Biology subjects

Stahlberg, H.

Publications and source records attributed to Stahlberg, H..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

ACE2 mimetic antibody potently neutralizes all SARS-CoV-2 variants and fully protects in XBB.1.5 challenged monkeys

The rapid evolution of SARS-CoV-2 to variants with improved transmission efficiency and reduced sensitivity to vaccine-induced humoral immunity has abolished the protective effect of licensed therapeutic human monoclonal antibodies (mAbs). To fill this unmet medical need and protect vulnerable patient populations, we isolated the P4J15 mAb from a previously infected, vaccinated donor, with <20 ng/ml neutralizing activity against all Omicron variants including the latest XBB.2.3 and EG.1 sub-lineages. Structural studies of P4J15 in complex with Omicron XBB.1 Spike show that the P4J15 epitope shares [~]93% of its buried surface area with the ACE2 contact region, consistent with an ACE2 mimetic antibody. Although SARS-CoV-2 mutants escaping neutralization by P4J15 were selected in vitro, these displayed lower infectivity, poor binding to ACE2, and the corresponding escape mutations are accordingly rare in public sequence databases. Using a SARS-CoV-2 XBB.1.5 monkey challenge model, we show that P4J15 confers complete prophylactic protection. We conclude that the P4J15 mAb has potential as a broad-spectrum anti-SARS-CoV-2 drug.

microbiology↗

bioRxiv2023

Structure of a membrane-bound menaquinol:organohalide oxidoreductase

Organohalide-respiring bacteria are key organisms for the bioremediation of soils and aquifers contaminated with halogenated organic compounds. The major players in this process are respiratory reductive dehalogenases, corrinoid enzymes that use organohalides as substrates and contribute to energy conservation. Here, we present the first structure of a functional menaquinol:organohalide oxidoreductase (MOOR) obtained by cryo-EM. The membrane-bound protein was isolated from Desulfitobacterium hafniense strain TCE1 as a PceA2B2 complex catalysing the dechlorination of tetrachloroethene (PCE). Two catalytic PceA subunits are anchored to the membrane by two small integral membrane PceB subunits. The structure reveals two menaquinone molecules bound at the interface of the two different subunits, which are the starting point of a chain of redox cofactors for electron transfer to the active site. This structure elucidates how energy is conserved during organohalide respiration in menaquinone-dependent organohalide-respiring bacteria.

microbiology↗

bioRxiv2023

Structure-function analysis of the cyclic beta-1,2-glucan synthase

The synthesis of complex sugars is a key aspect of microbial biology. Cyclic {beta}-1,2-glucan (C{beta}G) is a circular polysaccharide critical for host interactions of many bacteria, including major pathogens of humans (Brucella) and plants (Agrobacterium). C{beta}G is produced by the cyclic glucan synthase (Cgs), a massive multi-domain membrane protein. So far, its structure as well as the mechanisms underlining the synthesis have not been clarified. Here we use cryo-electron microscopy (cryo-EM) and functional approaches to study Cgs from A. tumefaciens. We were able to determine the structure of this complex protein machinery and clarify key aspects of C{beta}G synthesis. Our research opens new possibilities for combating pathogens that rely on polysaccharide virulence factors and can lead to new synthetic biology approaches for producing complex cyclic sugars.

molecular biology↗

bioRxiv2022

Rapid iPSC inclusionopathy models shed light on formation, consequence and molecular subtype of alpha-synuclein inclusions

Intracellular inclusions accompanying neurodegeneration are histopathologically and ultrastructurally heterogeneous but the significance of this heterogeneity is unclear. iPSC models, while promising for disease modeling, do not form inclusions in a reasonable timeframe and suffer from limited tractability. Here, we developed an iPSC toolbox utilizing piggyBac-based or targeted transgenes to rapidly induce CNS cells with concomitant expression of aggregation-prone proteins. This system is amenable to screening and longitudinal tracking at single-cell and single-inclusion resolution. For proof-of-principle, cortical neuron -synuclein "inclusionopathy" models were engineered to form inclusions through exogenous seeding or -synuclein mutation. These models recapitulated known fibril- and lipid-rich inclusion subtypes, uncovering dynamic interactions between them, and refined the classification of inclusions in postmortem brain. Genetic-modifier and protein-interaction screens pinpointed proteins like RhoA whose sequestration into specific inclusion subtypes is likely to be toxic. This iPSC platform should enhance our understanding of proteinaceous pathologies in neurodegeneration and facilitate therapeutics development.

