Publications by authors named "Iga Kucharska"

12 Publications

  • Page 1 of 1

Structural details of monoclonal antibody m971 recognition of the membrane-proximal domain of CD22.

J Biol Chem 2021 Aug 14;297(2):100966. Epub 2021 Jul 14.

Program in Molecular Medicine, The Hospital for Sick Children Research Institute, Toronto, Ontario, Canada; Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada; Department of Immunology, University of Toronto, Toronto, Ontario, Canada. Electronic address:

Cluster of differentiation-22 (CD22) belongs to the sialic acid-binding immunoglobulin (Ig)-like lectin family of receptors that is expressed on the surface of B cells. It has been classified as an inhibitory coreceptor for the B-cell receptor because of its function in establishing a baseline level of B-cell inhibition. The restricted expression of CD22 on B cells and its inhibitory function make it an attractive target for B-cell depletion in cases of B-cell malignancies. Genetically modified T cells with chimeric antigen receptors (CARs) derived from the m971 antibody have shown promise when used as an immunotherapeutic agent against B-cell acute lymphoblastic leukemia. A key aspect of the efficacy of this CAR-T was its ability to target a membrane-proximal epitope on the CD22 extracellular domain; however, the molecular details of m971 recognition of CD22 have thus far remained elusive. Here, we report the crystal structure of the m971 fragment antigen-binding in complex with the two most membrane-proximal Ig-like domains of CD22 (CD22). The m971 epitope on CD22 resides at the most proximal Ig domain (d7) to the membrane, and the antibody paratope contains electrostatic surfaces compatible with interactions with phospholipid head groups. Together, our data identify molecular details underlying the successful transformation of an antibody epitope on CD22 into an effective CAR immunotherapeutic target.
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http://dx.doi.org/10.1016/j.jbc.2021.100966DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8353475PMC
August 2021

Multivalency transforms SARS-CoV-2 antibodies into ultrapotent neutralizers.

Nat Commun 2021 06 16;12(1):3661. Epub 2021 Jun 16.

Program in Molecular Medicine, The Hospital for Sick Children Research Institute, Toronto, ON, Canada.

SARS-CoV-2, the virus responsible for COVID-19, has caused a global pandemic. Antibodies can be powerful biotherapeutics to fight viral infections. Here, we use the human apoferritin protomer as a modular subunit to drive oligomerization of antibody fragments and transform antibodies targeting SARS-CoV-2 into exceptionally potent neutralizers. Using this platform, half-maximal inhibitory concentration (IC) values as low as 9 × 10 M are achieved as a result of up to 10,000-fold potency enhancements compared to corresponding IgGs. Combination of three different antibody specificities and the fragment crystallizable (Fc) domain on a single multivalent molecule conferred the ability to overcome viral sequence variability together with outstanding potency and IgG-like bioavailability. The MULTi-specific, multi-Affinity antiBODY (Multabody or MB) platform thus uniquely leverages binding avidity together with multi-specificity to deliver ultrapotent and broad neutralizers against SARS-CoV-2. The modularity of the platform also makes it relevant for rapid evaluation against other infectious diseases of global health importance. Neutralizing antibodies are a promising therapeutic for SARS-CoV-2.
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http://dx.doi.org/10.1038/s41467-021-23825-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8209050PMC
June 2021

Structural ordering of the circumsporozoite protein repeats by inhibitory antibody 3D11.

Elife 2020 11 30;9. Epub 2020 Nov 30.

Program in Molecular Medicine, The Hospital for Sick Children Research Institute, Toronto, Canada.

Plasmodium sporozoites express circumsporozoite protein (CSP) on their surface, an essential protein that contains central repeating motifs. Antibodies targeting this region can neutralize infection, and the partial efficacy of RTS,S/AS01 - the leading malaria vaccine against (Pf) - has been associated with the humoral response against the repeats. Although structural details of antibody recognition of PfCSP have recently emerged, the molecular basis of antibody-mediated inhibition of other Plasmodium species via CSP binding remains unclear. Here, we analyze the structure and molecular interactions of potent monoclonal antibody (mAb) 3D11 binding to CSP (PbCSP) using molecular dynamics simulations, X-ray crystallography, and cryoEM. We reveal that mAb 3D11 can accommodate all subtle variances of the PbCSP repeating motifs, and, upon binding, induces structural ordering of PbCSP through homotypic interactions. Together, our findings uncover common mechanisms of antibody evolution in mammals against the CSP repeats of Plasmodium sporozoites.
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http://dx.doi.org/10.7554/eLife.59018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7704109PMC
November 2020

Recognition of Semaphorin Proteins by P. sordellii Lethal Toxin Reveals Principles of Receptor Specificity in Clostridial Toxins.

