (TRLs), LPL facilitates TG utilization and regulates circulating TG and TRL concentrations. Until very recently, structural information for LPL was limited to homology models, presumably due to the propensity of LPL to unfold and aggregate. By coexpressing LPL with a soluble variant of its accessory protein glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) and with its chaperone protein lipase maturation factor 1 (LMF1), we obtained a stable and homogenous LPL/GPIHBP1 complex that was suitable for structure determination. We report here X-ray crystal structures of human LPL in complex with human GPIHBP1 at 2.5–3.0 Å resolution, including a structure with a novel inhibitor bound to LPL. Binding of the inhibitor resulted in ordering of the LPL lid and lipid-binding regions and thus enabled determination of the first crystal structure of LPL that includes these important regions of the protein. It was assumed for many years that LPL was only active as a homodimer. The structures and additional biochemical data reported here are consistent with a new report that LPL, in complex with GPIHBP1, can be active as a monomeric 1:1 complex. The crystal structures illuminate the structural basis for LPL-mediated TRL lipolysis as well as LPL stabilization and transport by GPIHBP1.

Crystal Structure of the Interaction of Human Urokinase Plasminogen Activitor with Its Receptor.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 683-683 ◽  
Author(s):  
Qing Huai ◽  
Andrew P. Mazar ◽  
Graham Parry ◽  
Alice Kuo ◽  
Douglas B. Cines ◽  
...  
Keyword(s):  
Ternary Complex ◽  
Hydrophobic Interactions ◽  
Structural Basis ◽  
Diffusion Method ◽  
Monoclinic Space Group ◽  
Fab Fragment ◽  
Data Set ◽  
Kringle Domain ◽  
Amino Terminal ◽  
High Affinity

Abstract Urokinase-type plasminogen activator (uPA) and its cellular receptor (uPAR) mediate plasminogen activation. The uPA binds to uPAR with high affinity (Kd 0.1–1nM), thus localizing the generation of plasmin from plasminogen on the surface of a variety of cells. uPA-uPAR binding is also involved in other cellular functions and diverse pathophysiological processes such as tissue remodeling during wound healing, atherosclerosis, angiogenesis and tumor metastasis. We have determined the structure of uPAR complexed with the amino terminal fragment (amino acid residues 1–143) of uPA (ATF), which contains the uPAR binding domain, at 1.9 Å resolution by X-ray crystallography. Soluble uPAR (suPAR) and the ATF were expressed separately in stably transfected Drosophila Schneider 2 (S2) cells. The suPAR-ATF complex was crystallized by the sitting-drop vapor diffusion method. However, the diffraction of this crystal was limited to 3.1 Å resolution. Therefore, the Fab fragment of an antibody raised against suPAR, ATN-615, was used to facilitate suPAR-ATF crystallization. Crystals of the suPAR-ATF-ATN615 ternary complex were generated by microdialysis with 4% PEG4K, 5% ethylene glycol, 5% methanol, 0.05% sodium azide, 50 mM cacodylate pH 6.5. A complete data set of the ternary complex to 1.9 Å was collected using synchrotron radiation at the Advanced Photon Source (APS), Argonne National Laboratory. The crystals belong to the monoclinic space group, with unit cell parameters a=51.79 Å, b=86.81 Å, c=124.69 Å and β=94.54°. uPAR is comprised of three consecutive domains (D1, D2 and D3) that form the shape of a thick-walled teacup with a diameter of about 52 Å and a height of 27 Å. At the center of teacup and surrounded by three suPAR domains is a cone shape cavity with a wide opening (25 Å) and large depth (14 Å). ATF consists of a growth factor domain (GFD) and a kringle domain. Both domains pack more tightly in the complex structure compared with their unbound state. The GFD domain of uPA occupies part of the uPAR cavity and is primarily responsible for uPAR binding. The D1 domain of uPAR forms three hydrogen bonds and many hydrophobic interactions with the GFD domain of uPA, thus playing an important role in the binding of uPA. However, D2 and D3 of uPAR also have direct interactions with the GFD domain of uPA. The kringle domain of uPA sits outside the uPAR pocket, but forms some direct contacts with the D1 domain of uPAR. Therefore, the three domains of uPAR and two domains of uPA form a complementary interaction, which describes the structural basis for the high affinity binding of uPA to uPAR. This structure presents the first high resolution view of uPA-uPAR interaction, and may provide a new platform to design de novo uPA-uPAR antagonists.