neuroscience↗

bioRxiv2022

Cryo-EM structures and binding of mouse ACE2 to SARS-CoV-2 variants of concern

Investigation 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.[1] 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.[2] 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.[3,4] 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. AUTHOR SUMMARYThe SARS-CoV-2 virus can infect different types of animals beyond humans. The virus uses its Spike protein on its surface to bind to cells. These cells have a protein called ACE2 that the Spike protein recognizes. Animals have slightly different ACE2 receptors compared to humans. Mice are widely used as a research animal and live in the same environments as humans so scientists are particularly interested. Understanding how Spike proteins binds to the mouse ACE2 receptor allows us to understand the impact of immune evading mutations found in new variants. We use a high resolution imaging technique called cryo-electron microscopy to look at how different Spike variants bind to the ACE2 receptor from mouse at a resolution where we can see the amino acids. We can see directly the individual amino acids and mutations on the Spike protein that interact with the mouse ACE2 receptor. Many of the mutations found in variants of concern also increase the strength of binding to the mouse ACE2 receptor. This result suggests that mutations in the Spike protein of future variants may have an additional effect in influencing how it binds to not only human ACE2 receptors but to mice and also different animals.

immunology↗

bioRxiv2022

De novo design of site-specific protein interactions with learned surface fingerprints

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 grows. This knowledge gap has been a major obstacle for the comprehensive understanding of cellular protein-protein interaction (PPI) networks and for the de novo design of protein binders that are crucial for synthetic biology and translational applications. We exploit a geometric deep learning framework operating on protein surfaces that generates fingerprints to describe geometric and chemical features critical to drive PPIs. We hypothesized 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 while others were purely generated 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 a novel approach for the de novo design of protein interactions and, more broadly, of artificial proteins with function.

bioengineering↗

bioRxiv2022

SARS-CoV-2 Omicron potently neutralized by a novel antibody with unique Spike binding properties

The SARS-CoV-2 Omicron variant exhibits very high levels of transmission, pronounced resistance to authorized therapeutic human monoclonal antibodies and reduced sensitivity to vaccine-induced immunity. Here we describe P2G3, a human monoclonal antibody (mAb) isolated from a previously infected and vaccinated donor, which displays picomolar-range neutralizing activity against Omicron BA.1, BA.1.1, BA.2 and all other current variants, and is thus markedly more potent than all authorized or clinically advanced anti-SARS-CoV-2 mAbs. Structural characterization of P2G3 Fab in complex with the Omicron Spike demonstrates unique binding properties to both down and up spike trimer conformations at an epitope that partially overlaps with the receptor-binding domain (RBD), yet is distinct from those bound by all other characterized mAbs. This distinct epitope and angle of attack allows P2G3 to overcome all the Omicron mutations abolishing or impairing neutralization by other anti-SARS-COV-2 mAbs, and P2G3 accordingly confers complete prophylactic protection in the SARS-CoV-2 Omicron monkey challenge model. Finally, although we could isolate in vitro SARS-CoV2 mutants escaping neutralization by P2G3 or by P5C3, a previously described broadly active Class 1 mAb, we found these viruses to be lowly infectious and their key mutations extremely rare in the wild, and we could demonstrate that P2G3/P5C3 efficiently cross-neutralized one anothers escapees. We conclude that this combination of mAbs has great prospects in both the prophylactic and therapeutic settings to protect from Omicron and other VOCs.

immunology↗

bioRxiv2021

Structural analysis of the Spike of the Omicron SARS-COV-2 variant by Cryo-EM and implications for immune evasion

immunology↗

bioRxiv2021

Cryo-EM structure of alpha-synuclein fibrils amplified by PMCA from PD and MSA patient brains

Synucleinopathies are neurodegenerative diseases related to the aggregation of the protein alpha-synuclein (aSyn). Among these diseases, Parkinsons disease (PD) and multiple system atrophy (MSA) are most prevalent. aSyn can readily form different fibrillar polymorphs, if exposed to an air-water interface or by templating with pre-existing fibrils. We here report the structures of three fibrillar polymorphs that were obtained after seeding monomeric aSyn with PD and MSA patients brain homogenates using protein misfolding cyclic amplification (PMCA). Seeding with a control brain homogenate did not produce fibrils, and seeding with other in vitro generated fibrillar polymorphs as a control faithfully produced polymorphs of a different type. The here determined fibril structures from PD and MSA brain tissue represent new folds, which partly resemble that of previously reported in vitro generated fibrils from Y39 phosphorylated aSyn protein. The relevance of these fibrils for synucleinopathies in humans remains to be further investigated. Impact StatementNeurodegenerative diseases such as Parkinsons disease (PD) and Multiple System Atrophy (MSA) are suspected to be causatively related to the prion-like propagation of aggregates of the protein alpha-synuclein (aSyn). The fibril structures reported here were obtained after seeding from diseased human brain homogenate and differ from all previously published aSyn fibril arrangements. In case these fibrils would turn out to be the long sought causative agents of these diseases, their structures might lead to the development of therapeutic strategies to modify these diseases and to a better understanding of the mechanistic processes that lead to neurodegeneration and spreading of the diseases.

