visualization-atp-synthase-dimers-mitochondria-electron-cryo

Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as ...

Max Planck Institute of Biophysics

In Bacillus subtilis, RNA polymerase becomes concentrated into regions of the nucleoid called transcription foci. With green fluorescent protein-tagged RNA polymerase, these structures are only observed at higher growth rates and have been shown to represent the sites of rRNA synthesis. There are 10 rRNA (rrn) operons distributed around nearly half of the chromosome. In this study we analyzed the rrn composition of transcription foci with fluorescently tagged loci and showed that they comprise the origin-proximal operon rrnO but not the more dispersed rrnE or rrnD. This suggests that transcription foci comprise only the seven origin-proximal operons rrnO, rrnA, rrnJ, rrnW, rrnI, rrnH, and rrnG. These results have important implications for our understanding of microbial chromosome structure.

Localization of rRNA synthesis in Bacillus subtilis: characterization of loci involved in transcription focus formation.

Bacterial RNA polymerases (RNAPs) are capable of producing full-length transcripts in the absence of additional factors using in vitro assays. However, in vivo RNAP can become stalled during the elongation phase of transcription due to the presence of various sequence motifs. Subsequently, a host of elongation factors are required to modulate the activity of RNAP. NusA, the most intensively studied elongation factor, plays a role in increasing RNAP pausing and termination. Conversely, it is also important in transcription of rRNA where it functions as an anti-termination factor, helping to ensure only full-length transcripts are produced. Here we show that NusA is closely associated with RNAP within the bacterial nucleoid and that it is preferentially recruited to sites of rRNA synthesis. In vivo and in vitro analyses indicate this results in a change in stoichiometry of NusA:RNAP from 1:1 to approximately 2:1 at the subcellular sites of rRNA synthesis. A model is presented showing how the ratio of NusA:RNAP could affect the activity of the elongation complex so that it functions as an anti-terminator complex during rRNA synthesis.

The NusA:RNA polymerase ratio is increased at sites of rRNA synthesis in Bacillus subtilis.

HupR is a response regulator that controls the synthesis of the membrane-bound [NiFe]hydrogenase of the photosynthetic bacterium Rhodobacter capsulatus. The protein belongs to the NtrC subfamily of response regulators and is the second protein of a two-component system. We have crystallized the full-length protein HupR in the unphosphorylated state in two dimensions using the lipid monolayer technique. The 3D structure of negatively stained HupR was calculated to a resolution of approximately 23 A from tilted electron microscope images. HupR crystallizes as a dimer, and forms an elongated V-shaped structure with extended arms. The dimensions of the dimer are about 80 A length, 40 A width and 85 A thick. The HupR monomer consists of three domains, N-terminal receiver domain, central domain and C-terminal DNA-binding domain. We have fitted the known 3D structure of the central domain from NtrC1 Aquifex aeolicus protein into our 3D model; we propose that contact between the dimers is through the central domain. The N-terminal domain is in contact with the lipid monolayer and is situated on the top of the V-shaped structure. The central domain alone has been expressed and purified; it forms a pentamer in solution and lacks ATPase activity.

Structural and functional studies of the response regulator HupR.

Hydrogen uptake protein regulator (HupR) is a member of the nitrogen regulatory protein C (NtrC) family of response regulators. These proteins activate transcription by RNA polymerase (RNAP) in response to a change in environment. This change is detected through the phosphorylation of their receiver domain as part of a two-component signalling pathway. HupR is an unusual member of this family as it activates transcription when unphosphorylated, and transcription is inhibited by phosphorylation. Also, HupR activates transcription through the more general sigma(70) transcription initiation factor, which does not require activation by ATPase, in contrast to other NtrC family members that utilise sigma(54). Hence, its mode of action is expected to be different from those of the more conventional NtrC family members. We have determined the structures of the unphosphorylated N-terminal receiver domain of wild-type HupR, the mutant HupR(D55E)(N) (which cannot be phosphorylated and down-regulated), and HupR in the presence of the phosphorylation mimic BeF(3)(-). The structures show a typical response regulator fold organised as a dimer whose interface involves alpha4-beta5-alpha5 elements. The interactions across the interface are slightly different between apo and phospho mimics, and these reflect a rearrangement of key conserved residues around the active site aspartate that have been implicated in domain activation in other receiver domain proteins. We also show that the wild-type HupR receiver domain forms a weak dimer in solution, which is strengthened in the presence of the phosphorylation mimic BeF(3)(-). The results indicate many features similar to those that have been observed in other systems, including NtrC (where phosphorylation is activatory), and indicate that recognition properties, which allow HupR to be active in the absence of phosphorylation, lie in the transmission of phosphorylation signals through the linker region to the other domains of the protein.

