Biology subjects

Zimmermann, I.

Publications and source records attributed to Zimmermann, I..

7 recordsLinked to original sources

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↗

bioRxiv2020

Structural insights into glycine reuptake inhibition

The human glycine transporter 1 (GlyT1) regulates glycine mediated neuronal excitation and inhibition through sodium- and chloride-dependent reuptake of the neurotransmitter1-3. Inhibition of glycine reuptake via GlyT1 prolongs neurotransmitter signaling and has long served as a key therapeutic development strategy for treatment of a broad range of central nervous system disorders including schizophrenia and cognitive impairments4. Using an inhibition state-selective sybody and serial synchrotron crystallography, we determined the structure of GlyT1 in complex with a benzoylpiperazine chemotype inhibitor at 3.4 [A] resolution. The inhibitor locks GlyT1 in an inward-open conformation and binds at the intracellular gate of the release pathway, overlapping with the glycine release site. The inhibitor likely reaches GlyT1 from the cytoplasmic leaflet of the plasma membrane. The study defines the mechanism of non-competitive inhibition and enables the rational design of new, clinically efficacious GlyT1 inhibitors.

neuroscience↗

bioRxiv2020

Highly potent bispecific sybodies neutralize SARS-CoV-2

The ongoing COVID-19 pandemic represents an unprecedented global health crisis. Here, we report the identification of a synthetic nanobody (sybody) pair (Sb#15 and Sb#68) that can bind simultaneously to the SARS-CoV-2 spike-RBD and efficiently neutralize pseudotyped and live-viruses by interfering with ACE2 interaction. Two spatially-discrete epitopes identified by cryo-EM translated into the rational design of bispecific and tri-bispecific fusions constructs, exhibiting up to 100- and 1000-fold increase in neutralization potency. Cryo-EM of the sybody-spike complex further revealed a novel up-out RBD conformation. While resistant viruses emerged rapidly in the presence of single binders, no escape variants were observed in presence of the bispecific sybody. The multivalent bispecific constructs further increased the neutralization potency against globally-circulating SARS- CoV-2 variants of concern. Our study illustrates the power of multivalency and biparatopic nanobody fusions for the development of clinically relevant therapeutic strategies that mitigate the emergence of new SARS-CoV-2 escape mutants.

biochemistry↗

bioRxiv2020

Synthetic nanobodies targeting the SARS-CoV-2 receptor-binding domain

The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, has resulted in a global health and economic crisis of unprecedented scale. The high transmissibility of SARS-CoV-2, combined with a lack of population immunity and prevalence of severe clinical outcomes, urges the rapid development of effective therapeutic countermeasures. Here, we report the generation of synthetic nanobodies, known as sybodies, against the receptor-binding domain (RBD) of SARS-CoV-2. In an expeditious process taking only twelve working days, sybodies were selected entirely in vitro from three large combinatorial libraries, using ribosome and phage display. We obtained six strongly enriched sybody pools against the isolated RBD and identified 63 unique anti-RBD sybodies which also interact in the context of the full-length SARS-CoV-2 spike ectodomain. Among the selected sybodies, six were found to bind to the viral spike with double-digit nanomolar affinity, and five of these also showed substantial inhibition of RBD interaction with human angiotensin-converting enzyme 2 (ACE2). Additionally, we identified a pair of anti-RBD sybodies that can simultaneously bind to the RBD. It is anticipated that compact binders such as these sybodies could feasibly be developed into an inhalable drug that can be used as a convenient prophylaxis against COVID-19. Moreover, generation of polyvalent antivirals, via fusion of anti-RBD sybodies to additional small binders recognizing secondary epitopes, could enhance the therapeutic potential and guard against escape mutants. We present full sequence information and detailed protocols for the identified sybodies, as a freely accessible resource.

biochemistry↗

bioRxiv2018

The extracellular gate shapes the energy profile of an ABC exporter

ABC exporters harness the energy of ATP to pump substrates across membranes. Extracellular gate opening and closure are key steps of the transport cycle, but the underlying mechanism is poorly understood. Here, we generated a synthetic single domain antibody (sybody) that recognizes the heterodimeric ABC exporter TM287/288 exclusively in the presence of ATP, which was essential to solve a 3.2 [A] crystal structure of the outward-facing transporter. The sybody binds to an extracellular wing and strongly inhibits ATPase activity by shifting the transporters conformational equilibrium towards the outward-facing state, as shown by double electron-electron resonance (DEER). Mutations that facilitate extracellular gate opening resulted in a comparable equilibrium shift and strongly reduced ATPase activity and drug transport. Using the sybody as conformational probe, we demonstrate that efficient extracellular gate closure is required to dissociate the NBD dimer after ATP hydrolysis to reset the transporter back to its inward-facing state.

biochemistry↗

bioRxiv2018

Engineered Peptide Barcodes for In-Depth Analyses of Binding Protein Ensembles

Binding protein generation relies on laborious screening cascades that process candidate molecules individually. To break with this paradigm, we developed NestLink, a binder selection and identification technology able to biophysically characterize thousands of library members at once without handling individual clones at any stage of the process. NestLink builds on genetically fused barcoding peptides, termed flycodes, which are designed for maximal detectability by mass spectrometry and serve as unique molecular identifiers for accurate deep sequencing. We applied NestLink to overcome current limitations of binder generation. Rare binders against an integral membrane protein were identified directly in the cellular environment of a human pathogen. Hundreds of binder candidates were simultaneously ranked according to kinetic parameters. Adverse effects of target immobilization were overcome by selecting nanobodies against an ABC transporter entirely in solution. NestLink may provide a basis for the selection of tailored binder characteristics directly in tissues or in living organisms.

bioengineering↗

bioRxiv2017

Synthetic single domain antibodies for the conformational trapping of membrane proteins

Single domain antibodies called nanobodies are excellent affinity reagents for membrane proteins. However, their generation relies on immunizations, which is only amenable to robust proteins and impedes selections in the presence of non-covalent or toxic ligands. To overcome these key limitations, we developed a novel in vitro selection platform, which builds on synthetic nanobodies called sybodies. Inspired by the shape diversity of natural nanobodies, three sybody libraries exhibiting different randomized surface shapes were engineered for high thermal stability. Using ribosome display, exceptionally large libraries were pre-enriched against membrane protein targets and subsequently funneled into a robust phage display process, thereby reducing selection bias. We successfully generated conformation-selective, high affinity sybodies against the human glycine transporter GlyT1, the human equilibrative nucleotide transporter ENT1 and a bacterial ABC transporter. Our platform builds exclusively on commercially available reagents and enables non-specialized labs to generate conformation-specific binders against previously intractable protein targets.

bioengineering↗