Joanna Pieprzyk

Centre for Structural Systems Biology

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Christian Löw

Centre for Structural Systems Biology

Thomas C. Marlovits

Centre for Structural Systems Biology

C. Loew

Centre for Structural Systems Biology

Andrzej Ożyhar

Wrocław University of Science and Technology

Ali Flayhan

Centre for Structural Systems Biology

Dmitri I. Svergun

European Molecular Biology Laboratory

Marek Orłowski

AGH University of Krakow

John K. Wald

The University of Texas at San Antonio

M. Killer

Samuel Pažický

Centre for Structural Systems Biology

Cooperative Institutions

European Molecular Biology Laboratory

Wrocław University of Science and Technology

Centre for Structural Systems Biology

Deutsches Elektronen-Synchrotron DESY

AGH University of Krakow

Karolinska Institutet

University Medical Center Utrecht

Karolinska University Hospital

Institute of Organic Chemistry

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Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2

bioRxiv (Cold Spring Harbor Laboratory) (2020)

Tânia F. Custódio Hrishikesh Das Daniel J. Sheward Leo Hanke Samuel Pažický

Abstract The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. However, traditional antibody production is hampered by long development times and costly production. Here, we report the rapid isolation and characterization of nanobodies from a synthetic library, known as sybodies (Sb), that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Several binders with low nanomolar affinities and efficient neutralization activity were identified of which Sb23 displayed high affinity and neutralized pseudovirus with an IC 50 of 0.6 µg/ml. A cryo-EM structure of the spike bound to Sb23 showed that Sb23 binds competitively in the ACE2 binding site. Furthermore, the cryo-EM reconstruction revealed a novel conformation of the spike where two RBDs are in the ‘up’ ACE2-binding conformation. The combined approach represents an alternative, fast workflow to select binders with neutralizing activity against newly emerging viruses.

Coronavirus

2019-20 coronavirus outbreak

Binding affinities

spike protein

10.1101/2020.06.23.165415

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Citations (26)

Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2

Nature Communications (2020)

Tânia F. Custódio Hrishikesh Das Daniel J. Sheward Leo Hanke Samuel Pažický

Abstract The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. However, traditional antibody production is hampered by long development times and costly production. Here, we report the rapid isolation and characterization of nanobodies from a synthetic library, known as sybodies (Sb), that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Several binders with low nanomolar affinities and efficient neutralization activity were identified of which Sb23 displayed high affinity and neutralized pseudovirus with an IC 50 of 0.6 µg/ml. A cryo-EM structure of the spike bound to Sb23 showed that Sb23 binds competitively in the ACE2 binding site. Furthermore, the cryo-EM reconstruction revealed an unusual conformation of the spike where two RBDs are in the ‘up’ ACE2-binding conformation. The combined approach represents an alternative, fast workflow to select binders with neutralizing activity against newly emerging viruses.

Coronavirus

2019-20 coronavirus outbreak

Binding affinities

Pillar

10.1038/s41467-020-19204-y

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Citations (157)

Structural snapshots of human PepT1 and PepT2 reveal mechanistic insights into substrate and drug transport across epithelial membranes

Science Advances (2021)

Maxime Killer Jiri Wald Joanna Pieprzyk Thomas C. Marlovits Christian Löw

Structures of PepT1 and PepT2 provide mechanistic insights into substrate and drug translocation across epithelial membranes.

10.1126/sciadv.abk3259

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Citations (69)

Cholesterol, phospholipids, and fatty acids of normal immature neutrophils: comparison with acute myeloblastic leukemia cells and normal neutrophils.

Journal of Lipid Research (1979)

John Klock Joanna Pieprzyk

The lipid composition of immature myeloid cells from the bone marrow of normal persons and myeloblasts from patients with acute myeloblastic leukemia was studied and compared with the lipid composition of normal mature human neutrophils.Total cholesterol, phospholipid, and fatty acid composition was determined on each cell type.The leukemic cells showed decreased total cholesterol and cholesterol-to-phospholipid ratio, increased phosphatidylcholine and phosphatidylinositol, decreased phosphatidylethanolamine, and an increased percentage of unsaturated fatty acids when compared to normal mature neutrophils.A nearly identical pattern was seen in the normal immature myeloid precursors from normal bone marrow.We conclude that the altered lipid composition of acute myeloblastic leukemia cells is related to unexplained factors related to cell age and not to malignancy per se.-Klock,J. C., and J. K. Pieprzyk.Cholesterol, phospholipids, and fatty acids of normal immature neutrophils: comparison with acute myeloblastic leukemia cells and normal neutrophils.

