Biology subjects

Hartmann, M. D.

Publications and source records attributed to Hartmann, M. D..

12 recordsLinked to original sources

bioRxiv2026

Conformational diversity in poly-HAMP arrays and its implications for signal transduction

Prokaryotic transmembrane receptors are built around a helical coiled-coil backbone, with specialized sensory, modulatory, and effector domains arranged along its length. The modulatory HAMP domain forms a four-helix parallel coiled coil that is structurally integrated into this backbone, typically connecting transmembrane segments with downstream cytosolic domains. In many systems, HAMP domains have been shown to transduce signals through axial rotation of their helices; however, it is not clear how broadly applicable this mechanism is. Here, we describe two families of soluble chemoreceptors and sensory kinases that contain long arrays of concatenated HAMP domains, which we term poly-HAMP. Although their poly-HAMP arrays have clearly evolved independently, both families share many sequence features consistent with convergence on a similar functional system. We determined the crystal structures of 4-HAMP and 6-HAMP segments from the poly-HAMP array of histidine kinase HskS of Myxococcus xanthus, revealing unusually tight packing between adjacent domains and conformational patterns compatible with the rotational signaling model. To assess whether these features are general and to define the broader conformational landscape of poly-HAMP arrays, we computed AlphaFold2 models of over 200 chemoreceptor- and kinase-associated arrays. The models were consistent with the HskS structures, yet revealed distinct preferences in the two array types: HAMP domains of chemoreceptor arrays were consistently predicted to adopt stable conformations along their lengths, whereas those of kinase arrays were predicted to be biased toward less favorable conformations. By modeling HAMP domains from kinase arrays in isolation from the neighboring domains, we show that they adopt alternative, stable conformations that are related to the array-embedded forms through axial helix rotation. Taken together, our results suggest that, despite their independent origins and structural diversity, HAMP-containing systems ranging from the poly-HAMP arrays studied here through multi-HAMP architectures to canonical single-HAMP receptors may have converged on the same conserved mode of signal transduction that involves axial helix rotation.

molecular biology↗

bioRxiv2025

Archaeal histone HTkC hypercompacts DNA

Histones are important organizers of chromatin in eukaryotes and archaea. In eukaryotes, the core histones assemble with DNA to form the octameric nucleosome. In archaea, histones form hypernucleosomes that are not restricted to an octameric histone core but can extend to variable lengths. We previously identified face-to-face (FtF) histones as a widely distributed group of archaeal histones that assemble into toroidal tetramer structures, distinct from nucleosomal histones. Here, we characterize the FtF histone HTkC from Thermococcus kodakarensis, which also encodes the canonical histones HTkA and HTkB. We show that HTkC wraps DNA around its toroidal tetramer and forms highly compact nucleoprotein complexes, achieving a level of compaction approximately twice that of hypernucleosomes. Consistent with a major chromatin-organizing role, htkC is among the most highly expressed genes in T. kodakarensis and its deletion leads to impaired growth. Together, these findings establish FtF histones as important organizers of archaeal chromatin alongside classical histones.

biochemistry↗

bioRxiv2025

DNA Wrapping by a Tetrameric Bacterial Histone

Histones are conserved DNA-packaging proteins found across all domains of life. In eukaryotes, canonical histones form octamers that wrap [~]147 base pairs of DNA into nucleosomes -- the fundamental units of chromatin. In archaea, histones form dimers that further multimerize into extended hypernucleosomes along DNA. Although bacteria were long thought to lack histones, recent studies have uncovered histone homologs in diverse bacterial lineages, many of which possess key DNA-binding features. We previously characterized HBb, a bacterial histone from Bdellovibrio bacteriovorus, which binds DNA as a dimer and induces bending. Here, we describe HLp from Leptospira perolatii, a representative of a distinct bacterial histone group. Crystallographic and biophysical analyses reveal that HLp forms stable tetramers. Like HBb, HLp binds DNA non-specifically; however, it adopts a different mode of interaction -- wrapping [~]60 base pairs of DNA around its tetrameric core. This wrapping mode, supported by molecular dynamics simulations and DNA-binding assays, promotes DNA compaction and alters its topology. When expressed heterologously in Escherichia coli, HLp reorganizes nucleoid morphology, consistent with a role in chromatin organization. These findings expand the known repertoire of histone-DNA interaction in bacteria and underscore the structural and functional diversity of histone-based genome organization across the tree of life.

