Biology subjects

Wohlkonig, A.

Publications and source records attributed to Wohlkonig, A..

5 recordsLinked to original sources

bioRxiv2024

A biosensor-based phage display selection method for automated, high-throughput Nanobody discovery

Biopanning methods to select target-specific Nanobodies(R) (Nbs) involve presenting the antigen, immobilized on plastic plates or magnetic beads, to Nb libraries displayed on phage. Most routines are operator-dependent, labor-intensive and often material- and time-consuming. Here we validate an improved panning strategy that uses biosensors to present the antigen to phage-displayed Nbs in a well. The use of automated Octet biolayer interferometry sensors (Sartorius) enables high throughput and precise control over each step. By playing with association and dissociation times and buffer composition, one can efficiently decrease the background of aspecific and low-affinity Nbs, reducing the rounds of panning needed for the enrichment of high-affinity binders. Octet panning also enables the use of unpurified target proteins and unpurified phage from a bacterial culture supernatant. Additionally, downscaling to a 384-well format significantly reduces the amount of protein required. Moreover, enrichment of binders can be quantified by monitoring phage binding to the target by interferometry, omitting additional phage titration steps. Routinely, up to three rounds of Octet panning can be performed in only five days to deliver target-specific binders, ready for screening and characterization using the same Octet instrument. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/600780v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@72eda4org.highwire.dtl.DTLVardef@14fe6bcorg.highwire.dtl.DTLVardef@b57bc9org.highwire.dtl.DTLVardef@d1ca82_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

Nanobody CDR3 mimetics enhance SOS1-catalyzed nucleotide exchange on RAS

RAS proteins control various intracellular signaling networks. Mutations at specific locations were shown to stabilize their active GTP-bound state, which is associated with the development of multiple cancers. An attractive approach to modulate RAS signaling is through its regulatory guanine nucleotide exchange factor (GEF) son of sevenless 1 (SOS1). With the recent discovery of Nanobody14, which potently enhances SOS1-catalyzed nucleotide exchange on RAS, we explored the feasibility of developing peptide mimetics by structurally mimicking the complementarity-determining region 3 (CDR3). Guided by a biochemical GEF assay and X-ray co-crystal structures, successive rounds of optimization and gradual conformational rigidification led to CDR3 mimetics showing half of the maximal activation potential of the native nanobody. Altogether, this study provides the first proof-of-concept that peptides able to functionally modulate a protein-protein interaction can be obtained by structural mimicry of a nanobody paratope.

bioengineering↗

bioRxiv2022

ChILL & DisCO to discover competitive, connective and allosteric Nanobodies that modulate the SOS1-RAS protein-protein interactions and tune the nucleotide exchange rate

Protein-protein interactions (PPIs) are central in cell metabolism but research tools for the structural and functional characterization of these PPIs are often missing. Here we introduce novel and broadly applicable immunization (Cross-link PPIs and immunize llamas, ChILL) and selection strategies (Display and co-selection, DisCO) for the discovery of diverse Nanobodies that either stabilize or disrupt PPIs in a single experiment. We applied ChILL and DisCO to identify competitive, connective or fully allosteric Nanobodies that inhibit or facilitate the formation of the SOS1*RAS complex and modulate the nucleotide exchange rate on this pivotal GTPase in vitro and RAS signalling in cellulo. One of these connective Nanobodies fills a cavity that was previously identified as the binding pocket for a series of therapeutic lead compounds. The long complementarity-determining region (CDR3) that penetrates this binding pocket serves as an innovative pharmacophore for extending the repertoire of potential leads.

bioengineering↗

bioRxiv2020

Crystal and cryo-EM structures of the cytosolic G protein alpha chaperone and guanine nucleotide exchange factor Ric-8A bound to Gαi1

Ric-8A is a cytosolic Guanine Nucleotide exchange Factor (GEF) that activates heterotrimeric G protein alpha subunits (G)1. Ric-8A is essential to life in multicellular eukaryotes by virtue of its chaperone activity that is required for G biogenesis and membrane localization2, 3. Ric-8A adopts an armadillo (ARM)/HEAT repeat domain architecture and is structurally unrelated to G Protein-Coupled Receptors (GPCR)4. Both GEF and chaperone activities are stimulated by Casein Kinase II phosphorylation5. The mechanisms by which Ric-8A catalyzes GDP release and GTP binding to G, or exerts chaperone activity are unknown. Here, we report the structure of the nanobody-stabilized complex of nucleotide-free Gi1 (isoform 1 of G family i) and phosphorylated Ric-8A at near atomic resolution by cryo-electron microscopy and X-ray crystallography. We find that Ric-8A envelops the GTPase domain of G, disrupting all three switch regions that convey G nucleotide-binding and signaling activity, and displaces the C-terminal helix and helical domain of G. These cooperative interactions dismantle the GDP binding site and promote GDP release, while protecting structural elements of G that are dynamic in the nucleotide-free state. The structures also show how in vivo phosphorylation stabilizes G-binding elements of Ric-8A, thereby enhancing its GEF and chaperone activities.

biochemistry↗

bioRxiv2019

Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM

Nanobodies (Nbs) are popular and versatile tools for structural biology because they have a compact single immunoglobulin domain organization. Nbs bind their target proteins with high affinities while reducing their conformational heterogeneity, and they stabilize multi-protein complexes. Here we demonstrate that engineered Nbs can also help overcome two major obstacles that limit the resolution of single-particle cryo-EM reconstructions: particle size and preferential orientation at the water-air interface. We have developed and characterised novel constructs, termed megabodies, by grafting Nbs into selected protein scaffolds to increase their molecular weight while retaining the full antigen binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we used a megabody to solve the 2.5 [A] resolution cryo-EM structure of a membrane protein that suffers from severe preferential orientation, the human GABAA {beta}3 homopentameric receptor bound to its small-molecule agonist histamine.

biochemistry↗