Biology subjects

Haubrich, K.

Publications and source records attributed to Haubrich, K..

6 recordsLinked to original sources

bioRxiv2025

Two-dimensional NMR from a Single Pulse: Reconstructing Heteronuclear 2D spectra via off-resonance decoupling and Deep Neural Networks

Nuclear Magnetic Resonance (NMR) spectroscopy is ubiquitous in many areas of science, including chemistry, material science, biophysics and structural biology. Heteronuclear two-dimensional (2D) NMR experiments form the basis of several NMR based investigations, especially those involving biomolecules like proteins. However, in solution, as the molecule of interest becomes larger, transverse relaxation times of the spins of interest become shorter. This makes it difficult to record 2D 1H-13C or 1H-15N correlation maps of large protein molecules using standard Fourier-transform based experiments as they contain transfer delays that are long compared to the short relaxation times. Herein, we explore the possibility of leveraging deep neural networks (DNNs) to obtain 2D correlation maps of proteins while eliminating transfer delays from experiments. In this study, we show that 2D methyl 1H-13C correlation maps can be obtained from experiments containing only a single 1H excitation pulse followed by off-resonance 13C continuous-wave decoupling. A DNN was successfully trained to reconstruct the 2D 1H-13C correlation map from datasets recorded with two 13C decoupling fields using protein samples enriched with (ILV) 13CHD2 methyl groups in a 2H background. The efficacy of this strategy is demonstrated by using the DNN to reconstruct methyl 1H-13C correlation maps of the [~]8 kDa FF domain from human HYPA/FBP11, the [~]18 kDa T4 phage lysozyme, as well as on the [~]360 kDa 77 particle from the Thermoplasma acidophilum proteasome. This study illustrates the potential for improving the sensitivity and resolution of NMR spectra using new experiments tailored for use with DNNs. Significance statementMultidimensional heteronuclear solution state nuclear magnetic resonance (NMR) spectroscopy underpins studies of protein dynamics and interactions. However, the experiments have thus far required magnetization-transfer periods and evolution delays, both of which rapidly erode the signal arising from large biomolecules. We introduce here a single-pulse strategy wherein these delays are replaced by off-resonance continuous-wave decoupling so that the two-dimensional correlation map can be reconstructed from the off-resonance decoupling data using a deep neural network. The approach yields high-quality methyl correlation spectra for proteins spanning [~]8-360 kDa. The co-design of NMR experiments along with deep neural networks establishes a general framework to acquire multidimensional NMR spectra using nontraditional strategies that can be extended to other spin systems and higher-dimensional experiments.

biophysics↗

bioRxiv2025

LRRC58 defines an E3 ubiquitin ligase complex sensitive to cysteine abundance

Adaptation to fluctuating nutrient supply is essential for organismal survival, but how cells sense and respond to changes in the abundance of many critical nutrients remains undefined. One such example is the amino acid cysteine, whose reactive thiol group is exploited for diverse cellular functions. Here, by characterizing the machinery required for the conditional degradation of cysteine dioxygenase type I (CDO1), the critical enzyme responsible for cysteine catabolism, we identify a Cul2 E3 ligase complex containing the uncharacterized substrate adaptor LRRC58 that is sensitive to cysteine abundance. When cysteine is replete, LRRC58 activity is restrained through auto-ubiquitination and proteasomal degradation; upon cysteine deprivation, LRRC58 is stabilized to permit CDO1 degradation. Through saturation mutagenesis stability profiling, we systematically validate a structural model of the CDO1-LRRC58 interaction and identify residues at the LRRC58 C-terminus required for cysteine-dependent instability. The LRRC58-mediated degradation of CDO1 is essential to prevent ferroptotic cell death under conditions of cysteine scarcity, and mutations in CDO1 which cause neurodevelopmental defects in humans encode dominant-active proteins refractory to LRRC58 recognition. Altogether, these data reveal the CDO1-LRRC58 axis as a critical regulator of cysteine homeostasis that safeguards neural development.

molecular biology↗

bioRxiv2024

Design of a Cereblon construct for crystallographic and biophysical studies of protein degraders