Nogo Receptor crystal structures with a native disulfide pattern suggest a novel mode of self-interaction

2017 ◽  
Vol 73 (11) ◽  
pp. 860-876 ◽  
Author(s):  
Matti F. Pronker ◽  
Roderick P. Tas ◽  
Hedwich C. Vlieg ◽  
Bert J. C. Janssen
Keyword(s):  
Crystal Structures ◽  
Disulfide Bonds ◽  
Crystal Packing ◽  
Surface Protein ◽  
Terminal Segment ◽  
Structural Basis ◽  
Crystal Forms ◽  
Nogo Receptor ◽  
Terminal Loop ◽  
Lrr Domain

The Nogo Receptor (NgR) is a glycophosphatidylinositol-anchored cell-surface protein and is a receptor for three myelin-associated inhibitors of regeneration: myelin-associated glycoprotein, Nogo66 and oligodendrocyte myelin glycoprotein. In combination with different co-receptors, NgR mediates signalling that reduces neuronal plasticity. The available structures of the NgR ligand-binding leucine-rich repeat (LRR) domain have an artificial disulfide pattern owing to truncated C-terminal construct boundaries. NgR has previously been shown to self-associateviaits LRR domain, but the structural basis of this interaction remains elusive. Here, crystal structures of the NgR LRR with a longer C-terminal segment and a native disulfide pattern are presented. An additional C-terminal loop proximal to the C-terminal LRR cap is stabilized by two newly formed disulfide bonds, but is otherwise mostly unstructured in the absence of any stabilizing interactions. NgR crystallized in six unique crystal forms, three of which share a crystal-packing interface. NgR crystal-packing interfaces from all eight unique crystal forms are compared in order to explore how NgR could self-interact on the neuronal plasma membrane.

High-Resolution Crystal Structures of Human Hemoglobin with Mutations at Tryptophan 37β:  Structural Basis for a High-Affinity T-State†,‡

Biochemistry ◽  
1998 ◽  
Vol 37 (13) ◽  
pp. 4358-4373 ◽  
Author(s):  
Jeffrey S. Kavanaugh ◽  
Jamie A. Weydert ◽  
Paul H. Rogers ◽  
Arthur Arnone
Keyword(s):  
High Resolution ◽  
Crystal Structures ◽  
Structural Basis ◽  
Human Hemoglobin ◽  
High Affinity ◽  
T State

scholarly journals The Structural Basis for BVMO Substrate Profile and Stereospecificity

2014 ◽  
Vol 70 (a1) ◽  
pp. C485-C485
Author(s):  
Brahm Yachnin ◽  
Michelle McEvoy ◽  
Roderick MacCuish ◽  
Krista Morley ◽  
Anthony Mittermaier ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Crystal Structures ◽  
Rational Design ◽  
Enzyme Mechanism ◽  
Structural Basis ◽  
Nmr Studies ◽  
X Ray Scattering ◽  
The Relationship ◽  
Villiger Oxidation

The Baeyer-Villiger monooxygenases (BVMOs) are a group of bacterial enzymes that are able to catalyze the synthetically useful Baeyer-Villiger oxidation reaction. As such, these enzymes have attracted considerable attention as potential industrial biocatalysts. The interest in these enzymes has led to a desire to be able to rationally design them for tailored biocatalytic applications. While recent years have seen the publication of a number of crystal structures (1-3), we have been lacking a structure of a BVMO that has its native substrate or product bound in a conformation that will allow the determination of substrate specificity and stereospecificity. Without such a structure, progress towards tailored BVMOs has been hampered. We have been able to solve two crystal structures of cyclohexanone monooxygenase (CHMO) with its lactone product, ε-caprolactone, bound. These structures place the lactone in an ideal position for the determination of its substrate specificity and stereospecificity. These structures have provided us with a better understanding of the structural basis for substrate binding, paving the way for the rational design of tailored BVMOs. At the same time, we have pursued small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) studies to better understand the dynamic nature of the enzyme. These studies have allowed us to explain the relationship between the various crystallized states of BVMOs and their complex, fourteen step enzyme mechanism.

