Biology subjects

Flygaard, R. K.

Publications and source records attributed to Flygaard, R. K..

5 recordsLinked to original sources

bioRxiv2024

Structure of a Ca2+ bound phosphoenzyme intermediate in the inward-to-outward transition of Ca2+-ATPase 1 from Listeria monocytogenes

Active transport by Ca2+-ATPases of the P-type ATPase family maintain a very low cytosolic calcium concentration and steep electrochemical gradients. Detailed mechanisms of this transport have been described from structures of mammalian sarco/endoplasmic reticulum Ca2+-ATPases (SERCA) stabilized by inhibitors at specific intermediate steps of the transport cycle. An essentially irreversible step is crucial to prevent reflux in active transport against steep gradients. Single-molecule FRET (smFRET) study of the bacterial Ca2+-ATPase LMCA1 revealed an intermediate of the transition between so-called [Ca]E1P and E2P states, suggesting that calcium release from this intermediate is the irreversible step. Here, we present a 3.5[A] cryo-EM structure for a four-glycine insertion mutant (G4-LMCA1) in a lipid nanodisc obtained under turnover conditions and adopting such a calcium-bound intermediate, denoted [Ca]E2P. The cytosolic domains are positioned in the E2P-like conformation, while the calcium-binding transmembrane (TM) domain is similar to calcium-bound E1P-ADP like conformation of SERCA. Missing density for the E292 residue at the calcium site (equivalent of SERCA1a E309) suggests flexibility and a site poised for calcium release and proton uptake. The structure suggests a mechanism for the inward-to-outward transition in Ca2+-ATPases, where ADP release and re-organisation of the cytoplasmic domains precede calcium release.

molecular biology↗

bioRxiv2023

Structure of mitoribosome reveals mechanism of mRNA binding, tRNA interactions with L1 stalk, roles of cofactors and rRNA modifications

The mitoribosome translates mitochondrial mRNAs and regulates energy conversion that is a signature of aerobic life forms. We present a 2.2 [A] resolution structure of human mitoribosome together with validated mitoribosomal RNA (rRNA) modifications, including aminoacylated CP-tRNAVal. The structure shows how mitoribosomal proteins stabilise binding of mRNA and tRNA helping to align it in the decoding center, whereas the GDP-bound mS29 stabilizes intersubunit communication. Comparison between different states, with respect to tRNA position, allowed to characterize a non-canonical L1 stalk, and molecular dynamics simulations revealed how it facilitates tRNA transition in a way that does not require interactions with rRNA. We also report functionally important polyamines that are depleted when cells are subjected to an antibiotic treatment. The structural, biochemical, and computational data illuminate the principal functional components of the translation mechanism in mitochondria and provide the most complete description so far of the structure and function of the human mitoribosome.

biochemistry↗

bioRxiv2022

Structural basis of mitochondrial membrane bending by I-II-III2-IV2 supercomplex

Mitochondrial energy conversion requires an intricate architecture of the inner mitochondrial membrane1. Here we show that in ciliates, the membrane curvature is provided by a supercomplex containing all four respiratory chain components. We report cryo-electron microscopy and cryo-tomography structures of the supercomplex that comprises 150 different proteins and 311 bound lipids, forming a stable 5.8-megadalton assembly. Due to subunit acquisition and extension, complex I associates with a complex IV dimer, generating a wedge-shaped gap that serves as a binding site for complex II. Together with a tilted complex III dimer association, it results in a curved membrane region. Using molecular dynamics simulations, we demonstrate that the divergent supercomplex actively contributes to the membrane curvature induction and cristae tubulation. Our findings explain how the architecture of the native I-II-III2-IV2 supercomplex reflects the functional specialization of bioenergetics by shaping the membrane.

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 function of the bacterial protein toxin phenomycin

Phenomycin is a bacterial mini-protein of 89 amino acids discovered more than 50 years ago with toxicity in the nanomolar regime towards mammalian cells. The protein inhibits the function of the eukaryotic ribosome in cell free systems and appears to target translation initiation. Several fundamental questions concerning the cellular activity of phenomycin have however remained unanswered. In this paper, we have used morphological profiling to show that direct inhibition of translation underlies the toxicity of phenomycin in cells. We have performed studies of the cellular uptake mechanism of phenomycin, showing that endosomal escape is the toxicity-limiting step, and we have solved a solution phase high-resolution structure of the protein using NMR spectroscopy. Through bioinformatic as well as functional comparisons between phenomycin and two homologs, we have identified a peptide segment, which constitutes one of two loops in the structure, that is critical for the toxicity of phenomycin.

cell biology↗