The ubiquitin E3 ligase cereblon (CRBN) is the target of therapeutic drugs thalidomide and lenalidomide and is recruited by most targeted protein degraders (PROTACs and molecular glues) in clinical development. Biophysical and structural investigation of CRBN has been limited by current constructs that either require co-expression with the adaptor DDB1 or inadequately represent full-length protein, with high-resolution structures of degraders ternary complexes remaining rare. We present the design of CRBNmidi, a construct that readily expresses from E. coli with high yields as soluble, stable protein without DDB1. We benchmark CRBNmidi for wild-type functionality through a suite of biophysical techniques and solve high-resolution co-crystal structures of its binary and ternary complexes with degraders. We qualify CRBNmidi as an enabling tool to accelerate structure-based discovery of the next generation of CRBN based therapeutics. One sentence summaryA novel Cereblon construct (CRBNmidi) allows structural and biophysical enablement of ligand and degrader design

biochemistry↗

bioRxiv2021

Mechanism of riboregulation of p62 protein oligomerisation by vault RNA1-1 in selective autophagy

Macroautophagy ensures the clearance of intracellular substrates ranging from single ubiquitinated proteins to large proteotoxic aggregates and defective organelles. The selective autophagy receptor p62 binds these targets and recruits them to double-membrane vesicles, which fuse with lysosomes to degrade their content. We recently uncovered that p62 function is riboregulated by the small non-coding vault RNA1-1. Here, we present detailed insight into the underlying mechanism. We show that the PB1 domain and adjacent linker region of p62 (aa 1-122) are necessary and sufficient for specific vault RNA1-1 binding, and identify lysine 7 and arginine 21 as key hinges for p62 riboregulation. Chemical structure probing of vault RNA1-1 further reveals a central flexible loop within the RNA that mediates the specific p62 interaction. Our data define molecular determinants that govern mammalian autophagy via the p62-vault RNA1-1 riboregulatory pair.

molecular biology↗

bioRxiv2021

Validation and classification of RNA binding proteins identified by mRNA interactome capture

RNA binding proteins (RBPs) take part in all steps of the RNA life cycle and are often essential for cell viability. Most RBPs have a modular organization and comprise a set of canonical RNA binding domains. However, in recent years a number of high-throughput mRNA interactome studies on yeast, mammalian cell lines and whole organisms have uncovered a multitude of novel mRNA interacting proteins that lack classical RNA binding domains. Whereas a few have been confirmed to be direct and functionally relevant RNA binders, biochemical and functional validation of RNA binding of most others is lacking. In this study, we employed a combination of NMR spectroscopy and biochemical studies to test the RNA binding properties of six putative RNA binding proteins. Half of the analysed proteins showed no interaction, whereas the other half displayed weak chemical shift perturbations upon titration with RNA. One of the candidates we found to interact weakly with RNA in vitro is Drosophila melanogaster End binding protein 1 (EB1), a master regulator of microtubule plus-end dynamics. Further analysis showed that EB1s RNA binding occurs on the same surface as that with which EB1 interacts with microtubules. RNA immunoprecipitation and colocalization experiments suggest that EB1 is a rather non-specific, opportunistic RNA binder. Our data suggest that care should be taken when embarking on an RNA binding study involving these unconventional, novel RBPs, and we recommend initial and simple in vitro RNA binding experiments.

biochemistry↗

bioRxiv2020

RNA binding regulates TRIM25-mediated RIG-I ubiquitylation

TRIM25 is a ubiquitin E3 ligase active in innate immunity and cell fate decisions. Mounting evidence suggests that TRIM25's E3 ligase activity is regulated by RNAs. However, while mutations affecting RNA binding have been described, neither the precise RNA binding site has been identified nor which domains are involved. Here, we present biophysical evidence for the presence of RNA binding sites on both TRIM25 PRY/SPRY and coiled-coil domains, and map the binding site on the PRY/SPRY with residue resolution. Cooperative RNA-binding of both domains enhances their otherwise transient interaction in solution and increases the E3 ligase activity of TRIM25. We also show that TRIM25 not only binds RNA in mammalian cells but that interfering with RNA binding has an effect on cellular RIG-I ubiquitination.

molecular biology↗