Biology subjects

Nygaard, R.

Publications and source records attributed to Nygaard, R..

4 recordsLinked to original sources

bioRxiv2026

Genetically engineered rapamycin responsive K2P channels

Establishment of electrical potentials across biological membranes is a universal feature of all cells. Tandem pore domain (K2P) potassium ion channels play pivotal roles in maintaining cellular membrane potentials, shaping physiological responses across a diverse range of cell types. With only a limited repertoire of high-aAinity and subtype-selective K2P modulators available for experimental or therapeutic use, we devised a strategy to genetically engineer K2P channels that are potently activated by rapamycin or non-immunomodulatory rapamycin analogs. Insertion of the FRB domain of mTOR into a short flexible cytoplasmic loop between the second and third transmembrane (TM) domains of the TREK1 K2P channel yielded fusion channels that are activated by nanomolar concentrations of rapamycin. Rapamycin-induced potentiation requires recruitment of an FKBP binding partner, from either the endogenous pool of FKBP within the cell or through fusion of FKBP to the C-terminus of TREK1. Formation of an FRB/rapamycin/FKBP ternary complex within the core of the TREK1 channel leads to an increase in TREK1 single-channel open probability and unitary current, mimicking positive modulatory eAects of conventional TREK1 activating cues. Cryo-EM structures demonstrate that rapamycin-induced ternary complex formation rigidifies the position of the FRB and stabilizes the TM2/TM3 loop in an active channel conformation. We demonstrate that FRB fusion can be employed to successfully activate several K2P channel isoforms, providing chemogenetically targetable tools for direct manipulation of cellular membrane potential.

bioengineering↗

bioRxiv2024

Bridging species boundaries: eDNA and genetic analysis reveal hybrid settlement at the very end of American eel distribution

Documenting species distributions and hybridization patterns is paramount for elucidating biogeography and understanding speciation processes. Here we combined genetic specimen analysis and environmental DNA (eDNA) to investigate the presence of American eel (Anguilla rotrata) and American x European eel (Anguilla anguilla) hybrids in Greenland freshwater. We further tested the use of eDNA to document hybridization by using European eel mtDNA as a proxy for hybrid occurrence. Overall, eDNA analysis detected mainly American eel but also European eel mtDNA. This finding was validated by DNA sequencing, which identified 3 out of 26 captured eels (14.3%) carrying European eel mtDNA. Five eels (19.2%), including all three with European mtDNA, were heterozygous for species-specific nuclear gene variants, supporting a hybrid ancestry. Further, eDNA successfully identified eels in lakes where they were caught by fyke net fishing, extending their confirmed northern range by 40 km, and indicated eel presence >200km further north. The study provides an empirical demonstration of the use of eDNA to document hybrid occurrence and extends the reported northern distribution of eels in Greenland significantly beyond previous observations. Further, the existence of hybrid eels in Greenland may be key for understanding the complex mechanisms of hybridization between American and European eels.

genetics↗

bioRxiv2024

Structural insights into terminal arabinosylation biosynthesis of the mycobacterial cell wall arabinan

The emergence of drug-resistant strains exacerbates the global challenge of tuberculosis caused by Mycobacterium tuberculosis (Mtb). Central to the pathogenicity of Mtb is its complex cell envelope, which serves as a barrier against both immune system and pharmacological attacks. Two key components of this envelope, arabinogalactan (AG) and lipoarabinomannan (LAM) are complex polysaccharides that contain integral arabinan domains important for cell wall structural and functional integrity. The arabinofuranosyltransferase AftB terminates the synthesis of these arabinan domains by catalyzing the addition of {beta}-(1[->]2)-linked terminal arabinofuranose residues. Here, we present the cryo-EM structures of Mycobacterium chubuense AftB in its apo and donor substrate analog-bound form, determined to 2.9 [A] and 3.4 [A] resolution, respectively. Our structures reveal that AftB has a GT-C fold transmembrane (TM) domain comprised of eleven TM helices and a periplasmic cap domain. AftB has an irregular tube-shaped cavity that bridges the two proposed substrate binding sites. By integrating structural analysis, biochemical assays, and molecular dynamics simulations, we elucidate the molecular basis of the reaction mechanism of AftB and propose a model for catalysis.

biochemistry↗

bioRxiv2024

Mechanistic studies of mycobacterial glycolipid biosynthesis by the mannosyltransferase PimE

Tuberculosis (TB), exceeded in mortality only by COVID-19 among global infectious diseases, is caused by Mycobacterium tuberculosis (Mtb). The pathogenicity of Mtb is largely attributed to its complex cell envelope, which includes a class of glycolipids called phosphatidyl-myo-inositol mannosides (PIMs), found uniquely in mycobacteria and its related corynebacterineae. These glycolipids maintain the integrity of the mycobacterial cell envelope, regulate its permeability, and mediate host-pathogen interactions. PIMs consist of a phosphatidyl-myo-inositol core decorated with one to six mannose residues and up to four acyl chains. The mannosyltransferase PimE catalyzes the transfer of the fifth PIM mannose residue from a polyprenyl phosphate-mannose (PPM) donor. This step in the biosynthesis of higher-order PIMs contributes to the proper assembly and function of the mycobacterial cell envelope; however, the structural basis for substrate recognition and the catalytic mechanism of PimE remain poorly understood. Here, we present the cryo-electron microscopy (cryo-EM) structures of PimE from Mycobacterium abscessus captured in its apo form and in a product-bound complex with the reaction product Ac1PIM5 and the by-product polyprenyl phosphate (PP), determined at 3.0 [A] and 3.5 [A], respectively. The structures reveal the active site within a distinctive binding cavity that accommodates both donor and acceptor substrates/products. Within the cavity, we identified residues involved in substrate coordination and catalysis, which we confirmed through in vitro enzymatic assays and further validated by in vivo complementation experiments. Molecular dynamics simulations were applied to identify the access pathways and the dynamics involved in substrate binding. Integrating structural, biochemical, genetic, and computational experiments, our study provides comprehensive insights into how PimE functions, opening potential avenues for development of novel anti-TB therapeutics.

biochemistry↗