neuroscience↗

bioRxiv2021

Cryo-EM structure of native human thyroglobulin

The thyroglobulin (Tg) protein is essential to thyroid hormone synthesis, playing a vital role in the regulation of metabolism, development and growth. Its structure is conserved among vertebrates. Tg is delivered through the secretory pathway of the thyroid follicular unit to the central colloid depository, where it is iodinated at specific tyrosine sites to form mono- or diiodotyrosine, which combine to produce triiodothyronine (T3) and thyroxine (T4), respectively. Synthesis of these hormones depends on the precise 3D structure of Tg, which has remained unknown despite decades of research. Here, we present the cryo-electron microscopy structure of human thyroglobulin (hTg) to a global resolution of 3.2 [A]. The structure provides detailed information on the location of the hTg hormonogenic sites and reveals the position as well as the role of many of its glycosylation sites. Our results offer structural insight into thyroid hormonogenesis and provide a fundamental understanding of clinically relevant hTg mutations, which can improve treatment of thyroid diseases.

biophysics↗

bioRxiv2021

Functional and structural characterization of Serratia marcescens ExbB: determinants of the interaction with HasB/TonB

ExbB and ExbD are cytoplasmic membrane proteins that associate with TonB to convey the energy of the proton-motive force to outer membrane receptors in Gram-negative bacteria for iron uptake. The opportunistic pathogen Serratia marcescens (Sm) possesses both TonB and a heme-specific TonB paralog, HasB. ExbBSm has a long periplasmic extension absent in other bacteria such as E. coli (Ec). Long ExbBs are found in several genera of Alphaproteobacteria, most often in correlation with a hasB gene. We investigated specificity determinants of ExbBSm and HasB. We determined the cryo-EM structures of ExbBSm and of the ExbB-ExbDSm complex from S. marcescens. ExbBSm alone is a stable pentamer, and its complex includes two ExbD monomers. We showed that ExbBSm extension interacts with HasB and is involved in heme acquisition and we identified key residues in the membrane domain of ExbBSm and ExbBEc, essential for function and likely involved in the interaction with TonB/HasB. Our results shed light on the new class of inner membrane energy machinery formed by ExbB,ExbD and HasB.

microbiology↗

bioRxiv2021

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

Lipopolysaccharides (LPS) 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 1. The last step of LPS insertion via the Lpt pathway is mediated by the LptD/E protein complex 2. Despite detailed insights from X-ray crystallography into the architecture of LptDE transporter complexes 3-5, no structure of a laterally open LptD transporter has been described, a transient state that occurs during LPS release 6. To facilitate the acquisition of hitherto unknown conformations we subjected LptDE of N. gonorrhoeae to cryo-EM analyses. In complex with newly designed rigid chaperones derived from nanobodies (Pro-Macrobodies, PMbs) we obtained a map of a partially opened LptDE transporter at 3.4 [A] resolution and in addition we captured a laterally fully opened LptDE complex from a subset of particles. Our work offers new insights into the mechanism of LPS insertion, provides a structural framework for the development of antibiotics targeting LptD and describes a novel, highly rigid and widely applicable chaperone scaffold to enable structural biology of challenging protein targets.

biochemistry↗

bioRxiv2021

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

ABCG2 is a multidrug transporter expressed widely in the human body. Its physiological substrates include steroid derivatives and uric acid. In addition, it extrudes many structurally diverse cytotoxic drugs from various cells, thus affecting drug pharmacokinetics and contributing to multidrug resistance of cancer cells. Previous studies have revealed structures of ABCG2 bound to transport substrates, nucleotides, small-molecule inhibitors and inhibitory antibodies. However, the transport mechanism is not well-understood because all previous structures described trapped states, where the reaction cycle was halted by the absence of substrates or ATP, mutation of catalytic residues, or the presence of inhibitors. Here we present cryo-EM structures of nanodisc-reconstituted human ABCG2 under turnover conditions containing either the endogenous substrate estrone-3-sulfate or the exogenous substrate topotecan. We found 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 cavity between the TMDs, turnover-2 features semi-closed NBDs and an almost fully occluded cavity between the TMDs. The transition from turnover-1 to turnover-2 includes conformational changes that link the binding of ATP by the NBDs to the closing of the cytoplasmic side of the TMDs. The size of the substrate appears to control which turnover state corresponds to the main state in the transport cycle. 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. Our results provide a structural basis of substrate specificity of ABCG2 and provide key insight to understand the transport cycle.

biophysics↗

bioRxiv2021

Structural basis of drug recognition by the multidrug transporter ABCG2

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 insight into how ABCG2 differentiates between inhibitors and substrates and may guide a rational design of new modulators and substrates.

