Dongchun Ni

Cryo-EM structures and binding of mouse and human ACE2 to SARS-CoV-2 variants of concern indicate that mutations enabling immune escape could expand host range

Kelvin Ka Ching Lau, Alexander Myasnikov, Sergey Nazarov, Dongchun Ni, Florence Pojer, Henning Paul-Julius Stahlberg, Didier Trono, Priscilla Turelli, Emiko Uchikawa

nvestigation of potential hosts of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is crucial to understanding future risks of spillover and spillback. SARS-CoV-2 has been reported to be transmitted from humans to various animals after requiring relatively few mutations. There is significant interest in describing how the virus interacts with mice as they are well adapted to human environments, are used widely as infection models and can be infected. Structural and binding data of the mouse ACE2 receptor with the Spike protein of newly identified SARS-CoV-2 variants are needed to better understand the impact of immune system evading mutations present in variants of concern (VOC). Previous studies have developed mouse-adapted variants and identified residues critical for binding to heterologous ACE2 receptors. Here we report the cryo-EM structures of mouse ACE2 bound to trimeric Spike ectodomains of four different VOC: Beta, Omicron BA.1, Omicron BA.2.12.1 and Omicron BA.4/5. These variants represent the oldest to the newest variants known to bind the mouse ACE2 receptor. Our high-resolution structural data complemented with bio-layer interferometry (BLI) binding assays reveal a requirement for a combination of mutations in the Spike protein that enable binding to the mouse ACE2 receptor.

2023

De novo design of protein interactions with learned surface fingerprints

Michael Bronstein, Stephen M Buckley, Bruno Emanuel Ferreira De Sousa Correia, Beat Fierz, Pablo Gainza Cirauqui, Sandrine Madeleine Suzanne Georgeon, Casper Alexander Goverde, Zander Harteveld, Anthony Marchand, Jane Marsden, Dongchun Ni, Elisa Oricchio, Aaron Simone Petruzzella, Charlène Mireille Raymonde Raclot, Stéphane Rosset, Andreas Scheck, Henning Paul-Julius Stahlberg, Freyr Sverrisson, Alexandra Teslenko, Didier Trono, Priscilla Turelli, Alexandra Krina Van Hall-Beauvais, Sarah Wehrle

Physical interactions between proteins are essential for most biological processes governing life. However, the molecular determinants of such interactions have been challenging to understand, even as genomic, proteomic and structural data increase. This knowledge gap has been a major obstacle for the comprehensive understanding of cellular protein–protein interaction networks and for the de novo design of protein binders that are crucial for synthetic biology and translational applications. Here we use a geometric deep-learning framework operating on protein surfaces that generates fingerprints to describe geometric and chemical features that are critical to drive protein–protein interactions10. We hypothesized that these fingerprints capture the key aspects of molecular recognition that represent a new paradigm in the computational design of novel protein interactions. As a proof of principle, we computationally designed several de novo protein binders to engage four protein targets: SARS-CoV-2 spike, PD-1, PD-L1 and CTLA-4. Several designs were experimentally optimized, whereas others were generated purely in silico, reaching nanomolar affinity with structural and mutational characterization showing highly accurate predictions. Overall, our surface-centric approach captures the physical and chemical determinants of molecular recognition, enabling an approach for the de novo design of protein interactions and, more broadly, of artificial proteins with function.

2023

Allosteric control of type I-A CRISPR-Cas3 complexes and establishment as effective nucleic acid detection and human genome editing tools

Dongchun Ni, Henning Paul-Julius Stahlberg

Type I CRISPR-Cas systems typically rely on a two-step process to degrade DNA. First, an RNA-guided complex named Cascade identifies the complementary DNA target. The helicase-nuclease fusion enzyme Cas3 is then recruited in trans for processive DNA degradation. Contrary to this model, here, we show that type I-A Cascade and Cas3 function as an integral effector complex. We provide four cryoelectron microscopy (cryo-EM) snapshots of the Pyrococcus furiosus (Pfu) type I-A effector complex in different stages of DNA recognition and degradation. The HD nuclease of Cas3 is autoinhibited inside the effector complex. It is only allosterically activated upon full R-loop formation, when the entire targeted region has been validated by the RNA guide. The mechanistic insights inspired us to convert Pfu Cascade-Cas3 into a high-sensitivity, low-background, and temperature-activated nucleic acid detection tool. Moreover, Pfu CRISPR-Cas3 shows robust bi-directional deletion-editing activity in human cells, which could find usage in allele-specific inactivation of disease-causing mutations.

