Biology subjects

Brunner, J. D.

Publications and source records attributed to Brunner, J. D..

8 recordsLinked to original sources

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

Themis and Grb2 form a constitutive structural hub in T cell receptor signalling

Positive selection of thymocytes is essential for laying the foundations of the mammalian immune system that include the T cell repertoire, self-tolerance, and prevention of autoimmunity. Themis, the archetypal member of a metazoan protein family featuring distinctive CABIT domains, crucially regulates thymocyte positive selection by linking signalling by the T cell receptor (TCR) to the linker of activation of TCR (LAT). Intriguingly, Themis has been proposed to function via a constitutive complex with the multifunctional adaptor Grb2. Although poised to represent a paradigm shift in our understanding of TCR signalling, the structural and mechanistic basis of such an assembly has remained enigmatic. Here, we present the cryo-EM structure of Themis in complex with Grb2, which reveals how the tandem CABIT domains of Themis engulf the C-terminal SH3 domain of Grb2 (Grb2SH3C) to enable its latching onto the proline rich sequence of Themis. The remaining two domains of Grb2 adopt at least three conformational poses set to interact with other binding partners such as Sos1. Structural insights from unbound Themis unmask the pronounced flexibility of the CABIT domains of Themis, which becomes ordered upon binding to Grb2 to create a binding hotspot for their constitutive complex. Indeed, Themis variants that abrogate interactions with Grb2 also fail to activate the tyrosine phosphatase SHP-1 after TCR stimulation, analogous to the functional phenotype of Themis-deficient cells. Collectively, our study draws the blueprint of the Themis-Grb2 complex as a dynamic structural hub in T cell development.

immunology↗

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

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↗

bioRxiv2020

Epigenome Prediction of Gene Expression using a Dynamical System Approach

Gene regulation is an important fundamental biological process. The regulation of gene expression is managed through a variety of methods including epigentic processes (e.g., DNA methylation). Understanding the role of epigenetic changes in gene expression is a fundamental question of molecular biology. Predictions of gene expression values from epigenetic data have tremendous research and clinical potential. Despite active research, studies to date have focused on using statistical models to predict gene expression from methylation data. In contrast, dynamical systems can be used to generate a model to predict gene expression using epigenetic data and a gene regulatory network (GRN) which can also serve as a mechanistic hypothesis. Here we present a novel stochastic dynamical systems model that predicts gene expression levels from methylation data of genes in a given GRN. Software for dataset preparation, model parameter fitting and prediction generation, and reporting are available at https://github.com/kordk/stoch_epi_lib.

systems biology↗

bioRxiv2020

Minimizing the number of optimizations for efficient community dynamic flux balance analysis.

Dynamic flux balance analysis uses a quasi-steady state assumption to calculate an organisms metabolic activity at each time-step of a dynamic simulation, using the well-know technique of flux balance analysis. For microbial communities, this calculation is especially costly and involves solving a linear constrained optimization problem for each member of the community at each time step. However, this is unnecessary and inefficient, as prior solutions can be used to inform future time steps. Here, we show that a basis for the space of internal fluxes can be chosen for each microbe in a community and this basis can be used to simulate forward by solving a relatively inexpensive system of linear equations at most time steps, instead of the full optimization problem. Using our method, we can use this solution as long as the resulting metabolic activity remains within the optimization problems constraints (i.e. the solution remains feasible). As the solution becomes infeasible, it first becomes a feasible but degenerate solution to the optimization problem, and we can solve a different but related optimization problem to choose an appropriate basis to continue forward simulation. We show using an eight species community that this is an efficient and robust method for computing dynamic flux balance analysis simulations, and so is capable of simulating communities of organisms. We demonstrate that the method gives an approximately 85% speed-up per organism over the standard and widely used method. Our method has been implemented in the Python language and source code is available at https://github.com/jdbrunner/surfin_fba and in the Python Package Index (PyPI) as surfinFBA. Author summaryThe standard method in the field for dynamic flux balance analysis carries a prohibitively high computational cost because it requires solving a linear optimization problem at each time-step. We have developed a novel method for producing solutions to this dynamical system which greatly reduces the number of optimization problems that must be solved. We prove mathematically that we can solve the optimization problem once and simulate the system forward as an ordinary differential equation for some time interval, and solutions to this ODE provide solutions to the optimization problem. Eventually, the system reaches an easily checkable condition which implies that another optimization problem must be solved. We compare our method with the classical method to validate that it provides equivalent solutions in much lower computational time.

systems biology↗

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↗