Cell 2020 07 25;182(2):345-356.e16. Epub 2020 Jun 25.

Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON M5S 3E1, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada; Molecular Architecture of Life Program, Canadian Institute for Advanced Research (CIFAR), Toronto, ON M5G 1M1, Canada. Electronic address:

Pathogenic clostridial species secrete potent toxins that induce severe host tissue damage. Paeniclostridium sordellii lethal toxin (TcsL) causes an almost invariably lethal toxic shock syndrome associated with gynecological infections. TcsL is 87% similar to C. difficile TcdB, which enters host cells via Frizzled receptors in colon epithelium. However, P. sordellii infections target vascular endothelium, suggesting that TcsL exploits another receptor. Here, using CRISPR/Cas9 screening, we establish semaphorins SEMA6A and SEMA6B as TcsL receptors. We demonstrate that recombinant SEMA6A can protect mice from TcsL-induced edema. A 3.3 Å cryo-EM structure shows that TcsL binds SEMA6A with the same region that in TcdB binds structurally unrelated Frizzled. Remarkably, 15 mutations in this evolutionarily divergent surface are sufficient to switch binding specificity of TcsL to that of TcdB. Our findings establish semaphorins as physiologically relevant receptors for TcsL and reveal the molecular basis for the difference in tissue targeting and disease pathogenesis between highly related toxins.
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http://dx.doi.org/10.1016/j.cell.2020.06.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7316060PMC
July 2020

De novo protein design enables the precise induction of RSV-neutralizing antibodies.

Science 2020 05;368(6492)

Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.

De novo protein design has been successful in expanding the natural protein repertoire. However, most de novo proteins lack biological function, presenting a major methodological challenge. In vaccinology, the induction of precise antibody responses remains a cornerstone for next-generation vaccines. Here, we present a protein design algorithm called TopoBuilder, with which we engineered epitope-focused immunogens displaying complex structural motifs. In both mice and nonhuman primates, cocktails of three de novo-designed immunogens induced robust neutralizing responses against the respiratory syncytial virus. Furthermore, the immunogens refocused preexisting antibody responses toward defined neutralization epitopes. Overall, our design approach opens the possibility of targeting specific epitopes for the development of vaccines and therapeutic antibodies and, more generally, will be applicable to the design of de novo proteins displaying complex functional motifs.
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http://dx.doi.org/10.1126/science.aay5051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7391827PMC
May 2020

Biochemical Reconstitution of HIV-1 Assembly and Maturation.

J Virol 2020 02 14;94(5). Epub 2020 Feb 14.

Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia, USA

The assembly of an orthoretrovirus such as HIV-1 requires the coordinated functioning of multiple biochemical activities of the viral Gag protein. These activities include membrane targeting, lattice formation, packaging of the RNA genome, and recruitment of cellular cofactors that modulate assembly. In most previous studies, these Gag activities have been investigated individually, which provided somewhat limited insight into how they functionally integrate during the assembly process. Here, we report the development of a biochemical reconstitution system that allowed us to investigate how Gag lattice formation, RNA binding, and the assembly cofactor inositol hexakisphosphate (IP6) synergize to generate immature virus particles The results identify an important rate-limiting step in assembly and reveal new insights into how RNA and IP6 promote immature Gag lattice formation. The immature virus-like particles can be converted into mature capsid-like particles by the simple addition of viral protease, suggesting that it is possible in principle to fully biochemically reconstitute the sequential processes of HIV-1 assembly and maturation from purified components. Assembly and maturation are essential steps in the replication of orthoretroviruses such as HIV-1 and are proven therapeutic targets. These processes require the coordinated functioning of the viral Gag protein's multiple biochemical activities. We describe here the development of an experimental system that allows an integrative analysis of how Gag's multiple functionalities cooperate to generate a retrovirus particle. Our current studies help to illuminate how Gag synergizes the formation of the virus compartment with RNA binding and how these activities are modulated by the small molecule IP6. Further development and use of this system should lead to a more comprehensive understanding of the molecular mechanisms of HIV-1 assembly and maturation and may provide new insights for the development of antiretroviral drugs.
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http://dx.doi.org/10.1128/JVI.01844-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7022372PMC
February 2020

Solution NMR Provides New Insight into Lipid-Protein Interaction.

