Henning Stahlberg

Structural biology. Proton-powered turbine of a plant motor.

ATP synthases are enzymes that can work in two directions to catalyse either the synthesis or breakdown of ATP, and they constitute the smallest rotary motors in biology. The flow of protons propels the rotation of a membrane-spanning complex of identical protein subunits, the number of which determines the efficiency of energy conversion. This proton-powered turbine is predicted to consist of 12 subunits, based on data for Escherichia coli. The yeast mitochondrial enzyme, however, has only 10 subunits. We have imaged the ATP synthase from leaf chloroplasts by using atomic force microscopy and, surprisingly, find that its turbine has 14 subunits, arranged in a cylindrical ring.

The fold of α-synuclein fibrils

The aggregation of proteins into amyloid fibrils is associated with several neurodegenerative diseases. In Parkinsons disease it is believed that the aggregation of α-synuclein (α-syn) from monomers by intermediates into amyloid fibrils is the toxic disease-causative mechanism. Here, we studied the structure of α-syn in its amyloid state by using various biophysical approaches. Quenched hydrogen/deuterium exchange NMR spectroscopy identified five β-strands within the fibril core comprising residues 35–96 and solid-state NMR data from amyloid fibrils comprising the fibril core residues 30–110 confirmed the presence of β-sheet secondary structure. The data suggest that β1-strand interacts with β2, β2 with β3, β3 with β4, and β4 with β5. High-resolution cryoelectron microscopy revealed the protofilament boundaries of ≈2 × 3.5 nm. Based on the combination of these data and published structural studies, a fold of α-syn in the fibrils is proposed and discussed.

Proton-powered turbine of a plant motor

ATP synthases are enzymes that can work in two directions to catalyse either the synthesis or breakdown of ATP, and they constitute the smallest rotary motors in biology. The flow of protons propels the rotation of a membrane-spanning complex of identical protein subunits, the number of which determines the efficiency of energy conversion. This proton-powered turbine is predicted to consist of 12 subunits, based on data for Escherichia coli. The yeast mitochondrial enzyme, however, has only 10 subunits. We have imaged the ATP synthase from leaf chloroplasts by using atomic force microscopy and, surprisingly, find that its turbine has 14 subunits, arranged in a cylindrical ring.

Coassembly of Mgm1 isoforms requires cardiolipin and mediates mitochondrial inner membrane fusion

The soluble and membrane-anchored isoforms of Mgm1 are only active when they work together (in trans).

Bacterial Na+‐ATP synthase has an undecameric rotor

Synthesis of adenosine triphosphate (ATP) by the F1F0 ATP synthase involves a membrane‐embedded rotary engine, the F0 domain, which drives the extra‐membranous catalytic F1 domain. The F0 domain consists of subunits a1b2 and a cylindrical rotor assembled from 9–14 α‐helical hairpin‐shaped c‐subunits. According to structural analyses, rotors contain 10 c‐subunits in yeast and 14 in chloroplast ATP synthases. We determined the rotor stoichiometry of Ilyobacter tartaricus ATP synthase by atomic force microscopy and cryo‐electron microscopy, and show the cylindrical sodium‐driven rotor to comprise 11 c‐subunits.

High resolution AFM topographs of the Escherichia coli water channel aquaporin Z.

Aquaporins form a large family of membrane channels involved in osmoregulation. Electron crystallography has shown monomers to consist of six membrane spanning α‐helices confirming sequence based predictions. Surface exposed loops are the least conserved regions, allowing differentiation of aquaporins. Atomic force microscopy was used to image the surface of aquaporin Z, the water channel of Escherichia coli. Recombinant protein with an N‐terminal fragment including 10 histidines was isolated as a tetramer by Ni‐affinity chromatography, and reconstituted into two‐dimensional crystals with p4212 symmetry. Small crystalline areas with p4 symmetry were found as well. Imaging both crystal types before and after cleavage of the N‐termini allowed the cytoplasmic surface to be identified; a drastic change of the cytoplasmic surface accompanied proteolytic cleavage, while the extracellular surface morphology did not change. Flexibility mapping and volume calculations identified the longest loop at the extracellular surface. This loop exhibited a reversible force‐induced conformational change.