The HupR receiver domain crystal structure in its nonphospho and inhibitory phospho states.

The self-assembly of supramolecular structures that are ordered on the nanometre scale is a key objective in nanotechnology. DNA and peptide nanotechnologies have produced various two- and three-dimensional structures, but protein molecules have been underexploited in this area of research. Here we show that the genetic fusion of subunits from protein assemblies that have matching rotational symmetry generates species that can self-assemble into well-ordered, pre-determined one- and two-dimensional arrays that are stabilized by extensive intermolecular interactions. This new class of supramolecular structure provides a way to manufacture biomaterials with diverse structural and functional properties.

Generation of protein lattices by fusing proteins with matching rotational symmetry.

We used electron cryotomography to study the molecular arrangement of large respiratory chain complexes in mitochondria from bovine heart, potato, and three types of fungi. Long rows of ATP synthase dimers were observed in intact mitochondria and cristae membrane fragments of all species that were examined. The dimer rows were found exclusively on tightly curved cristae edges. The distance between dimers along the rows varied, but within the dimer the distance between F(1) heads was constant. The angle between monomers in the dimer was 70° or above. Complex I appeared as L-shaped densities in tomograms of reconstituted proteoliposomes. Similar densities were observed in flat membrane regions of mitochondrial membranes from all species except Saccharomyces cerevisiae and identified as complex I by quantum-dot labeling. The arrangement of respiratory chain proton pumps on flat cristae membranes and ATP synthase dimer rows along cristae edges was conserved in all species investigated. We propose that the supramolecular organization of respiratory chain complexes as proton sources and ATP synthase rows as proton sinks in the mitochondrial cristae ensures optimal conditions for efficient ATP synthesis.

Macromolecular organization of ATP synthase and complex I in whole mitochondria.

GRecon: a method for the lipid reconstitution of membrane proteins.

We used electron cryotomography of mitochondrial membranes from wild-type and mutant Saccharomyces cerevisiae to investigate the structure and organization of ATP synthase dimers in situ. Subtomogram averaging of the dimers to 3.7 nm resolution revealed a V-shaped structure of twofold symmetry, with an angle of 86° between monomers. The central and peripheral stalks are well resolved. The monomers interact within the membrane at the base of the peripheral stalks. In wild-type mitochondria ATP synthase dimers are found in rows along the highly curved cristae ridges, and appear to be crucial for membrane morphology. Strains deficient in the dimer-specific subunits e and g or the first transmembrane helix of subunit 4 lack both dimers and lamellar cristae. Instead, cristae are either absent or balloon-shaped, with ATP synthase monomers distributed randomly in the membrane. Computer simulations indicate that isolated dimers induce a plastic deformation in the lipid bilayer, which is partially relieved by their side-by-side association. We propose that the assembly of ATP synthase dimer rows is driven by the reduction in the membrane elastic energy, rather than by direct protein contacts, and that the dimer rows enable the formation of highly curved ridges in mitochondrial cristae.

Structure of the yeast F1Fo-ATP synthase dimer and its role in shaping the mitochondrial cristae.

To truly understand bioenergetic processes such as ATP synthesis, membrane-bound substrate transport or flagellar rotation, systems need to be analysed in a cellular context. Cryo-ET (cryo-electron tomography) is an essential part of this process, as it is currently the only technique which can directly determine the spatial organization of proteins at the level of both the cell and the individual protein complexes. The need to assess bioenergetic processes at a cellular level is becoming more and more apparent with the increasing interest in mitochondrial diseases. In recent years, cryo-ET has contributed significantly to our understanding of the molecular organization of mitochondria and chloroplasts. The present mini-review first describes the technique of cryo-ET and then discusses its role in membrane bioenergetics specifically in chloroplasts and mitochondrial research.

Karen M. Davies

Department of Structural Biology

Max Planck Institute of Biophysics

Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography

Karen M. Davies

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Structural Biology

Max Planck Institute of Biophysics

Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography

Department of Structural Biology