Crystallization and preliminary X-ray analysis of birch-pollen allergen Bet v 1 in complex with a murine monoclonal IgG Fab′ fragment

1999 ◽  
Vol 55 (12) ◽  
pp. 2035-2036 ◽  
Author(s):  
Michael D. Spangfort ◽  
Osman Mirza ◽  
L. Anders Svensson ◽  
Jørgen N. Larsen ◽  
Michael Gajhede ◽  
...  
Keyword(s):  
Immunoglobulin E ◽  
Birch Pollen ◽  
Pollen Allergen ◽  
Structural Basis ◽  
Type I ◽  
X Rays ◽  
Fab Fragment ◽  
Cell Parameters ◽  
Bet V 1 ◽  
Birch Pollen Allergen

The human type I allergic response is characterized by the presence of allergen-specific serum immunoglobulin E (IgE). Allergen-mediated cross-linking of receptor-bound IgE on the surface of mast cells and circulating basophils triggers the release of mediators, resulting in the development of the clinical symptoms of allergy. In order to study the structural basis of allergen–antibody interaction, a complex between the major birch-pollen allergen Bet v 1 and a Fab′ fragment isolated from the murine monoclonal Bet v 1 antibody BV16 has been crystallized. Complex crystals belong to space group P1, with unit-cell parameters a = 91.65, b = 99.14, c = 108.90 Å, α = 105.7, β = 98.32, γ = 97.62°, and diffract to 2.9 Å resolution when analyzed at 100 K using synchrotron-generated X-rays.

scholarly journals Structural basis for HCMV Pentamer recognition by antibodies and neuropilin 2

2021 ◽  
Author(s):  
Daniel Wrapp ◽  
Xiaohua Ye ◽  
Zhiqiang Ku ◽  
Hang Su ◽  
Harrison G Jones ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Neutralizing Antibodies ◽  
Cell Surface Receptor ◽  
Structural Basis ◽  
Calcium Dependent ◽  
High Affinity ◽  
Molecular Determinants ◽  
Surface Receptor ◽  
Affinity Interaction

Human cytomegalovirus (HCMV) encodes for multiple surface glycoproteins and glycoprotein complexes. One of these complexes, the HCMV Pentamer (gH, gL, UL128, UL130 and UL131), mediates tropism to both epithelial and endothelial cells by interacting with the cell surface receptor neuropilin 2 (NRP2). Despite the critical nature of this interaction, the molecular determinants that govern NRP2 recognition remain unclear. Here we describe the cryo-EM structure of NRP2 bound to the HCMV Pentamer. The high-affinity interaction between these proteins is calcium-dependent and differs from the canonical C-terminal arginine (CendR) binding that NRP2 typically utilizes. The interaction is primarily mediated by NRP2 domains a2 and b2, which interact with UL128 and UL131. We also determine the structures of four human-derived neutralizing antibodies in complex with the HCMV Pentamer to define susceptible epitopes. The two most potent antibodies recognize a novel epitope yet do not compete with NRP2 binding. Collectively, these findings provide a structural basis for HCMV tropism and antibody-mediated neutralization, and serve as a guide for the development of HCMV treatments and vaccines.

scholarly journals Structural Basis for the High-Affinity Interaction between CASK and Mint1

Structure ◽  
2020 ◽  
Vol 28 (6) ◽  
pp. 664-673.e3
Author(s):  
Xiandeng Wu ◽  
Qixu Cai ◽  
Yiyun Chen ◽  
Shihan Zhu ◽  
Jing Mi ◽  
...  
Keyword(s):  
Structural Basis ◽  
High Affinity ◽  
Affinity Interaction
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