biochemistry↗

bioRxiv2025

Functional relevance of CASP16 nucleic acid predictions as evaluated by structure providers

Accurate biomolecular structure prediction enables the prediction of mutational effects, the speculation of function based on predicted structural homology, the analysis of ligand binding modes, experimental model building and many other applications. Such algorithms to predict essential functional and structural features remain out of reach for biomolecular. Here, we report quantitative and qualitative evaluation of nucleic acid structures for the CASP16 blind prediction challenge by 12 of the experimental groups who provided nucleic acid targets. Blind predictions accurately model secondary structure and some aspects of tertiary structure, including reasonable global folds for some complex RNAs, however, predictions often lack accuracy in the regions of highest functional importance. All models have inaccuracies in non-canonical regions where, e.g., the nucleic-acid backbone bends or a base forms a non-standard hydrogen bond. These bends and non-canonical interactions are integral to form functionally important regions such as RNA enzymatic active sites. Additionally, the modeling of conserved and functional interfaces between nucleic acids and ligands, proteins, or other nucleic acids remains poor. For some targets, the experimental structures may not represent the only structure the biomolecular complex occupies in solution or in its functional life-cycle, posing a future challenge for the community.

biophysics↗

bioRxiv2025

Native metabolomics identifies pteridines as CutA ligands and modulators of copper binding

CutA, a conserved protein across all domains of life, has long been linked to copper tolerance in Escherichia coli, though recent studies question this association. To clarify its function, we studied cutA knockout mutants from two phylogenetically distant species, E. coli and Synechococcus elongatus PCC 7942, using phenotyping combined with targeted and untargeted metabolomics. Native metabolomics of cell extracts revealed the lumazine dehydroxyxanthopterin B2, a previously uncharacterized pteridine, to bind CutA in both species. Based on these results, we identified other pteridines, including the essential cofactor tetrahydrobiopterin, as ligands of CutA proteins. In the presence of pterins, we observed higher affinity of CutA to copper ions. These findings, alongside the known role of pteridines as redox shuttles, suggest a previously unrecognized role for CutA in coordinating copper homeostasis and redox balance via pteridine metabolism. SignificanceWe identified the molecular class of pteridines as natural ligands of CutA, including the so far unknown lumazine dehydroxyxanthopterin B2. Pteridines are known redox shuttles involved in various cellular processes such as cofactors for redox enzymes. Our data showed increased copper binding to CutA in the presence of pteridines. Together, these results suggest that pteridines are physiological ligands of CutA that may modulate copper binding and redox homeostasis.

microbiology↗

bioRxiv2024

Discovery of a synthetic small molecule targeting the central regulator of Salmonella pathogenicity

The enteric pathogen Salmonella enterica serovar Typhimurium relies on the activity of effector proteins to invade, replicate, and disseminate into host epithelial cells and other tissues, thereby causing disease. Secretion and injection of effector proteins into host cells is mediated by dedicated secretion systems, which hence represent major virulence determinants. Here, we report the identification of a synthetic small molecule with drug-like properties, C26, which suppresses the secretion of effector proteins, and consequently hinders bacterial invasion of eukaryotic cells. C26 binds to and inhibits HilD, the transcriptional regulator of the major secretion systems. While sharing the same binding pocket as the previously described long-chain fatty acid ligands, C26 inhibits HilD with a unique binding mode and a distinct mechanism. We provide evidence for target engagement within infected eukaryotic cells and present analogs with improved potency and suitability as scaffolds to develop anti-virulence agents against Salmonella infections in humans and animals.

microbiology↗

bioRxiv2023

Atomic resolution structure of a DNA-binding histone protein from the bacterium Bdellovibrio bacteriovorus

