Rosalba Rothnagel

3D structure of the Yersinia entomophaga toxin complex and implications for insecticidal activity

Toxin complex (Tc) proteins are a class of bacterial protein toxins that form large, multisubunit complexes. Comprising TcA, B, and C components, they are of great interest because many exhibit potent insecticidal activity. Here we report the structure of a novel Tc, Yen-Tc, isolated from the bacterium Yersinia entomophaga MH96, which differs from the majority of bacterially derived Tcs in that it exhibits oral activity toward a broad range of insect pests, including the diamondback moth (Plutella xylostella). We have determined the structure of the Yen-Tc using single particle electron microscopy and studied its mechanism of toxicity by comparative analyses of two variants of the complex exhibiting different toxicity profiles. We show that the A subunits form the basis of a fivefold symmetric assembly that differs substantially in structure and subunit arrangement from its most well characterized homologue, the Xenorhabdus nematophila toxin XptA1. Histopathological and quantitative dose response analyses identify the B and C subunits, which map to a single, surface-accessible region of the structure, as the sole determinants of toxicity. Finally, we show that the assembled Yen-Tc has endochitinase activity and attribute this to putative chitinase subunits that decorate the surface of the TcA scaffold, an observation that may explain the oral toxicity associated with the complex.

The structure of bacterial RNA polymerase in complex with the essential transcription elongation factor NusA.

There are three stages of transcribing DNA into RNA. These stages are initiation, elongation and termination, and they are well‐understood biochemically. However, despite the plethora of structural information made available on RNA polymerase in the last decade, little is available for RNA polymerase in complex with transcription elongation factors. To understand the mechanisms of transcriptional regulation, we describe the first structure, to our knowledge, for a bacterial RNA polymerase in complex with an essential transcription elongation factor. The resulting structure formed between the RNA polymerase and NusA from Bacillus subtilis provides important insights into the transition from an initiation complex to an elongation complex, and how NusA is able to modulate transcription elongation and termination.

Three-dimensional structure of AAA ATPase Vps4: advancing structural insights into the mechanisms of endosomal sorting and enveloped virus budding.

Vps4 is a AAA ATPase that mediates endosomal membrane protein sorting. It is also a host factor hijacked by a diverse set of clinically important viruses, including HIV and Ebola, to facilitate viral budding. Here we present the three-dimensional structure of the hydrolysis-defective Vps4p(E233Q) mutant. Single-particle analysis, multiangle laser light scattering, and the docking of independently determined atomic models of Vps4 monomers reveal a complex with C6 point symmetry, distinguishing between a range of previously suggested oligomeric states (8-14 subunits). The 3D reconstruction also reveals a tail-to-tail subunit organization between the two rings of the complex and identifies the location of domains critical to complex assembly and interaction with partner proteins. Our refined Vps4 structure is better supported by independent lines of evidence than those previously proposed, and provides insights into the mechanism of endosomal membrane protein sorting and viral envelope budding.

Structure of the dengue virus glycoprotein non-structural protein 1 by electron microscopy and single-particle analysis

The flavivirus non-structural protein 1 (NS1) is a glycoprotein that is secreted as a soluble hexameric complex during the course of natural infection. Growing evidence indicates that this secreted form of NS1 (sNS1) plays a significant role in immune evasion and modulation during infection. Attempts to determine the crystal structure of NS1 have been unsuccessful to date and relatively little is known about the macromolecular organization of the sNS1 hexamer. Here, we have applied single-particle analysis to images of baculovirus-derived recombinant dengue 2 virus NS1 obtained by electron microscopy to determine its 3D structure to a resolution of 23 Å. This structure reveals a barrel-like organization of the three dimeric units that comprise the hexamer and provides further insights into the overall organization of oligomeric sNS1.

Bacterial Mechanosensitive Channels: Models for Studying Mechanosensory Transduction

SIGNIFICANCE Sensations of touch and hearing are manifestations of mechanical contact and air pressure acting on touch receptors and hair cells of the inner ear, respectively. In bacteria, osmotic pressure exerts a significant mechanical force on their cellular membrane. Bacteria have evolved mechanosensitive (MS) channels to cope with excessive turgor pressure resulting from a hypo-osmotic shock. MS channel opening allows the expulsion of osmolytes and water, thereby restoring normal cellular turgor and preventing cell lysis. RECENT ADVANCES As biological force-sensing systems, MS channels have been identified as the best examples of membrane proteins coupling molecular dynamics to cellular mechanics. The bacterial MS channel of large conductance (MscL) and MS channel of small conductance (MscS) have been subjected to extensive biophysical, biochemical, genetic, and structural analyses. These studies have established MscL and MscS as model systems for mechanosensory transduction. CRITICAL ISSUES In recent years, MS ion channels in mammalian cells have moved into focus of mechanotransduction research, accompanied by an increased awareness of the role they may play in the pathophysiology of diseases, including cardiac hypertrophy, muscular dystrophy, or Xerocytosis. FUTURE DIRECTIONS A recent exciting development includes the molecular identification of Piezo proteins, which function as nonselective cation channels in mechanosensory transduction associated with senses of touch and pain. Since research on Piezo channels is very young, applying lessons learned from studies of bacterial MS channels to establishing the mechanism by which the Piezo channels are mechanically activated remains one of the future challenges toward a better understanding of the role that MS channels play in mechanobiology.