Biochemistry 2017 08 1;56(33):4291-4292. Epub 2017 Jun 1.

Center for Membrane and Cell Physiology and Department of Molecular Physiology and Biological Physics, University of Virginia , Charlottesville, Virginia 22903, United States.

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http://dx.doi.org/10.1021/acs.biochem.7b00336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5568481PMC
August 2017

Refinement of OprH-LPS Interactions by Molecular Simulations.

Biophys J 2017 Jan;112(2):346-355

Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania; Bioengineering Program, Lehigh University, Bethlehem, Pennsylvania. Electronic address:

The outer membrane (OM) of Gram-negative bacteria is composed of lipopolysaccharide (LPS) in the outer leaflet and phospholipids in the inner leaflet. The outer membrane protein H (OprH) of Pseudomonas aeruginosa provides an increased stability to the OMs by directly interacting with LPS. Here we report the influence of various P. aeruginosa and, for comparison, Escherichia coli LPS environments on the physical properties of the OMs and OprH using all-atom molecular dynamics simulations. The simulations reveal that although the P. aeruginosa OMs are thinner hydrophobic bilayers than the E. coli OMs, which is expected from the difference in the acyl chain length of their lipid A, this effect is almost imperceptible around OprH due to a dynamically adjusted hydrophobic match between OprH and the OM. The structure and dynamics of the extracellular loops of OprH show distinct behaviors in different LPS environments. Including the O-antigen greatly reduces the flexibility of the OprH loops and increases the interactions between these loops and LPS. Furthermore, our study shows that the interactions between OprH and LPS mainly depend on the secondary structure of OprH and the chemical structure of LPS, resulting in distinctive patterns in different LPS environments.
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http://dx.doi.org/10.1016/j.bpj.2016.12.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5266143PMC
January 2017

Molecular Interactions of Lipopolysaccharide with an Outer Membrane Protein from Pseudomonas aeruginosa Probed by Solution NMR.

Biochemistry 2016 09 31;55(36):5061-72. Epub 2016 Aug 31.

Center for Membrane and Cell Physiology and Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine , Charlottesville, Virginia 22908, United States.

Pseudomonas aeruginosa is an opportunistic human pathogen causing pneumonias that are particularly severe in cystic fibrosis and immunocompromised patients. The outer membrane (OM) of P. aeruginosa is much less permeable to nutrients and other chemical compounds than that of Escherichia coli. The low permeability of the OM, which also contributes to Pseudomonas' significant antibiotic resistance, is augmented by the presence of the outer membrane protein H (OprH). OprH directly interacts with lipopolysaccharides (LPS) that constitute the outer leaflet of the OM and thus contributes to the structural stability of the OM. In this study, we used solution NMR spectroscopy to characterize the interactions between LPS and OprH in molecular detail. NMR chemical shift perturbations observed upon the addition of LPS to OprH in DHPC micelles indicate that this interaction is predominantly electrostatic and localized to the extracellular loops 2 and 3 and a number of highly conserved basic residues near the extracellular barrel rim of OprH. Single-site mutations of these residues were not enough to completely abolish binding, but OprH with cumulative mutations of Lys70, Arg72, and Lys103 no longer binds LPS. The dissociation constant (∼200 μM) measured by NMR is sufficient to efficiently bind LPS to OprH in the OM. This work highlights that solution NMR is suitable to study specific interactions of lipids with integral membrane proteins and provides a detailed molecular model for the interaction of LPS with OprH; i.e., an interaction that contributes to the integrity of the OM of P. aeruginosa under low divalent cation and antibiotic stress conditions. These methods should thus be useful for screening antibiotics that might disrupt OprH-LPS interactions and thereby increase the permeability of the OM of P. aeruginosa.
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http://dx.doi.org/10.1021/acs.biochem.6b00630DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5441840PMC
September 2016

Protein rethreading: A novel approach to protein design.

Sci Rep 2016 05 27;6:26847. Epub 2016 May 27.

Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, Virginia, 22903, United States.

Protein engineering is an important tool for the design of proteins with novel and desirable features. Templates from the protein databank (PDB) are often used as initial models that can be modified to introduce new properties. We examine whether it is possible to reconnect a protein in a manner that generates a new topology yet preserves its structural integrity. Here, we describe the rethreading of dihydrofolate reductase (DHFR) from E. coli (wtDHFR). The rethreading process involved the removal of three native loops, and the introduction of three new loops with alternate connections. The structure of the rethreaded DHFR (rDHFR-1) was determined to 1.6 Å, demonstrating the success of the rethreading process. Both wtDHFR and rDHFR-1 exhibited similar affinities towards methotrexate. However, rDHFR-1 showed no reducing activity towards dihydrofolate, and exhibited about ~6-fold lower affinity towards NADPH than wtDHFR. This work demonstrates that protein rethreading can be a powerful tool for the design of a large array of proteins with novel structures and topologies, and that by careful rearrangement of a protein sequence, the sequence to structure relationship can be expanded substantially.
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http://dx.doi.org/10.1038/srep26847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4882587PMC
May 2016

OprG Harnesses the Dynamics of its Extracellular Loops to Transport Small Amino Acids across the Outer Membrane of Pseudomonas aeruginosa.

Structure 2015 Dec 19;23(12):2234-2245. Epub 2015 Nov 19.

Department of Molecular Physiology and Biological Physics, Center for Membrane Biology, University of Virginia, Charlottesville, VA 22908, USA. Electronic address:

OprG is an outer membrane protein of Pseudomonas aeruginosa whose function as an antibiotic-sensitive porin has been controversial and not well defined. Circumstantial evidence led to the proposal that OprG might transport hydrophobic compounds by using a lateral gate in the barrel wall thought to be lined by three conserved prolines. To test this hypothesis and to find the physiological substrates of OprG, we reconstituted the purified protein into liposomes and found it to facilitate the transport of small amino acids such as glycine, alanine, valine, and serine, which was confirmed by Pseudomonas growth assays. The structures of wild-type and a critical proline mutant were determined by nuclear magnetic resonance in dihexanoyl-phosphatidylcholine micellar solutions. Both proteins formed eight-stranded β-barrels with flexible extracellular loops. The interfacial prolines did not form a lateral gate in these structures, but loop 3 exhibited restricted motions in the inactive P92A mutant but not in wild-type OprG.
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http://dx.doi.org/10.1016/j.str.2015.10.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4699568PMC
December 2015

Optimizing nanodiscs and bicelles for solution NMR studies of two β-barrel membrane proteins.

J Biomol NMR 2015 Apr 10;61(3-4):261-74. Epub 2015 Feb 10.

Center for Membrane Biology and Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA, 22908, USA.

Solution NMR spectroscopy has become a robust method to determine structures and explore the dynamics of integral membrane proteins. The vast majority of previous studies on membrane proteins by solution NMR have been conducted in lipid micelles. Contrary to the lipids that form a lipid bilayer in biological membranes, micellar lipids typically contain only a single hydrocarbon chain or two chains that are too short to form a bilayer. Therefore, there is a need to explore alternative more bilayer-like media to mimic the natural environment of membrane proteins. Lipid bicelles and lipid nanodiscs have emerged as two alternative membrane mimetics that are compatible with solution NMR spectroscopy. Here, we have conducted a comprehensive comparison of the physical and spectroscopic behavior of two outer membrane proteins from Pseudomonas aeruginosa, OprG and OprH, in lipid micelles, bicelles, and nanodiscs of five different sizes. Bicelles stabilized with a fraction of negatively charged lipids yielded spectra of almost comparable quality as in the best micellar solutions and the secondary structures were found to be almost indistinguishable in the two environments. Of the five nanodiscs tested, nanodiscs assembled from MSP1D1ΔH5 performed the best with both proteins in terms of sample stability and spectral resolution. Even in these optimal nanodiscs some broad signals from the membrane embedded barrel were severely overlapped with sharp signals from the flexible loops making their assignments difficult. A mutant OprH that had two of the flexible loops truncated yielded very promising spectra for further structural and dynamical analysis in MSP1D1ΔH5 nanodiscs.
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http://dx.doi.org/10.1007/s10858-015-9905-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4397663PMC
April 2015
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