ATP-Dependent Conformational Changes and Translocation of Substrates in Clpap Protease as Revealed by Cryo-Electron Microscopy

2000 ◽  
Vol 6 (S2) ◽  
pp. 260-261
Author(s):  
Takashi Ishikawa ◽  
Fabienne Beuron ◽  
Martin Kessel ◽  
Sue Wickner ◽  
Michael R. Maurizi ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Conformational Changes ◽  
Layered Structure ◽  
Bacteriophage P1 ◽  
E Coli ◽  
Protein Substrates ◽  
Cryo Electron Microscopy ◽  
Clpap Protease ◽  
Image Averaging

ClpAP, an ATP-dependent protease of E. coli, recognizes and unfolds protein substrates via ClpA, its chaperonelike ATPase component, and digests them in ClpP, its protease component . ClpA forms hexameric rings with a two-layered structure, and stacks axially on either face of the double heptameric rings of ClpP. Protein substrates can bind to ClpAP in the presence of ATPγS, which is not hydrolyzed by ClpA, but are not degraded unless ATP is added. This property makes it possible to synchronize degradation in vitro by forming enzymesubstrate complexes in the presence of ATPγS and then adding ATP to trigger subsequent steps. We have used image averaging of electron micrographs of frozen hydrated and negatively stained specimens to characterize interactions of ClpA and ClpAP complexes with the model substrate, bacteriophage P1 protein, RepA.

scholarly journals Insights into Actin-Myosin Interactions within Muscle from 3-D Electron Microscopy

2019 ◽  
Author(s):  
Kenneth A. Taylor ◽  
Hamidreza Ramani ◽  
Robert J. Edwards ◽  
Michael K. Reedy
Keyword(s):  
Electron Microscopy ◽  
Conformational Changes ◽  
Flight Muscle ◽  
X Ray Crystallography ◽  
In Vitro Motility ◽  
Cryo Electron Microscopy ◽  
History Of ◽  
Cross Bridges

Much has been learned about the interaction between myosin and actin through biochemistry, in vitro motility assays and cryo-electron microscopy of F-actin decorated with myosin heads. Comparatively less is known about actin-myosin interactions within the filament lattice of muscle, where myosin heads function as independent force generators and thus most measurements report an average signal from multiple biochemical and mechanical states. All of the 3-D imaging by electron microscopy that has revealed the interplay of the regular array of actin subunits and myosin heads within the filament lattice has been accomplished using the flight muscle of the large waterbug Lethocerus sp. Lethocerus flight muscle possesses a particularly favorable filament arrangement that enables all the myosin cross-bridges contacting the actin filament to be visualized in a thin section. This review covers the history of this effort and the progress toward visualizing the complex set of conformational changes that myosin heads make when binding to actin in several static states as well as fast frozen actively contracting muscle. The efforts have revealed a consistent pattern of changes to the myosin head structures determined by X-ray crystallography needed to explain the structure of the different acto-myosin interactions observed in situ.

scholarly journals Supramolecular assembly of the E. coli LdcI upon acid stress

2020 ◽  
Author(s):  
Matthew Jessop ◽  
Clarissa Liesche ◽  
Jan Felix ◽  
Ambroise Desfosses ◽  
Megghane Baulard ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Acid Stress ◽  
Super Resolution ◽  
Low Ph ◽  
Lysine Decarboxylase ◽  
E Coli ◽  
Cryo Electron Microscopy ◽  
Supramolecular Organisation

AbstractPathogenic and commensal bacteria often have to resist the harsh acidity of the host stomach. The inducible lysine decarboxylase LdcI buffers the cytosol and the local extracellular environment to ensure enterobacterial survival at low pH. Here, we investigate the acid-stress response regulation of E. coli LdcI by combining biochemical and biophysical characterisation with negative stain and cryo-electron microscopy, and wide-field and super-resolution fluorescence imaging. Due to deleterious effects of fluorescent protein fusions on native LdcI decamers, we opt for three-dimensional localisation of nanobody-labelled endogenous wild-type LdcI in acid-stressed E. coli cells, and show that it organises into distinct patches at the cell periphery. Consistent with recent hypotheses that in vivo clustering of metabolic enzymes often reflects their polymerisation as a means of stimulus-induced regulation, we show that LdcI assembles into filaments in vitro at physiologically relevant low pH. We solve the structures of these filaments and of the LdcI decamer formed at neutral pH by cryo-electron microscopy, and reveal the molecular determinants of LdcI polymerisation, confirmed by mutational analysis. Finally, we propose a model for LdcI function inside the enterobacterial cell, providing a structural and mechanistic basis for further investigation of the role of its supramolecular organisation in the acid stress response.Significance statementBacteria possess a sophisticated arsenal of defence mechanisms that allow them to survive in adverse conditions. Adaptation to acid stress and hypoxia is crucial for the enterobacterial transmission in the gastrointestinal tract of their human host. When subjected to low pH, E. coli and many other enterobacteria activate a proton-consuming resistance system based on the acid-stress inducible lysine decarboxylase LdcI. Here we develop generally-applicable tools to uncover the spatial localisation of LdcI inside the cell by super-resolution fluorescence microscopy, and investigate the in vitro supramolecular organisation of this enzyme by cryo-EM. We build on these results to propose a mechanistic model for LdcI function and offer tools for further in vivo investigations.