CELL PRESS2022

Cryo-EM structures of a LptDE transporter in complex with Pro-macrobodies offer insight into lipopolysaccharide translocation

Denis Bucher, Dongchun Ni, Henning Paul-Julius Stahlberg, Iwan Zimmermann

Lipopolysaccharides are major constituents of the extracellular leaflet in the bacterial outer membrane and form an effective physical barrier for environmental threats and for antibiotics in Gram-negative bacteria. The last step of LPS insertion via the Lpt pathway is mediated by the LptD/E protein complex. Detailed insights into the architecture of LptDE transporter complexes have been derived from X-ray crystallography. However, no structure of a laterally open LptD transporter, a transient state that occurs during LPS release, is available to date. Here, we report a cryo-EM structure of a partially opened LptDE transporter in complex with rigid chaperones derived from nanobodies, at 3.4 Å resolution. In addition, a subset of particles allows to model a structure of a laterally fully opened LptDE complex. Our work offers insights into the mechanism of LPS insertion, provides a structural framework for the development of antibiotics targeting LptD and describes a highly rigid chaperone scaffold to enable structural biology of challenging protein targets.

2022

Mechanism ofcyclic beta-glucan export by ABC transporter Cgt of Brucella

Dongchun Ni, Henning Paul-Julius Stahlberg, Na Wu

Polysaccharides play critical roles in bacteria, including the formation of protective capsules and biofilms and establishing specific host cell interactions. Their transport across membranes is often mediated by ATP-binding cassette (ABC) transporters, which utilize ATP to translocate diverse molecules. Cyclic beta-glucans (C beta Gs) are critical for host interaction of the Rhizobiales, including the zoonotic pathogen Brucella. C beta Gs are exported into the periplasmic space by the cyclic glucan transporter (Cgt). The interaction of an ABC transporter with a polysaccharide substrate has not been visualized so far. Here we use single-particle cryoelectron microscopy to elucidate the structures of Cgt from Brucella abortus in four conformational states. The substrate-bound structure reveals an unusual binding pocket at the height of the cytoplasmic leaflet, whereas ADP-vanadate models hint at an alternative mechanism of substrate release. Our work provides insights into the translocation of large, heterogeneous substrates and sheds light on protein-polysaccharide interactions in general.

NATURE PORTFOLIO2022

Structural basis of drug recognition by the multidrug transporter ABCG2

Dongchun Ni, Henning Paul-Julius Stahlberg

ABCG2 is an ATP-binding cassette (ABC) transporter whose function affects the pharmacokinetics of drugs and contributes to multidrug resistance of cancer cells. While its interaction with the endogenous substrate estrone-3-sulfate (E1S) has been elucidated at a structural level, the recognition and recruitment of exogenous compounds is not understood at sufficiently high resolution. Here we present three cryo-EM structures of nanodisc-reconstituted, human ABCG2 bound to anticancer drugs tariquidar, topotecan and mitoxantrone. To enable structural insight at high resolution, we used Fab fragments of the ABCG2-specific monoclonal antibody 5D3, which binds to the external side of the transporter but does not interfere with drug-induced stimulation of ATPase activity. We observed that the binding pocket of ABCG2 can accommodate a single tariquidar molecule in a C-shaped conformation, similar to one of the two tariquidar molecules bound to ABCB1, where tariquidar acts as an inhibitor. We also found single copies of topotecan and mitoxantrone bound between key phenylalanine residues. Mutagenesis experiments confirmed the functional importance of two residues in the binding pocket, F439 and N436. Using 3D variability analyses, we found a correlation between substrate binding and reduced dynamics of the nucleotide binding domains (NBDs), suggesting a structural explanation for drug-induced ATPase stimulation. Our findings provide additional insight into how ABCG2 differentiates between inhibitors and substrates and may guide a rational design of new modulators and substrates.

