Biology subjects

Bickel, D.

Publications and source records attributed to Bickel, D..

7 recordsLinked to original sources

bioRxiv2024

Cryo-EM structures of the MnmE-MnmG complex reveal large conformational changes and provide new insights into the mechanism of tRNA modification

MnmE and MnmG form a conserved protein complex responsible for the addition of a 5-carboxymethylaminomethyl (cmnm5) group onto the wobble uridine of several tRNAs. Within this complex, both proteins collaborate intensively to catalyze a tRNA modification reaction that involves glycine as a substrate in addition to three different cofactors, with FAD and NADH binding to MnmG and methylenetetrahydrofolate (5,10-CH2-THF) to MnmE. Without structures of the MnmEG complex it remained enigmatic how these substrates and co-factors can be brought together in a concerted manner. Prior SAXS data suggested that the MnmE (2) and MnmG ({beta}2) homodimers can adopt either an 2{beta}2 or 4{beta}2 complex, depending on the nucleotide state of MnmE. Here, we report the cryo-EM structures of the MnmEG complex in the 2{beta}2 and 4{beta}2 oligomeric states. These structures reveal that MnmE undergoes large conformational changes upon interaction with MnmG, resulting in an asymmetric MnmE dimer. In particular, the functionally important C-terminal helix of MnmE relocates from the 5,10-CH2-THF binding pocket of MnmE to the FAD binding pocket of MnmG, thus suggesting a mechanism for the transfer of an activated methylene group from one active site to the other. Together, these findings provide crucial new insights into the MnmEG-catalyzed reaction. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/625835v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@144e41forg.highwire.dtl.DTLVardef@1dce537org.highwire.dtl.DTLVardef@14c9359org.highwire.dtl.DTLVardef@5dec04_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Identification of non-charged 7.44 analogs interacting with the NHR2 domain of RUNX1-ETO and exhibiting an improved, selective antiproliferative effect in RUNX-ETO positive cells

The RUNX1/ETO fusion protein is a chimeric transcription factor in acute myeloid leukemia (AML) created by chromosomal translocation t(8;21)(q22;q22). t(8;21) abnormality is associated with 12% of de novo AML cases and up to 40% in the AML subtype M2. Previously, we identified the small-molecule inhibitor 7.44, which specifically interferes with NHR2 domain tetramerization of RUNX1/ETO, restores gene expression down-regulated by RUNX1/ETO, inhibits proliferation, and reduces RUNX1/ETO-related tumor growth in a mouse model. However, despite generally favorable physicochemical, pharmacokinetic, and toxicological properties, 7.44 is negatively charged at physiological pH and was predicted to have low to medium membrane permeability. Here, we identified M23, M27, and M10 as non-charged analogs of 7.44 using ligand-based virtual screening, in vivo hit identification, biophysical and in vivo hit validation, and integrative modeling and ADMET predictions. All three compounds interact with the NHR2 domain and show KD,app values of 39-114 {micro}M in Microscale Thermophoresis experiments as well as IC50 values of 33-77 M as to cell viability in RUNX1/ETO-positive KASUMI cells, i.e., are [~]5 to 10-fold more potent than 7.44. M23 is [~]10-fold more potent than 7.44 in inhibiting cell proliferation of RUNX1/ETO-positive cells. M23 and M27 are negligibly protonated or in a [~]1:1 ratio at physiological pH, while M10 has no (de-)protonatable group. The non-protonated species are predicted to be highly membrane-permeable, along with other favorable pharmacokinetic and toxicological properties. These compounds might serve as lead structures for the optimization of binding affinity, bioavailability, and anti-leukemic effects of compounds inhibiting RUNX1/ETO oncogenic function in t(8;21) AML.

pharmacology and toxicology↗

bioRxiv2024

Effects of phosphorylation on protein backbone dynamics and conformational preferences