Histones are essential for genome compaction and transcription regulation in eukaryotes, where they assemble into octamers to form the nucleosome core. In contrast, archaeal histones assemble into dimers that form hypernucleosomes upon DNA binding. Although histone homologs have been identified in bacteria recently, their DNA-binding characteristics remain largely unexplored. Our study reveals that the bacterial histone HBb (Bd0055) is indispensable for the survival of Bdellovibrio bacteriovorus, suggesting critical roles in DNA organization and gene regulation. By determining crystal structures of free and DNA-bound HBb, we unveil its distinctive dimeric assembly, diverging from those of eukaryotic and archaeal histones, while also elucidating how it binds and bends DNA through interaction interfaces reminiscent of eukaryotic and archaeal histones. Building on this, by employing various biophysical and biochemical approaches, we further substantiated the ability of HBb to bind and compact DNA by bending in a sequence-independent manner. Finally, using DNA affinity purification and sequencing, we reveal that HBb binds along the entire genomic DNA of B. bacteriovorus without sequence specificity. These distinct DNA-binding properties of bacterial histones, showcasing remarkable similarities yet significant differences from their archaeal and eukaryotic counterparts, highlight the diverse roles histones play in DNA organization across all domains of life. SummaryHistones, traditionally known for organizing and regulating DNA in eukaryotes and archaea, have recently been discovered in bacteria, opening up a new frontier in our understanding of genome organization across the domains of life. Our study investigates the largely unexplored DNA-binding properties of bacterial histones, focusing on HBb in Bdellovibrio bacteriovorus. We reveal that HBb is essential for bacterial survival and exhibits DNA-binding properties similar to archaeal and eukaryotic histones. However, unlike eukaryotic and archaeal histones, which wrap DNA, HBb bends DNA without sequence specificity. This work not only broadens our understanding of DNA organization across different life forms but also suggests that bacterial histones may have diverse roles in genome organization.

molecular biology↗

bioRxiv2023

HilE represses the activity of HilD via a mechanism distinct from that of intestinal long-chain fatty acids.

The expression of virulence factors essential for the invasion of host cells by Salmonella enterica is tightly controlled by a network of transcription regulators. The AraC/XylS transcription factor HilD is the main integration point of environmental signals into this regulatory network, with many factors affecting HilD activity. Long chain fatty acids (LCFAs), which are highly abundant throughout the host intestine directly bind to, and repress HilD, acting as environmental cues to coordinate virulence gene expression. The regulatory protein HilE also negatively regulates HilD activity, through a protein-protein interaction. Both of these regulators inhibit HilD dimerisation, preventing HilD from binding to target DNA. We investigated the structural basis of these mechanisms of HilD repression. LCFAs bind to a conserved pocket in HilD, in a comparable manner to that reported for other AraC/XylS regulators, whereas HilE forms a stable heterodimer with HilD by binding to the HilD dimerisation interface. Our results highlight two distinct mechanisms by which HilD activity is repressed, which could be exploited for the development of new antivirulence leads.

biochemistry↗

bioRxiv2022

Carbon signaling protein SbtB possesses redox-regulated apyrase activity to facilitate regulation of bicarbonate transporter SbtA

The PII superfamily consists of widespread signal transduction proteins found in all domains of life. In addition to canonical PII proteins involved in C/N sensing, structurally similar PII-like proteins evolved to fulfill diverse, yet poorly understood cellular functions. In cyanobacteria, the bicarbonate transporter SbtA is expressed with the conserved PII-like protein, SbtB, to augment intracellular Ci levels for efficient CO2-fixation. We identified SbtB as a sensor of various adenine nucleotides including the second messenger nucleotides cAMP, known as carbon-status indicator, and c-di-AMP, involved in global cellular homeostasis. Moreover, many SbtB proteins possess a C-terminal extension with a disulfide bridge. We previously implied a redox-regulatory function of this extension, which we now call R-loop. Here, we reveal an unusual ATP/ADP apyrase (diphosphohydrolase) activity of SbtB that is controlled by the R-loop. We followed the sequence of the hydrolysis reactions from ATP over ADP to AMP in crystal-lographic snapshots and reveal the structural mechanism by which changes of the R-loop redox state modulate apyrase activity. We further gathered evidence that this redox state is controlled by thioredoxin TrxA, suggesting that it is generally linked to cellular metabolism. Finally, we present a refined model of how SbtB regulates SbtA activity, in which both the apyrase activity and its redox regulation play a central role. This highlights SbtB as a central switch-point in cyanobacterial cell physiology, integrating not only signals from the energy state (adenyl-nucleotide binding) and the carbon supply via cAMP binding, but also from the day/night status reported by the C-terminal redox-switch.