2021

Structures of ABCG2 under turnover conditions reveal a key step in the drug transport mechanism

Dongchun Ni, Henning Paul-Julius Stahlberg

ABCG2 is a multidrug transporter that affects drug pharmacokinetics and contributes to multidrug resistance of cancer cells. In previously reported structures, the reaction cycle was halted by the absence of substrates or ATP, mutation of catalytic residues, or the presence of small-molecule inhibitors or inhibitory antibodies. Here we present cryo-EM structures of ABCG2 under turnover conditions containing either the endogenous substrate estrone-3-sulfate or the exogenous substrate topotecan. We find two distinct conformational states in which both the transport substrates and ATP are bound. Whereas the state turnover-1 features more widely separated NBDs and an accessible substrate cavity between the TMDs, turnover-2 features semi-closed NBDs and an almost fully occluded substrate cavity. Substrate size appears to control which turnover state is mainly populated. The conformational changes between turnover-1 and turnover-2 states reveal how ATP binding is linked to the closing of the cytoplasmic side of the TMDs. The transition from turnover-1 to turnover-2 is the likely bottleneck or rate-limiting step of the reaction cycle, where the discrimination of substrates and inhibitors occurs. ABCG2 is a transporter contributing to multidrug resistance of cancer cells. Here, structures of human ABCG2 under turnover conditions reveal distinct conformational states, provide insight into the transport cycle and suggest a mechanism of discrimination between substrates and inhibitors.

NATURE RESEARCH2021

New insights on the structure of alpha-synuclein fibrils using cryo-electron microscopy

Ricardo Cesar Guerrero Ferreira, Dongchun Ni, Henning Paul-Julius Stahlberg

Fibrils of alpha-synuclein are significant components of cellular inclusions associated with several neuropathological disorders including Parkinson’s disease, multiple system atrophy and dementia with Lewy bodies. In recent years, technological advances in the field of transmission electron microscopy and image processing have made it possible to solve the structure of alpha-synuclein fibrils at high resolution. This review discusses the results of structural studies using cryo-electron microscopy, which revealed that in-vitro produced fibrils vary in diameter from 5 nm for single-protofilament fibrils, to 10 nm for two-protofilament fibrils. In addition, the atomic models hint at contributions of the familial Parkinson’s disease mutation sites to inter-protofilament interaction and the locations where post-translational modifications take place. Here, we propose a nomenclature system that allows identifying the existing alpha-synuclein polymorphs and that will allow to incorporate additional high-resolution structures determined in the future.

2020

Dongchun Ni

Cryo-EM structures and binding of mouse and human ACE2 to SARS-CoV-2 variants of concern indicate that mutations enabling immune escape could expand host range

De novo design of protein interactions with learned surface fingerprints

Allosteric control of type I-A CRISPR-Cas3 complexes and establishment as effective nucleic acid detection and human genome editing tools

Cryo-EM structures of a LptDE transporter in complex with Pro-macrobodies offer insight into lipopolysaccharide translocation

It takes two times two to tango - cryoEM structure resolution of the membrane-bound tetrachloroethene reductive dehalogenase complex

Mechanism ofcyclic beta-glucan export by ABC transporter Cgt of Brucella

Insight into Lipopolysaccharide Translocation by Cryo-EM structures of a LptDE Transporter in Complex with Pro-Macrobodies

Structural basis of drug recognition by the multidrug transporter ABCG2

Structures of ABCG2 under turnover conditions reveal a key step in the drug transport mechanism

New insights on the structure of alpha-synuclein fibrils using cryo-electron microscopy

It takes two times two to tango - cryoEM structure resolution of the membrane-bound tetrachloroethene reductive dehalogenase complex

Cryo-EM structures and binding of mouse and human ACE2 to SARS-CoV-2 variants of concern indicate that mutations enabling immune escape could expand host range

Insight into Lipopolysaccharide Translocation by Cryo-EM structures of a LptDE Transporter in Complex with Pro-Macrobodies

Structural basis of drug recognition by the multidrug transporter ABCG2

Structures of ABCG2 under turnover conditions reveal a key step in the drug transport mechanism

New insights on the structure of alpha-synuclein fibrils using cryo-electron microscopy

De novo design of protein interactions with learned surface fingerprints

Allosteric control of type I-A CRISPR-Cas3 complexes and establishment as effective nucleic acid detection and human genome editing tools

Cryo-EM structures of a LptDE transporter in complex with Pro-macrobodies offer insight into lipopolysaccharide translocation

Mechanism ofcyclic beta-glucan export by ABC transporter Cgt of Brucella