Phosphorylations are the most common and extensively studied post-translational modification (PTM) of proteins in eukaryotes. They constitute a major regulatory mechanism, modulating protein function, protein-protein interactions, as well as subcellular localization. Phosphorylation sites are preferably located in intrinsically disordered regions and have been shown to trigger structural rearrangements and order-to-disorder transitions. They can therefore have a significant effect on protein backbone dynamics or conformation, but only sparse experimental data are available. To obtain a more general description of how and when phosphorylations have a significant effect on protein behavior, molecular dynamics (MD) currently provides the only suitable framework to study these effects at a large scale in atomistic detail. This study develops a systematic MD simulation framework to explore the influence of phosphorylations on the local backbone dynamics and conformational propensities of proteins. Through a series of glycine-backbone peptides, we studied the effects of amino acid residues including the three most common phosphorylations (Ser, Thr, and Tyr), on local backbone dynamics and conformational propensities. We further extended our study to investigate the interactions of all such residues between position i to positions i+1, i+2, i+3, and i+4 in such peptides. The final dataset is comprised of structural ensembles for 3,393 sequences with more than 1 {micro}s of sampling for each ensemble. To validate the relevance of the results, the structural and conformational properties extracted from the MD simulations are compared to NMR data from the Biological Magnetic Resonance Data Bank. The systematic nature of this study enables the projection of the gained knowledge onto any phosphorylation-site in the proteome and provides a general framework for the study of further PTMs. The full dataset is publicly available, as a training and reference set.

bioinformatics↗

bioRxiv2023

Data-driven probabilistic definition of the low energy conformational states of protein residues

Protein dynamics and related conformational changes are essential for their function but difficult to characterise and interpret. Amino acids in a protein behave according to their local energy landscape, which is determined by their local structural context and environmental conditions. The lowest energy state for a given residue can correspond to sharply defined conformations, e.g., in a stable helix, or can cover a wide range of conformations, e.g., in intrinsically disordered regions. A good definition of such low energy states is therefore important to describe the behavior of a residue and how it changes with its environment. We propose a data-driven probabilistic definition of six low energy conformational states typically accessible for amino acid residues in proteins. This definition is based on solution NMR information of 1,322 proteins through a combined analysis of structure ensembles with interpreted chemical shifts. We further introduce a conformational state variability parameter that captures, based on an ensemble of protein structures from molecular dynamics or other methods, how often a residue moves between these conformational states. The approach enables a different perspective on the local conformational behavior of proteins that is complementary to their static interpretation from single structure models.

bioinformatics↗

bioRxiv2021

Biophysical and pharmacokinetic characterization of a small-molecule inhibitor of RUNX1/ETO tetramerization with anti-leukemic effects

Acute myeloid leukemia (AML) is a malignant disease of immature myeloid cells and the most prevalent acute leukemia among adults. The oncogenic homo-tetrameric fusion protein RUNX1/ETO results from the chromosomal translocation t(8;21) and is found in AML patients. The nervy homology region 2 (NHR2) domain of ETO mediates tetramerization; this oligomerization is essential for oncogenic activity. Previously, we identified the first-in-class small-molecule inhibitor of NHR2 tetramer formation, 7.44, which was shown to specifically interfere with NHR2, restore gene expression down-regulated by RUNX1/ETO, inhibit the proliferation of RUNX1/ETO-depending SKNO-1 cells, and reduce the RUNX1/ETO-related tumor growth in a mouse model. However, no biophysical and structural characterization of 7.44 binding to the NHR2 domain has been reported. Likewise, the compound has not been characterized as to physicochemical, pharmacokinetic, and toxicological properties. Here, we characterize the interaction between the NHR2 domain of RUNX1/ETO and 7.44 by biophysical assays and show that 7.44 interferes with NHR2 tetramer stability and leads to an increase in the dimer population of NHR2. The affinity of 7.44 with respect to binding to NHR2 is Klig = 3.95 {+/-} 1.28 M. By NMR spectroscopy combined with molecular dynamics simulations, we show that 7.44 binds with both heteroaromatic moieties to NHR2 and interacts with or leads to conformational changes in the N-termini of the NHR2 tetramer. Finally, we demonstrate that 7.44 has favorable physicochemical, pharmacokinetic, and toxicological properties. Together with biochemical, cellular, and in vivo assessments, the results reveal 7.44 as a lead for further optimization towards targeted therapy of t(8;21) AML.