Interaction of complexes I, III, and IV within the bovine respirasome by single particle cryoelectron tomography

The respirasome is a multisubunit supercomplex of the respiratory chain in mitochondria. Here we report the 3D reconstruction of the bovine heart respirasome, composed of dimeric complex III and single copies of complex I and IV, at about 2.2-nm resolution, determined by cryoelectron tomography and subvolume averaging. Fitting of X-ray structures of single complexes I, III2, and IV with high fidelity allows interpretation of the model at the level of secondary structures and shows how the individual complexes interact within the respirasome. Surprisingly, the distance between cytochrome c binding sites of complexes III2 and IV is about 10 nm. Modeling indicates a loose interaction between the three complexes and provides evidence that lipids are gluing them at the interfaces.

Domain structure of secretin PulD revealed by limited proteolysis and electron microscopy

Secretins, a superfamily of multimeric outer membrane proteins, mediate the transport of large macromolecules across the outer membrane of Gram‐negative bacteria. Limited proteolysis of secretin PulD from the Klebsiella oxytoca pullulanase secretion pathway showed that it consists of an N‐terminal domain and a protease‐resistant C‐terminal domain that remains multimeric after proteolysis. The stable C‐terminal domain starts just before the region in PulD that is highly conserved in the secretin superfamily and apparently lacks the region at the C‐terminal end to which the secretin‐specific pilot protein PulS binds. Electron microscopy showed that the stable fragment produced by proteolysis is composed of two stacked rings that encircle a central channel and that it lacks the peripheral radial spokes that are seen in the native complex. Moreover, the electron microscopic images suggest that the N‐terminal domain folds back into the large cavity of the channel that is formed by the C‐terminal domain of the native complex, thereby occluding the channel, consistent with previous electrophysiological studies showing that the channel is normally closed.

GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death

Pyroptosis is a lytic type of cell death that is initiated by inflammatory caspases. These caspases are activated within multi‐protein inflammasome complexes that assemble in response to pathogens and endogenous danger signals. Pyroptotic cell death has been proposed to proceed via the formation of a plasma membrane pore, but the underlying molecular mechanism has remained unclear. Recently, gasdermin D (GSDMD), a member of the ill‐characterized gasdermin protein family, was identified as a caspase substrate and an essential mediator of pyroptosis. GSDMD is thus a candidate for pyroptotic pore formation. Here, we characterize GSDMD function in live cells and in vitro. We show that the N‐terminal fragment of caspase‐1‐cleaved GSDMD rapidly targets the membrane fraction of macrophages and that it induces the formation of a plasma membrane pore. In vitro, the N‐terminal fragment of caspase‐1‐cleaved recombinant GSDMD tightly binds liposomes and forms large permeability pores. Visualization of liposome‐inserted GSDMD at nanometer resolution by cryo‐electron and atomic force microscopy shows circular pores with variable ring diameters around 20 nm. Overall, these data demonstrate that GSDMD is the direct and final executor of pyroptotic cell death.

Characterization of the motion of membrane proteins using high-speed atomic force microscopy

For cells to function properly, membrane proteins must be able to diffuse within biological membranes. The functions of these membrane proteins depend on their position and also on protein-protein and protein-lipid interactions. However, so far, it has not been possible to study simultaneously the structure and dynamics of biological membranes. Here, we show that the motion of unlabelled membrane proteins can be characterized using high-speed atomic force microscopy. We find that the molecules of outer membrane protein F (OmpF) are widely distributed in the membrane as a result of diffusion-limited aggregation, and while the overall protein motion scales roughly with the local density of proteins in the membrane, individual protein molecules can also diffuse freely or become trapped by protein-protein interactions. Using these measurements, and the results of molecular dynamics simulations, we determine an interaction potential map and an interaction pathway for a membrane protein, which should provide new insights into the connection between the structures of individual proteins and the structures and dynamics of supramolecular membranes.