Biology subjects

Kuehlbrandt, W.

Publications and source records attributed to Kuehlbrandt, W..

5 recordsLinked to original sources

bioRxiv2025

Structures of Chaetomium thermophilum TOM complexes with bound preproteins

Mitochondria import most of their proteins from the cytoplasm through the TOM complex. Preproteins containing targeting signals are recognized by the TOM receptor subunits, and translocated by Tom40 across the outer mitochondrial membrane. We present four structures of the preprotein-bound and preprotein-free TOM core and holo complex from the thermophilic fungus Chaetomium thermophilum, obtained by single-particle electron cryomicroscopy. Our structures reveal the symmetric arrangement of two copies of the Tom20 receptor subunit in the TOM holo complex. Several different conformations of Tom20 within the TOM holo complex highlight the dynamic nature of the receptor. The structure of preprotein-bound Tom20 provides insight into the early stages of protein translocation.

biophysics↗

bioRxiv2023

Two conformations of the Tom20 preprotein receptor in the TOM holo complex

The TOM complex is the main entry point for precursor proteins into mitochondria. Precursor proteins containing targeting sequences are recognized by the TOM complex and imported into the mitochondria. We have determined the structure of the TOM core complex from Neurospora crassa by single-particle cryoEM at 3.3 [A] resolution, showing its interaction with a bound presequence at 4 [A] resolution, and of the TOM holo complex including the Tom20 receptor at 6-7 [A] resolution. TOM is a transmembrane complex consisting of two {beta}-barrels, three receptor subunits and three short transmembrane subunits. Tom20 has a transmembrane helix and a receptor domain on the cytoplasmic side. We propose that Tom20 acts as a dynamic gatekeeper, guiding precursor proteins into the pores of the TOM complex. We analyze the interactions of Tom20 with other TOM subunits, present insights into the structure of the TOM holo complex, and suggest a translocation mechanism.

biophysics↗

bioRxiv2022

Conformational changes in mitochondrial complex I from the thermophilic eukaryote Chaetomium thermophilum

Mitochondrial complex I is a redox-driven proton pump that generates proton-motive force across the inner mitochondrial membrane, powering oxidative phosphorylation and ATP synthesis in eukaryotes. We report the structure of complex I from the thermophilic fungus Chaetomium thermophilum, determined by cryoEM up to 2.4 [A] resolution. We show that the complex undergoes a transition between two conformations, which we refer to as form 1 and 2. The conformational switch is manifest in a twisting movement of the peripheral arm relative to the membrane arm, but most notably in substantial rearrangements of the Q-binding cavity and the E-channel, resulting in a continuous aqueous passage from the E-channel to subunit ND5 at the far end of the membrane arm. The conformational changes in the complex interior resemble those reported for mammalian complex I, suggesting a highly conserved, universal mechanism of coupling electron transport to proton pumping.

biochemistry↗

bioRxiv2019

The resolution revolution in cryoEM requires new sample preparation procedures: A rapid pipeline to high resolution maps of yeast FAS

Single-particle electron cryo-microscopy (cryoEM) has undergone a \"resolution revolution\" that makes it possible to characterize megadalton (MDa) complexes at atomic resolution without crystals. To fully exploit the new opportunities in molecular microscopy, new procedures for the cloning, expression and purification of macromolecular complexes need to be explored. Macromolecular assemblies are often unstable, and invasive construct design or inadequate purification conditions or sample preparation methods can result in disassembly or denaturation. The structure of the 2.6 MDa yeast fatty acid synthase (FAS) has been studied by electron microscopy since the 1960s. We report a new, streamlined protocol for the rapid production of purified yeast FAS for structure determination by high-resolution cryoEM. Together with a companion protocol for preparing cryoEM specimens on a hydrophilized graphene layer, our new protocol has yielded a 3.1 [A] map of yeast FAS from 15,000 automatically picked particles within a day. The high map quality enabled us to build a complete atomic model of an intact fungal FAS.

biochemistry↗

bioRxiv2019

Mechanism of electroneutral sodium/proton antiporter from transition-path shooting

Na+/H+ antiporters exchange sodium ions (Na+) and protons (H+) on opposite sides of lipid membranes, using the gradient of one ion to drive the uphill transport of the other. The electroneutral Na+/H+ antiporter NhaP from archaea Pyrococcus abyssi (PaNhaP) is a functional homolog of the human Na+/H+ exchanger NHE1, which is an important drug target. Here we resolve the Na+ and H+ transport cycle of PaNhaP in continuous and unbiased molecular dynamics trajectories that cover the entire transport cycle. We overcome the enormous time-scale gap between seconds-scale ion exchange and microseconds simulations by transition-path shooting. In this way, we selectively capture the rare events in which the six-helix-bundle transporter domain spontaneously moves up and down to shuttle protons and ions across the membrane. The simulations reveal two hydrophobic gates above and below the ion-binding sites that open and close in response to the bundle motion. Weakening the outside gate by mutagenesis makes the transporter faster, suggesting that the gate balances competing demands of fidelity and efficiency. Transition-path sampling and a committor-based reaction coordinate optimization identify the essential motions and interactions that realize conformational alternation between the two access states in transporter function.

biophysics↗