Biology subjects

Kragelund, B. B.

Publications and source records attributed to Kragelund, B. B..

22 records · Page 2Linked to original sources

bioRxiv2020

Dynamic NHE1-Calmodulin complexes of varying stoichiometry and structure regulate Ca2+-dependent NHE1 activation

Calmodulin (CaM) engages in Ca2+-dependent interactions with numerous proteins, including human Na+/H+-exchanger NHE1. Using nuclear magnetic resonance (NMR) spectroscopy, isothermal titration calorimetry, and fibroblasts expressing wildtype and mutant NHE1, we discovered multiple accessible states of this important complex existing in different NHE1:CaM stoichiometries and structures. We solved the NMR solution structure of a ternary complex in which CaM links two NHE1 cytosolic tails. In vitro, stoichiometries and affinities were tunable by variations in NHE1:CaM ratio and calcium ([Ca2+]) and by phosphorylation of S648 in the first CaM-binding -helix. In cells, Ca2+-CaM-induced NHE1 activity was reduced by mimicking S648 phosphorylation or mutating the first CaM-binding helix, whereas Ca2+-induced NHE1 activity was unaffected by inhibition of Akt, one of several kinases phosphorylating S648. Our results reveal the diversity of NHE1:CaM interactions and suggest that CaM may contribute to NHE1 dimerization. We propose that similar structural diversity is relevant to other CaM complexes.

cell biology↗

bioRxiv2020

Order and disorder - an integrative structure of the full-length human growth hormone receptor

Despite the many physiological and pathophysiological functions of the human growth hormone receptor (hGHR), a detailed understanding of its modus operandi is hindered by the lack of structural information of the entire receptor at the molecular level. Due to its relatively small size (70 kDa) and large content of structural disorder (>50%), this membrane protein falls between the cracks of conventional high-resolution structural biology methods. Here, we study the structure of the full-length hGHR in nanodiscs with small angle-X-ray scattering (SAXS) as the foundation. We developed an approach in which we combined SAXS, X-ray diffraction and NMR spectroscopy obtained on the individual domains and integrated the data through molecular dynamics simulations to interpret SAXS data on the full-length hGHR in nanodiscs. The structure of the hGHR was determined in its monomeric state and provides the first experimental model of any full-length cytokine receptor in a lipid membrane. Combined, our results highlight that the three domains of the hGHR are free to reorient relative to each other, resulting in a broad structural ensemble. Our work exemplifies how integrating experimental data from several techniques computationally, may enable the characterization of otherwise inaccessible structures of membrane proteins with long disordered regions, a widespread phenomenon in biology. To understand orchestration of cellular signaling by disordered chains, the hGHR is archetypal and its structure emphasizes that we need to take a much broader, ensemble view on signaling.

biophysics↗

bioRxiv2019

Mutations in a single signaling pathway allow growth on a different solvent than water

Since life is completely dependent on water, it is difficult to gauge the impact of solvent change. To analyze the role of water as a solvent in biology, we replaced water with heavy water (D2O), and investigated the biological effects by a wide range of techniques, using the fission yeast Schizosaccharomyces pombe as model organism. We show that high concentrations of D2O lead to altered glucose metabolism, growth retardation, and inhibition of meiosis. However, mitosis and overall cell viability were only slightly affected. After prolonged incubation in D2O, cells displayed gross morphological changes, thickened cell walls as well as aberrant septa and cytoskeletal organization. RNA sequencing revealed that D2O causes a strong downregulation of most tRNAs and triggers activation of the general stress response pathway. Genetic screens identified several D2O sensitive mutants, while mutants compromised in the cell integrity pathway, including the protein kinase genes pmk1, mkh1, pek1 and pck2, that control cell wall biogenesis, were more tolerant to D2O. We speculate that D2O affects the phospholipid membrane or cell wall glycans causing an activation of the cell integrity pathway. In conclusion, the effects of solvent replacement are pleiotropic but the D2O-triggered activation of the cell integrity pathway and subsequent increased deposition of cell wall material and septation problems appear most critical for the cell growth defects.

genetics↗

bioRxiv2018

The Arabidopsis autophagy cargo receptors ATI1 and ATI2 bind to ATG8 via intrinsically disordered regions and are post-translationally modified upon ATG8 interaction

Selective autophagy has emerged as an important mechanism by which eukaryotic cells control the abundance of specific proteins. This mechanism relies on cargo recruitment to autophagosomes by receptors that bind to both the ubiquitin-like AUTOPHAGY8 (ATG8) protein through ATG8 interacting motifs (AIMs) and to the cargo to be degraded. In plants, two autophagy cargo receptors, ATG8 Interacting Protein 1 (ATI1) and 2 (ATI2), were identified early on, but their molecular properties remain poorly understood. Here, we show that ATI1 and ATI2 are transmembrane proteins with long N-terminal intrinsically disordered regions (IDRs). The N-terminal IDRs contain the functional AIMs, and we use nuclear magnetic resonance spectroscopy to directly observe the disorder-order transition of the AIM upon ATG8 binding. Our analyses also show that the IDRs of ATI1 and ATI2 are not equivalent, because ATI2 has properties of a fully disordered polypeptide, while ATI1 has properties consistent with a collapsed pre-molten globule-like conformation Interestingly, wild type ATI1 and ATI2 exist as distinct post-translationally modified forms. Specifically, different forms are detectable upon mutation of the AIM, suggesting that interaction of ATI1 and ATI2 to ATG8 is coupled to a change in their post-translational modification.

molecular biology↗