biophysics↗

bioRxiv2021

Respiratory and C4-photosynthetic NAD-malic enzyme coexist in bundle sheath cells mitochondria and evolved via association of differentially adapted subunits

In different lineages of Cleomaceae, NAD-malic enzyme (NAD-ME) was independently co-opted to participate in C4 photosynthesis. In the C4 Cleome species Gynandropsis gynandra and Cleome angustifolia, all NAD-ME genes (NAD-ME, NAD-ME{beta}1, and NAD-ME{beta}2) were affected by C4 evolution and are expressed at higher levels than their orthologs in the C3 Cleome species Tarenaya hassleriana. In the latter C3 species, the NAD-ME housekeeping function is performed by two heteromers, NAD-ME/{beta}1 and NAD-ME/{beta}2, with similar biochemical properties. In both C4 species analyzed, this role is restricted the NAD-ME/{beta}2 heteromer. In the C4 species, NAD-ME/{beta}1 is exclusively present in the leaves, where it accounts for most of the enzymatic activity. GgNAD-ME/{beta}1 exhibits high catalytic efficiency and is differentially activated by the C4 intermediate aspartate, confirming its role as the C4-decarboxylase. During C4 evolution, GgNAD-ME{beta}1and CaNAD-ME{beta}1 lost their catalytic activity; their contribution to enzymatic activity results from a stabilizing effect on the associated -subunit. We conclude that in bundle sheath cell mitochondria of C4 Cleome species, the functions of NAD-ME as C4 photosynthetic decarboxylase and as a tricarboxylic acid cycle-associated housekeeping enzyme coexist and are performed by isoforms that combine the same subunit with differentially adapted {beta} subunits.

plant biology↗

bioRxiv2020

Indigenous plants promote insect biodiversity in urban greenspaces

The contribution of urban greenspaces to support biodiversity and provide benefits for people is increasingly recognised. However, ongoing management practices still favour (1) vegetation oversimplification, often limiting greenspaces to lawns and tree canopy rather than multi-layered vegetation that includes under and midstorey; and (2) the use of nonnative plant species. These practices likely hinder the potential of greenspaces to sustain indigenous biodiversity, particularly for taxa like insects, that rely on plants for food and habitat. Yet, little is known about which plant species may maximise positive outcomes for taxonomically and functionally diverse insect communities in urban greenspaces. Additionally, while urban environments are expected to experience high rates of introductions, quantitative assessments of the relative occupancy of indigenous vs. introduced insect species in greenspace are rare - hindering understanding of how greenspace management may promote indigenous biodiversity while limiting the establishment of introduced insects. Using a hierarchically replicated study design across 15 public parks, we recorded occurrence data from 552 insect species on 133 plant species - differing in planting design element (lawn, midstorey and tree canopy), midstorey growth form (forbs, lilioids, graminoids and shrubs) and origin (nonnative, native and indigenous) - to assess: (1) the relative contributions of indigenous and introduced insect species and (2) which plant species sustained the highest number of indigenous insects. Our data indicates that the insect community was predominately composed of indigenous rather than introduced species. Our findings further highlight the core role of multi-layered vegetation in sustaining high insect biodiversity in urban areas, with indigenous midstorey and canopy representing key elements to maintain rich and functionally diverse indigenous insect communities. Intriguingly, graminoids supported the highest indigenous insect richness across all studied growth forms by plant origin groups. Taken together, our study emphasise the opportunity posed by indigenous understory and midstorey plants, particularly indigenous graminoids in our study area, to promote indigenous insect biodiversity in urban greenspaces. Our work provides a blueprint and stimulus for built-environment professionals to incorporate into their practice plant species palettes that foster a larger presence of indigenous over regionally native or nonnative plant species, whilst incorporating a broader mixture of midstorey growth forms.

ecology↗