Biology subjects

Karlsen, J. L.

Publications and source records attributed to Karlsen, J. L..

5 recordsLinked to original sources

bioRxiv2025

Structure and function of a fungal AB toxin-like chimerolectin involved in anti-nematode defense

Fungal defense against predators largely relies on protein toxins, many of which are lectins. We previously showed that the production of the nematotoxin CCTX2 is upregulated in the Agaricomycete Coprinopsis cinerea upon predation by nematodes. Here, we classify CCTX2 as the founding member of a family of fungal chimerolectins. Cryo-EM analysis to 3.2 [A] resolution reveals five domains. The four N-terminal {beta}-trefoil fold (BTF) domains cradle a C-terminal domain, which exhibits a novel +{beta} protein fold. Mutational analysis shows that both N-terminal and C-terminal domains are required for nematotoxicity. While the biochemical function of the C-terminal domain remains unclear, the first two BTF domains enable CCTX2 to bind to glycosphingolipids with LacNAc or LacdiNAc glycoepitopes on nematode intestinal epithelial cells. Experiments in the model nematode Caenorhabditis elegans demonstrate that the chimerolectin CCTX2 exploits the endocytic and retrograde trafficking machinery of the target cell to exert its toxicity and obtain access to the yet-to-be-identified intracellular target of the non-lectin domain. The structure and mode of action of CCTX2 is reminiscent of bacterial and plant AB toxins.

microbiology↗

bioRxiv2022

Structural remodelling of the carbon-phosphorus lyase machinery by a dual ABC ATPase

Phosphorus is an essential macronutrient for all microorganisms and can be extracted from the environment by several metabolic pathways. In Escherichia coli, the 14-cistron phn operon encoding the carbon-phosphorus (C-P) lyase enzymatic machinery allows for extraction of phosphorus from a wide range of phosphonates characterised by the highly stable C-P bond.1, 2 As part of a complex, multi-step pathway, the PhnJ subunit was proposed to cleave the C-P bond via a radical reaction, however, the details of the mechanism were not immediately compatible with the structure of the 220 kDa PhnGHIJ C-P lyase core complex, leaving a significant gap in our understanding of phosphonate breakdown in bacteria.3, 4 Here we show using single-particle cryogenic-electron microscopy that PhnJ mediates binding of a unique double dimer of ATP-binding cassette (ABC) proteins, PhnK and PhnL to the core complex. ATP hydrolysis by PhnK induces drastic structural remodelling leading to opening of the core and reconfiguration of a metal-binding site located at the interface between the PhnI and PhnJ subunits. Our results offer new insights into the mechanism underlying C-P lyase and uncover a hitherto unknown configuration of ABCs that have wide-ranging implications for our understanding of the role of this module in biological systems.

biochemistry↗

bioRxiv2021

Cryo-EM structure of the human NKCC1 transporter reveals mechanisms of ion coupling and specificity

The sodium-potassium-chloride transporter NKCC1 (SLC12A2) performs Na+-dependent Cl- and K+ ion uptake across plasma membranes. NKCC1 is important for regulating e.g. cell volume, hearing, blood pressure, and chloride gradients defining GABAergic and glycinergic signaling in brain. Here, we present a 2.6 [A] resolution cryo-electron microscopy (cryo-EM) structure of human NKCC1 in the substrate-loaded (Na+, K+, 2 Cl-) and inward-facing conformation adopting an occluded state that has also been observed for the SLC6 type transporters MhsT and LeuT. Cl- binding at the Cl1 site together with the nearby K+ ion provide a crucial bridge between the LeuT-fold scaffold and bundle domains. Cl- ion binding at the Cl2 site seems to undertake a structural role similar to a conserved glutamate of SLC6 transporters and may allow for chloride-sensitive regulation of transport. Supported by functional studies in mammalian cells and computational simulations we describe the Na+ binding site and a putative Na+ release pathway along transmembrane helix 5. The results provide insight into the structure-function relationship of NKCC1 with broader implications for other SLC12 family members.

biochemistry↗

bioRxiv2019

Structure and autoregulation of a P4-ATPase lipid flippase

P4-ATPases are lipid flippases that drive active transport of phospholipids from the exoplasmic or lumenal to the cytosolic leaflets of eukaryotic membranes to maintain their asymmetric lipid composition. The molecular architecture of P4-ATPases and how they work in lipid recognition and transport has remained elusive. Using cryo-electron microscopy we have determined the structures of a P4-ATPase, specifically of the Saccharomyces cerevisiae Drs2p-Cdc50p, which is a phosphatidylserine and phosphatidylethanolamine specific lipid flippase. Drs2p-Cdc50p is autoinhibited by the Drs2p C-terminal tail and activated by phosphatidylinositol-4 phosphate (PI4P). We present three structures representing an autoinhibited, an intermediate, and a fully activated state. The analysis highlights specific features of P4-ATPases and reveals sites of auto-inhibition and PI4P-dependent activation. We observe the opening of a putative flippase pathway engaging conserved residues Ile508 of transmembrane segment 4 and Lys1018 and polar residues of transmembrane segment 5 in the centre of the lipid bilayer.

molecular biology↗

bioRxiv2018

Namdinator - Automatic Molecular Dynamics flexible fitting of structural models into cryo-EM and crystallography experimental maps

Model building into experimental maps is a key element of structural biology, but can be both time consuming and error-prone. Here we present Namdinator, an easy-to-use tool that enables the user to run a Molecular Dynamics Flexible Fitting (MDFF) simulation in an automated manner through a pipeline system. Namdinator will modify an atomic model to fit within cryo-EM or crystallography density maps, and can be used advantageously for both the initial fitting of models, and for a geometrical optimization step to correct outliers, clashes and other model problems. We have benchmarked Namdinator against 39 deposited models and maps from cryo-EM and observe model improvements in 34 of these cases (87%). Clashes between atoms were reduced, and model-to-map fit and overall model geometry were improved, in several cases substantially. We show that Namdinator is able to model large scale conformational changes compared to the starting model. Namdinator is a fast and easy way to create suitable initial models for both cryo-EM and crystallography. It can fix model errors in the final steps of model building, and is usable for structural model builders at all skill levels. Namdinator is available as a web service (https://namdinator.au.dk), or can be run locally as a command-line tool.\n\nSynopsisA pipeline tool called Namdinator is presented that enables the user to run a Molecular Dynamics Flexible Fitting (MDFF) simulation in a fully automated manner, both online and locally. This provides a fast and easy way to create suitable initial models for both cryo-EM and crystallography and help fix errors in the final steps of model building.

biophysics↗