biophysics↗

bioRxiv2020

Structural basis of the activation of the CC chemokine receptor 5 by a chemokine agonist

The human CC chemokine receptor 5 (CCR5) is a G protein-coupled receptor (GPCR) that plays a major role in inflammation and is involved in the pathology of cancer, HIV, and COVID-19. Despite its significance as a drug target, the activation mechanism of CCR5, i.e. how chemokine agonists transduce the activation signal through the receptor, is yet unknown. Here, we report the cryo-EM structure of wild-type CCR5 in an active conformation bound to the chemokine super-agonist [6P4]CCL5 and the heterotrimeric Gi protein. The structure provides the rationale for the sequence-activity relation of agonist and antagonist chemokines. The N-terminus of agonist chemokines pushes onto an aromatic connector that transmits activation to the canonical GPCR microswitch network. This activation mechanism differs significantly from other CC chemokine receptors that bind shorter chemokines in a shallow binding mode and have unique sequence signatures and a specialized activation mechanism. One-sentence summaryThe structure of CCR5 in complex with the chemokine agonist [6P4]CCL5 and the heterotrimeric Gi protein reveals its activation mechanism

biophysics↗

bioRxiv2020

Cryo-EM structural studies of the agonist complexed human TRPV4 ion-channel reveals novel structural rearrangements resulting in an open-conformation

The human transient receptor potential vanilloid 4 (hTRPV4) ion channel plays a critical role in a variety of biological processes. Whilst the activation of hTRPV4 gating properties has been reported for a broad spectrum of stimuli, including synthetic 4-phorbols, the molecular basis of the activation is poorly understood. Here we report the novel cryo-EM structure of the hTRPV4 determined in the presence of the archetypical phorbol acid agonist, 4-PDD. Complementary mutagenesis experiments support the EM-identified binding site as well as allowing rationalization of disruptive mutants located outside of the 4-PDD binding site. This work represents the first structural information of hTRPV4 in a ligand-induced open conformation. Together, our data reveal the underlying molecular mechanisms resulting in the opening of the central pore and ion-channel activation and provide a structural template for designing inhibitors targeting the open-state conformation of hTRPV4.

molecular biology↗

bioRxiv2020

Structural investigation of ACE2 dependent disassembly of the trimeric SARS-CoV-2 Spike glycoprotein

The human membrane protein Angiotensin-converting enzyme 2 (hACE2) acts as the main receptor for host cells invasion of the new coronavirus SARS-CoV-2. The viral surface glycoprotein Spike binds to hACE2, which triggers virus entry into cells. As of today, the role of hACE2 for virus fusion is not well understood. Blocking the transition of Spike from its prefusion to post-fusion state might be a strategy to prevent or treat COVID-19. Here we report a single particle cryo-electron microscopy analysis of SARS-CoV-2 trimeric Spike in presence of the human ACE2 ectodomain. The binding of purified hACE2 ectodomain to Spike induces the disassembly of the trimeric form of Spike and a structural rearrangement of its S1 domain to form a stable, monomeric complex with hACE2. This observed hACE2 dependent dissociation of the Spike trimer suggests a mechanism for the therapeutic role of recombinant soluble hACE2 for treatment of COVID-19.

biophysics↗

bioRxiv2020

The architecture of the centriole cartwheel-containing region revealed by cryo-electron tomography

Centrioles are evolutionarily conserved barrels of microtubule triplets that form the core of the centrosome and the base of the cilium. In the proximal region of the centriole, nine microtubule triplets attach to each other via A-C linkers and encircle a central cartwheel structure, which directs the early events of centriole assembly. While the crucial role of the proximal region in centriole biogenesis has been well documented in many species, its native architecture and evolutionary conservation remain relatively unexplored. Here, using cryo-electron tomography of centrioles from four evolutionarily distant species, including humans, we report on the architectural diversity of the centriolar proximal cartwheel-bearing region. Our work reveals that the cartwheel central hub, previously reported to have an 8.5 nm periodicity in Trichonympha, is constructed from a stack of paired rings with an average periodicity of [~]4 nm. In all four examined species, cartwheel inner densities are found inside the hubs ring-pairs. In both Paramecium and Chlamydomonas, the repeating structural unit of the cartwheel has a periodicity of 25 nm and consists of three ring-pairs with 6 radial spokes emanating and merging into a single bundle that connects to the triplet microtubule via the pinhead. Finally, we identified that the cartwheel is indirectly connected to the A-C linker through a flexible triplet-base structure extending from the pinhead. Together, our work provides unprecedented evolutionary insights into the architecture of the centriole proximal region, which underlies centriole biogenesis.

cell biology↗