Biology subjects

Schenck, S.

Publications and source records attributed to Schenck, S..

7 recordsLinked to original sources

bioRxiv2026

Antibiotics promote susceptibility to C. difficile infection through a CCR5-dependent immune response

Antibiotics (ABXs) represent the current standard of care for treating Clostridioides difficile infection (CDI). Paradoxically, ABX-induced dysbiosis is the primary risk factor for CDI, as disruption of the colonic microbial ecosystem creates an opportunity for C. difficile colonization. Given that ABXs can also alter immune responses, we investigated whether ABXs prime the colonic immune milieu for CDI susceptibility. Here, we implicate ABXs in driving CDI severity through the emergence of pathogenic CCR5-reliant immune populations in the mouse colon. High-throughput immune cell profiling revealed that ABXs shift the colonic immune compartment toward a CCR5-associated type I immunity signature, marked by an expansion of CCR5+ ILC1s and CCR5+ Th1 cells. A partial genetic deletion of CCR5 reversed CDI severity, alleviating colonic inflammation and improving survival. Pharmacological inhibition of the CCL3/4/5-CCR5 circuit also recapitulated these favorable disease outcomes, which we attribute to reduced colonic CCR5+ ILC1, CCR5+ Th1, and CCR5+ CD8 T cell populations during CDI. Together, our findings extend beyond dysbiosis as the canonical CDI risk factor and establish ABX-induced immune imbalance as an underappreciated determinant of CDI susceptibility.

immunology↗

bioRxiv2025

Stereoselectivity and functional plasticity of a common ligand-binding pocket in TRPM3

The transient receptor potential melastatin 3 (TRPM3) channel is a key mediator of peripheral pain signaling, and pathogenic mutations in TRPM3 are linked to neurodevelopmental delay and epilepsy. Despite the therapeutic promise of TRPM3 modulators, the molecular mechanisms by which ligands modulate channel gating remain poorly understood. Here, we combine cryo-electron microscopy (cryo-EM) with functional analyses to characterize a promiscuous ligand-binding pocket formed by transmembrane helices S1-S4. This pocket accommodates several chemically diverse plant-derived and synthetic agonists and antagonists. We uncover unanticipated stereoselectivity of TRPM3 for the non-natural enantiomer of the flavonoid antagonist isosakuranetin and the (R)-enantiomer of the synthetic agonist CIM0216. Mutations within this pocket--including newly identified patient variants--alter ligand affinity and, in some cases, invert the functional outcome of ligand binding. These findings reveal the stereoselectivity and functional plasticity of the TRPM3 ligand-binding pocket, highlighting how subtle changes in the molecular interactions can produce divergent effects on channel gating, with important ramifications for TRPM3-targeted drug development and therapy.

biochemistry↗

bioRxiv2025

MISO: Microfluidic protein isolation enables single particle cryo-EM structure determination from a single cell colony

Single particle cryo-EM enables reconstructing near-atomic or even atomic resolution 3D maps of proteins by visualizing thousands to a few million purified protein particles embedded in nanometer- thick vitreous ice. This corresponds to picograms of purified protein, which can potentially be isolated from a few thousand cells. Hence, cryo-EM holds the potential of one of the most sensitive analytical methods that deliver a high-resolution protein structure as a readout. In practice, more than a million times more starting biological material is required to prepare cryo-EM grids. To close the gap, we developed a micro isolation (MISO) method that combines microfluidics-based protein purification with cryo-EM grid preparation. We validated the method on soluble bacterial and eukaryotic membrane proteins. We showed that MISO enables protein structure determination starting from below one microgram of a target protein and going from cells to cryo-EM grids within a few hours. This scales down the purification by a factor of a few hundred to a few thousand and opens possibilities for the structural characterization of hitherto inaccessible proteins.

biophysics↗

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↗

bioRxiv2024

Structures of native SV2A reveal the binding mode for tetanus neurotoxin and anti-epileptic racetams

The synaptic vesicle glycoprotein 2A (SV2A) is a bona fide synaptic vesicle (SV) constituent of controversial function with homology to the major facilitator superfamily (MFS) and essential in vertebrate neurotransmission. Despite its high medical relevance as the target of the anti-epileptic drug Levetiracetam (LEV) and as receptor for clostridial neurotoxins (CNTs), among them several botulinum neurotoxin (BoNT) serotypes and potentially tetanus neurotoxin (TeNT), we lack detailed insight about these molecular interactions. We purified native SV2A from brain and subjected it to a structural analysis to advance our understanding of drug-binding to this enigmatic protein and explore structurally uncharacterized toxin-SV2A interfaces. Our analysis uncovered that TeNT binds SV2 proteins strikingly different from BoNT/A and delivers visual evidence for the dual receptor hypothesis through structurally resolved, co-purified gangliosides in the complex. The structures provide compelling support for SV2A as the protein receptor for TeNT in central neurons, recapitulate the geometry of CNT binding to dipartite SV2-ganglioside receptors on neuronal surfaces in a membrane-like constellation and have implications for toxin-engineering through a previously unknown SV2A-toxin interface. Further, a LEV-bound structure of SV2A reveals the drug-interacting residues and delineates a putative substrate pocket in SV2A. Our work provides an explanation for the SV2-isoform-specificity of LEV and its derivatives and paves the way for improved design of anti-convulsant drugs in epilepsy treatment.

biochemistry↗

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↗

bioRxiv2018

Structural basis for ion selectivity in TMEM175 K+ channels

The TMEM175 family constitutes recently discovered K+ channels that lack signatures for a P-loop selectivity filter, a hallmark of all known K+ channels. This raises the question how selectivity in TMEM175 channels is achieved. Here we report the X-ray structure of a bacterial TMEM175 family member in complex with a novel chaperone built of a nanobody fusion-protein. The structure of the channel in a non-conductive conformation was solved at 2.4 [A] and revealed bound K+ ions along the channel pore. A hydrated K+ ion at the extracellular pore entrance that could be substituted with Cs+ and Rb+ is coordinated by backbone-oxygens forming a cation-selective filter at the tip of the pore-lining helices. Another K+ ion within the pore indicates the passage of dehydrated ions. Unexpectedly, a highly conserved threonine residue deeper in the pore conveys the K+ selectivity. The position of this threonine in the non-conductive state suggests major conformational rearrangements of the pore-lining helices for channel opening, possibly involving iris-like motions.

biochemistry↗