Cryoelectron Microscopy Map of Atadenovirus Reveals Cross-Genus Structural Differences from Human Adenovirus

ABSTRACT A three-dimensional (3D) cryoelectron microscopy reconstruction of the prototype Atadenovirus (OAdV [an ovine adenovirus isolate]) showing information at a 10.6-Å resolution (0.5 Fourier shell correlation) was derived by single-particle analysis. This is the first 3D structure solved for any adenovirus that is not a Mastadenovirus, allowing cross-genus comparisons between structures and the assignment of genus-specific capsid proteins. Viable OAdV mutants that lacked the genus-specific LH3 and p32k proteins in purified virions were also generated. Negatively stained 3D reconstructions of these mutants were used to identify the location of protein LH3 and infer that of p32k within the capsid. The key finding was that LH3 is a critical protein that holds the outer capsid of the virus together. In its absence, the outer viral capsid is unstable. LH3 is located in the same position among the hexon subunits as its protein IX equivalent from mastadenoviruses but sits on top of the hexon trimers, forming prominent “knobs” on the virion surface that visually distinguish OAdV from other known AdVs. Electron density was also assigned to hexon and penton subunits and to proteins IIIa and VIII. There was good correspondence between OAdV density and human AdV hexon structures, which also validated the significant differences that were observed between the penton base protein structures.

RAZA: A Rapid 3D z-crossings Algorithm to segment electron tomograms and extract organelles and macromolecules

Resolving the 3D architecture of cells to atomic resolution is one of the most ambitious challenges of cellular and structural biology. Central to this process is the ability to automate tomogram segmentation to identify sub-cellular components, facilitate molecular docking and annotate detected objects with associated metadata. Here we demonstrate that RAZA (Rapid 3D z-crossings algorithm) provides a robust, accurate, intuitive, fast, and generally applicable segmentation algorithm capable of detecting organelles, membranes, macromolecular assemblies and extrinsic membrane protein domains. RAZA defines each continuous contour within a tomogram as a discrete object and extracts a set of 3D structural fingerprints (major, middle and minor axes, surface area and volume), enabling selective, semi-automated segmentation and object extraction. RAZA takes advantage of the fact that the underlying algorithm is a true 3D edge detector, allowing the axes of a detected object to be defined, independent of its random orientation within a cellular tomogram. The selectivity of object segmentation and extraction can be controlled by specifying a user-defined detection tolerance threshold for each fingerprint parameter, within which segmented objects must fall and/or by altering the number of search parameters, to define morphologically similar structures. We demonstrate the capability of RAZA to selectively extract subgroups of organelles (mitochondria) and macromolecular assemblies (ribosomes) from cellular tomograms. Furthermore, the ability of RAZA to define objects and their contours, provides a basis for molecular docking and rapid tomogram annotation.

Structure of a PSI–LHCI–cyt b6f supercomplex in Chlamydomonas reinhardtii promoting cyclic electron flow under anaerobic conditions

Significance To optimize photosynthetic performance and minimize photooxidative damage, photosynthetic organisms evolved to efficiently balance light energy absorption and electron transport with cellular energy requirements under constantly changing light conditions. The regulation of linear electron flow (LEF) and cyclic electron flow (CEF) contributes to this fine-tuning. Here we present a model of the formation and structural molecular organization of a CEF-performing photosystem I (PSI)–light harvesting complex I (LHCI)–cytochrome (cyt) b6f supercomplex from the green alga Chlamydomonas reinhardtii. Such a structural arrangement could modulate the distinct operation of LEF and CEF to optimize light energy utilization, despite the same individual structural units contributing to these two different functional modes. Photosynthetic linear electron flow (LEF) produces ATP and NADPH, while cyclic electron flow (CEF) exclusively drives photophosphorylation to supply extra ATP. The fine-tuning of linear and cyclic electron transport levels allows photosynthetic organisms to balance light energy absorption with cellular energy requirements under constantly changing light conditions. As LEF and CEF share many electron transfer components, a key question is how the same individual structural units contribute to these two different functional modes. Here, we report the structural identification of a photosystem I (PSI)–light harvesting complex I (LHCI)–cytochrome (cyt) b6f supercomplex isolated from the unicellular alga Chlamydomonas reinhardtii under anaerobic conditions, which induces CEF. This provides strong evidence for the model that enhanced CEF is induced by the formation of CEF supercomplexes, when stromal electron carriers are reduced, to generate additional ATP. The additional identification of PSI–LHCI–LHCII complexes is consistent with recent findings that both CEF enhancement and state transitions are triggered by similar conditions, but can occur independently from each other. Single molecule fluorescence correlation spectroscopy indicates a physical association between cyt b6f and fluorescent chlorophyll containing PSI–LHCI supercomplexes. Single particle analysis identified top-view projections of the corresponding PSI–LHCI–cyt b6f supercomplex. Based on molecular modeling and mass spectrometry analyses, we propose a model in which dissociation of LHCA2 and LHCA9 from PSI supports the formation of this CEF supercomplex. This is supported by the finding that a Δlhca2 knockout mutant has constitutively enhanced CEF.