scholarly journals Substrate-engaged 26S proteasome structures reveal mechanisms for ATP-hydrolysis–driven translocation

Science ◽  
2018 ◽  
Vol 362 (6418) ◽  
pp. eaav0725 ◽  
Author(s):  
Andres H. de la Peña ◽  
Ellen A. Goodall ◽  
Stephanie N. Gates ◽  
Gabriel C. Lander ◽  
Andreas Martin
Keyword(s):  
Electron Microscopy ◽  
Conformational Changes ◽  
Adenosine Triphosphatase ◽  
Atp Hydrolysis ◽  
26S Proteasome ◽  
Phosphate Release ◽  
Protein Substrates ◽  
Cellular Processes ◽  
Cryo Electron Microscopy ◽  
Aaa Atpases

The 26S proteasome is the primary eukaryotic degradation machine and thus is critically involved in numerous cellular processes. The heterohexameric adenosine triphosphatase (ATPase) motor of the proteasome unfolds and translocates targeted protein substrates into the open gate of a proteolytic core while a proteasomal deubiquitinase concomitantly removes substrate-attached ubiquitin chains. However, the mechanisms by which ATP hydrolysis drives the conformational changes responsible for these processes have remained elusive. Here we present the cryo–electron microscopy structures of four distinct conformational states of the actively ATP-hydrolyzing, substrate-engaged 26S proteasome. These structures reveal how mechanical substrate translocation accelerates deubiquitination and how ATP-binding, -hydrolysis, and phosphate-release events are coordinated within the AAA+ (ATPases associated with diverse cellular activities) motor to induce conformational changes and propel the substrate through the central pore.

scholarly journals The novel asymmetric entry intermediate of a picornavirus captured with nanodiscs

2016 ◽  
Vol 2 (8) ◽  
pp. e1501929 ◽  
Author(s):  
Hyunwook Lee ◽  
Kristin L. Shingler ◽  
Lindsey J. Organtini ◽  
Robert E. Ashley ◽  
Alexander M. Makhov ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Conformational Changes ◽  
Viral Proteins ◽  
Receptor Interaction ◽  
Icosahedral Symmetry ◽  
The Novel ◽  
Structural Studies ◽  
Cryo Electron Microscopy ◽  
Unique Site

Many nonenveloped viruses engage host receptors that initiate capsid conformational changes necessary for genome release. Structural studies on the mechanisms of picornavirus entry have relied on in vitro approaches of virus incubated at high temperatures or with excess receptor molecules to trigger the entry intermediate or A-particle. We have induced the coxsackievirus B3 entry intermediate by triggering the virus with full-length receptors embedded in lipid bilayer nanodiscs. These asymmetrically formed A-particles were reconstructed using cryo-electron microscopy and a direct electron detector. These first high-resolution structures of a picornavirus entry intermediate captured at a membrane with and without imposing icosahedral symmetry (3.9 and 7.8 Å, respectively) revealed a novel A-particle that is markedly different from the classical A-particles. The asymmetric receptor binding triggers minimal global capsid expansion but marked local conformational changes at the site of receptor interaction. In addition, viral proteins extrude from the capsid only at the site of extensive protein remodeling adjacent to the nanodisc. Thus, the binding of the receptor triggers formation of a unique site in preparation for genome release.

scholarly journals The Core and Holoenzyme Forms of RNA Polymerase from Mycobacterium smegmatis

2018 ◽  
Vol 201 (4) ◽  
Author(s):  
Tomáš Kouba ◽  
Jiří Pospíšil ◽  
Jarmila Hnilicová ◽  
Hana Šanderová ◽  
Ivan Barvík ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rna Polymerase ◽  
Conformational Changes ◽  
Mycobacterium Smegmatis ◽  
Drug Target ◽  
Conformational Dynamics ◽  
Three Dimensional ◽  
Cryo Electron Microscopy ◽  
Bacterial Rna ◽  
High Degree