molecular biology↗

bioRxiv2021

Assessing the utility of CASP14 models for molecular replacement

The assessment of CASP models for utility in molecular replacement is a measure of their use in a valuable real-world application. In CASP7, the metric for molecular replacement assessment involved full likelihood-based molecular replacement searches; however, this restricted the assessable targets to crystal structures with only one copy of the target in the asymmetric unit, and to those where the search found the correct pose. In CASP10, full molecular replacement searches were replaced by likelihood-based rigid-body refinement of models superimposed on the target using the LGA algorithm, with the metric being the refined likelihood (LLG) score. This enabled multi-copy targets and very poor models to be evaluated, but a significant further issue remained: the requirement of diffraction data for assessment. We introduce here the relative-expected-LLG (reLLG), which is independent of diffraction data. This reLLG is also independent of any crystal form, and can be calculated regardless of the source of the target, be it X-ray, NMR or cryo-EM. We calibrate the reLLG against the LLG for targets in CASP14, showing that it is a robust measure of both model and group ranking. Like the LLG, the reLLG shows that accurate coordinate error estimates add substantial value to predicted models. We find that refinement by CASP groups can often convert an inadequate initial model into a successful MR search model. Consistent with findings from others, we show that the AlphaFold2 models are sufficiently good, and reliably so, to surpass other current model generation strategies for attempting molecular replacement phasing.

bioinformatics↗

bioRxiv2020

The proteasome-related Connectase is a fast and specific protein ligase

Genetic methods allow the recombinant production of any protein of interest, but yield the full-length construct in one step and are limited to native amino acids. For the "on demand" generation of chimeric, immobilized, fluorophore-conjugated or segmentally labeled proteins, these proteins must be modified using chemical1,2, (split) intein2,3, split domain4 or enzymatic methods5. While each of these options comes with its own advantages and drawbacks, ligase enzymes are often used where small ligation motifs and good chemoselectivity are required. However, applications with the reference enzyme Sortase A are impeded by poor catalytic efficiencies, low substrate specificities and side reactions6,7. Here, we present the discovery of Connectase, a monomeric proteasome homolog that ligates substrates via a highly conserved KDPGA motif originally identified in methyltransferase A (MtrA), a key enzyme in archaeal methanogenesis. Connectase displays nanomolar affinity and thus great specificity for its substrates, allowing efficient protein-protein ligations even in complex solutions and at low substrate concentrations. Compared to an optimized Sortase variant, Connectase catalyzes such ligations at substantially higher rates, with higher yields but without detectable side reactions and thus presents a valuable new tool for protein conjugations.

biochemistry↗

bioRxiv2019

Structural diversity of oligomeric β-propellers with different numbers of identical blades

{beta}-Propellers arise through the amplification of a supersecondary structure element called a blade. This process produces toroids of between four and twelve repeats, which are almost always arranged sequentially in a single polypeptide chain. We found that new propellers evolve continuously by amplification from single blades. We therefore investigated whether such nascent propellers can fold as homo-oligomers before they have been fully amplified within a single chain. One-to six-bladed building blocks derived from two seven-bladed WD40 propellers yielded stable homo-oligomers with six to nine blades, depending on the size of the building block. High-resolution structures for tetramers of two blades, trimers of three blades, and dimers of four and five blades, respectively, show structurally diverse propellers and include a novel fold, highlighting the inherent flexibility of the WD40 blade. Our data support the hypothesis that subdomain-sized fragments can provide structural versatility in the evolution of new proteins.

biochemistry↗