Acute myeloblastic leukemia

10.1016/s0022-2275(20)40020-3

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Citations (49)

Structural Analyses of Ordered and Disordered Regions in Ecdysteroid Receptor

Springer eBooks (2015)

Marek Orłowski Piotr Dobryszycki Anna Zoglowek Joanna Pieprzyk K Bielska

Ecdysteroid

10.1007/978-3-319-18729-7_6

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Citations (1)

Molecular determinants ofDrosophilaimmunophilin FKBP39 nuclear localization

Biological Chemistry (2018)

Marek Orłowski Katarzyna Popławska Joanna Pieprzyk Aleksandra Szczygieł-Sommer Anna Wiȩch

Abstract FK506-binding proteins (FKBPs) belong to a distinct class of immunophilins that interact with immunosuppressants. They use their peptidyl-prolyl isomerase (PPIase) activity to catalyze the cis-trans conversion of prolyl bonds in proteins during protein-folding events. FKBPs also act as a unique group of chaperones. The Drosophila melanogaster peptidyl-prolyl cis-trans isomerase FK506-binding protein of 39 kDa (FKBP39) is thought to act as a transcriptional modulator of gene expression in 20-hydroxyecdysone and juvenile hormone signal transduction. The aim of this study was to analyze the molecular determinants responsible for the subcellular distribution of an FKBP39-yellow fluorescent protein (YFP) fusion construct (YFP-FKBP39). We found that YFP-FKBP39 was predominantly nucleolar. To identify the nuclear localization signal (NLS), a series of YFP-tagged FKBP39 deletion mutants were prepared and examined in vivo . The identified NLS signal is located in a basic domain. Detailed mutagenesis studies revealed that residues K188 and K191 are crucial for the nuclear targeting of FKBP39 and its nucleoplasmin-like (NPL) domain contains the sequence that controls the nucleolar-specific translocation of the protein. These results show that FKBP39 possesses a specific NLS in close proximity to a putative helix-turn-helix (HTH) motif and FKBP39 may bind DNA in vivo and in vitro .

FKBP

Prolyl isomerase

NLS

10.1515/hsz-2017-0251

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Citations (6)

Multispecific Substrate Recognition in a Proton-Dependent Oligopeptide Transporter

Structure (2018)

Maria Martinez Molledo E.M. Quistgaard Ali Flayhan Joanna Pieprzyk Christian Löw

Proton-dependent oligopeptide transporters (POTs) are important for uptake of dietary di- and tripeptides in many organisms, and in humans are also involved in drug absorption. These transporters accept a wide range of substrates, but the structural basis for how different peptide side chains are accommodated has so far remained obscure. Twenty-eight peptides were screened for binding to PepTSt from Streptococcus thermophilus, and structures were determined of PepTSt in complex with four physicochemically diverse dipeptides, which bind with millimolar affinity: Ala-Leu, Phe-Ala, Ala-Gln, and Asp-Glu. The structures show that PepTSt can adapt to different peptide side chains through movement of binding site residues and water molecules, and that a good fit can be further aided by adjustment of the position of the peptide itself. Finally, structures were also determined in complex with adventitiously bound HEPES, polyethylene glycol, and phosphate molecules, which further underline the adaptability of the binding site.

Tripeptide

Oligopeptide

Side chain

10.1016/j.str.2018.01.005

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Citations (74)

HsPepT2 bound to Ala-Phe in the inward facing partially occluded conformation

M. Killer John K. Wald Joanna Pieprzyk Thomas C. Marlovits C. Loew

10.2210/pdb7pmy/pdb

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Citations (0)

Probing the Architecture of a Multi-PDZ Domain Protein: Structure of PDZK1 in Solution