ABSTRACT Bacterial RNA polymerase (RNAP) is essential for gene expression and as such is a valid drug target. Hence, it is imperative to know its structure and dynamics. Here, we present two as-yet-unreported forms of Mycobacterium smegmatis RNAP: core and holoenzyme containing σA but no other factors. Each form was detected by cryo-electron microscopy in two major conformations. Comparisons of these structures with known structures of other RNAPs reveal a high degree of conformational flexibility of the mycobacterial enzyme and confirm that region 1.1 of σA is directed into the primary channel of RNAP. Taken together, we describe the conformational changes of unrestrained mycobacterial RNAP. IMPORTANCE We describe here three-dimensional structures of core and holoenzyme forms of mycobacterial RNA polymerase (RNAP) solved by cryo-electron microscopy. These structures fill the thus-far-empty spots in the gallery of the pivotal forms of mycobacterial RNAP and illuminate the extent of conformational dynamics of this enzyme. The presented findings may facilitate future designs of antimycobacterial drugs targeting RNAP.

scholarly journals The U4 Antibody Epitope on Human Papillomavirus 16 Identified by Cryo-electron Microscopy

2015 ◽  
Vol 89 (23) ◽  
pp. 12108-12117 ◽  
Author(s):  
Jian Guan ◽  
Stephanie M. Bywaters ◽  
Sarah A. Brendle ◽  
Hyunwook Lee ◽  
Robert E. Ashley ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibody ◽  
Human Papillomavirus ◽  
Conformational Changes ◽  
Vaccine Development ◽  
Contact Point ◽  
Human Papillomavirus 16 ◽  
Cryo Electron Microscopy ◽  
C Terminus ◽  
L1 Protein

ABSTRACTThe human papillomavirus (HPV) major structural protein L1 composes capsomers that are linked together through interactions mediated by the L1 C terminus to constitute a T=7 icosahedral capsid. H16.U4 is a type-specific monoclonal antibody recognizing a conformation-dependent neutralizing epitope of HPV thought to include the L1 protein C terminus. The structure of human papillomavirus 16 (HPV16) complexed with H16.U4 fragments of antibody (Fab) was solved by cryo-electron microscopy (cryo-EM) image reconstruction. Atomic structures of virus and Fab were fitted into the corresponding cryo-EM densities to identify the antigenic epitope. The antibody footprint mapped predominately to the L1 C-terminal arm with an additional contact point on the side of the capsomer. This footprint describes an epitope that is presented capsid-wide. However, although the H16.U4 epitope suggests the presence of 360 potential binding sites exposed in the capsid valley between each capsomer, H16.U4 Fab bound only to epitopes located around the icosahedral five-fold vertex of the capsid. Thus, the binding characteristics of H16.U4 defined in this study showed a distinctive selectivity for local conformation-dependent interactions with specific L1 invading arms between five-fold related capsomers.IMPORTANCEHuman papillomavirus 16 (HPV16) is the most prevalent oncogenic genotype in HPV-associated anogenital and oral cancers. Here we use cryo-EM reconstruction techniques to solve the structures of the HPV16 capsid complexes using H16.U4 fragment of antibody (Fab). Different from most other antibodies directed against surface loops, H16.U4 monoclonal antibody is unique in targeting the C-terminal arm of the L1 protein. This monoclonal antibody (MAb) is used throughout the HPV research community in HPV serological and vaccine development and to define mechanisms of HPV uptake. The unique binding mode of H16.U4 defined here shows important conformation-dependent interactions within the HPV16 capsid. By targeting an important structural and conformational epitope, H16.U4 may identify subtle conformational changes in different maturation stages of the HPV capsid and provide a key probe to analyze the mechanisms of HPV uptake during the early stages of virus infection. Our analyses precisely define important conformational epitopes on HPV16 capsids that are key targets for successful HPV prophylactic vaccines.

scholarly journals Conserved mechanisms of microtubule-stimulated ADP release, ATP binding, and force generation in transport kinesins

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Joseph Atherton ◽  
Irene Farabella ◽  
I-Mei Yu ◽  
Steven S Rosenfeld ◽  
Anne Houdusse ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Conformational Changes ◽  
Catalytic Site ◽  
Fundamental Question ◽  
Force Generation ◽  
Atp Binding ◽  
Cellular Functions ◽  
Microtubule Binding ◽  
Cryo Electron Microscopy ◽  
Adp Release

Kinesins are a superfamily of microtubule-based ATP-powered motors, important for multiple, essential cellular functions. How microtubule binding stimulates their ATPase and controls force generation is not understood. To address this fundamental question, we visualized microtubule-bound kinesin-1 and kinesin-3 motor domains at multiple steps in their ATPase cycles—including their nucleotide-free states—at ∼7 Å resolution using cryo-electron microscopy. In both motors, microtubule binding promotes ordered conformations of conserved loops that stimulate ADP release, enhance microtubule affinity and prime the catalytic site for ATP binding. ATP binding causes only small shifts of these nucleotide-coordinating loops but induces large conformational changes elsewhere that allow force generation and neck linker docking towards the microtubule plus end. Family-specific differences across the kinesin–microtubule interface account for the distinctive properties of each motor. Our data thus provide evidence for a conserved ATP-driven mechanism for kinesins and reveal the critical mechanistic contribution of the microtubule interface.

scholarly journals Fabrication & Characterization of Chitosan Coated Biologically Synthesized TiO2 Nanoparticles against PDR E. coli of Veterinary Origin

2020 ◽  
Vol 2020 ◽  
pp. 1-13 ◽  
Author(s):  
Naheed Zafar ◽  
Bushra Uzair ◽  
Muhammad Bilal Khan Niazi ◽  
Shamaila Sajjad ◽  
Ghufrana Samin ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Antibacterial Activity ◽  
Titanium Dioxide ◽  
Morphological Changes ◽  
Titanium Dioxide Nanoparticles ◽  
Chitosan Nanoparticles ◽  
Diffusion Method ◽  
Potential Threat ◽  
E Coli

Treatment of pandrug resistant (PDR) Escherichia coli strain is the leading causative agent of bovine mastitis worldwide. Hence, becoming a potential threat to veterinary and public health. Therefore, to control the infection new nontoxic, biocompatible antimicrobial formulation with enhanced antibacterial activity is massively required. Current study was planned to synthesize chitosan coated titanium dioxide nanoparticles (CS-NPs coated TiO2). Coating was being done by chitosan nanoparticles (CS-NPs) using ionic gelation method. Aqueous solution of Moringa concanensis leaf extract was used to synthesize titanium dioxide nanoparticles (TiO2 NPs). The synthesized nanoformulations were characterized by using XRD, SEM, and FTIR. X-ray diffraction (XRD) analysis indicated the crystalline phase of TiO2 NPs and CS-NPs coated TiO2 NPs. Scanning Electron Microscopy (SEM) confirmed spherical shaped nanoparticles size of chitosan NPs ranging from 19–25 nm and TiO2 NPs 35–50 nm. Thesize of CS-NPs coated TiO2 NPs was in the range of 65–75 nm. The UV-Vis Spectra and band gap values illustrated the red shift in CS-NPs coated TiO2 NPs. Fourier transform infrared (FTIR) spectroscopy confirmed the linkages between TiO2 NPs and chitosan biopolymer, Zeta potential confirmed the stability of CS-NPs coated TiO2 NPs by showing 95 mV peak value. In-vitro antibacterial activity of CS-NPs coated TiO2 NPs and Uncoated TiO2 NPs was evaluated by disc diffusion method against PDR strain of E. coli isolated from mastitic milk samples. The antibacterial activity of all the synthesized nanoformulations were noted and highest antibacterial activity was shown by CS-NPs coated TiO2-NPs against pandrug resistant (PDR) E. coli strain with the prominent zone of inhibition of 23 mm. Morphological changes of E. coli cells after the treatment with MIC concentration (0.78 μg/ml) of CS-NPs coated TiO2 NPs were studied by transmission electron microscopy TEM showedrigorous morphological defectand has distorted the general appearance of the E. coli cells. Cytotoxicity (HepG2 cell line) and hemolytic (human blood) studies confirmed nontoxic/biocompatible nature of CS-NPs coated biologically synthesized TiO2 NPs. The results suggested that biologically synthesized and surface modified TiO2 NPs by mucoadhesive polysaccharides (e.g. chitosan) coating would be an effective and non-toxic alternative therapeutic agent to be used in livestock industry to control drug resistant veterinary pathogens.

scholarly journals Cryo-EM structure and polymorphism of Aβ amyloid fibrils purified from Alzheimer’s brain tissue

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Marius Kollmer ◽  
William Close ◽  
Leonie Funk ◽  
Jay Rasmussen ◽  
Aref Bsoul ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Brain Tissue ◽  
Amyloid Fibrils ◽  
Structural Basis ◽  
Amyloid Angiopathy ◽  
Cryo Electron Microscopy ◽  
Aβ Amyloid ◽  
Cerebral Amyloid ◽  
Aβ Fibrils

Abstract The formation of Aβ amyloid fibrils is a neuropathological hallmark of Alzheimer’s disease and cerebral amyloid angiopathy. However, the structure of Aβ amyloid fibrils from brain tissue is poorly understood. Here we report the purification of Aβ amyloid fibrils from meningeal Alzheimer’s brain tissue and their structural analysis with cryo-electron microscopy. We show that these fibrils are polymorphic but consist of similarly structured protofilaments. Brain derived Aβ amyloid fibrils are right-hand twisted and their peptide fold differs sharply from previously analyzed Aβ fibrils that were formed in vitro. These data underscore the importance to use patient-derived amyloid fibrils when investigating the structural basis of the disease.

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