scholarly journals Structural mechanism of laminin recognition by integrin

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Takao Arimori ◽  
Naoyuki Miyazaki ◽  
Emiko Mihara ◽  
Mamoru Takizawa ◽  
Yukimasa Taniguchi ◽  
...  
Keyword(s):  
Metal Ion ◽  
Electrostatic Interactions ◽  
High Mobility ◽  
Laminin Receptor ◽  
Integrin Receptors ◽  
Unique Role ◽  
Structural Mechanism ◽  
Cryo Electron Microscopy ◽  
Alternatively Spliced ◽  
Anchor Points

AbstractRecognition of laminin by integrin receptors is central to the epithelial cell adhesion to basement membrane, but the structural background of this molecular interaction remained elusive. Here, we report the structures of the prototypic laminin receptor α6β1 integrin alone and in complex with three-chain laminin-511 fragment determined via crystallography and cryo-electron microscopy, respectively. The laminin-integrin interface is made up of several binding sites located on all five subunits, with the laminin γ1 chain C-terminal portion providing focal interaction using two carboxylate anchor points to bridge metal-ion dependent adhesion site of integrin β1 subunit and Asn189 of integrin α6 subunit. Laminin α5 chain also contributes to the affinity and specificity by making electrostatic interactions with large surface on the β-propeller domain of α6, part of which comprises an alternatively spliced X1 region. The propeller sheet corresponding to this region shows unusually high mobility, suggesting its unique role in ligand capture.

scholarly journals A new topology of the HK97-like fold revealed in Bordetella bacteriophage by cryoEM at 3.5 Å resolution

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Xing Zhang ◽  
Huatao Guo ◽  
Lei Jin ◽  
Elizabeth Czornyj ◽  
Asher Hodes ◽  
...  
Keyword(s):  
Phage Display ◽  
Electrostatic Interactions ◽  
Whooping Cough ◽  
Atomic Structures ◽  
Complex Interactions ◽  
Cryo Electron Microscopy ◽  
Naturally Occurring ◽  
Feasible Alternative ◽  
Β Sheet ◽  
Cement Protein

Bacteriophage BPP-1 infects and kills Bordetella species that cause whooping cough. Its diversity-generating retroelement (DGR) provides a naturally occurring phage-display system, but engineering efforts are hampered without atomic structures. Here, we report a cryo electron microscopy structure of the BPP-1 head at 3.5 Å resolution. Our atomic model shows two of the three protein folds representing major viral lineages: jellyroll for its cement protein (CP) and HK97-like (‘Johnson’) for its major capsid protein (MCP). Strikingly, the fold topology of MCP is permuted non-circularly from the Johnson fold topology previously seen in viral and cellular proteins. We illustrate that the new topology is likely the only feasible alternative of the old topology. β-sheet augmentation and electrostatic interactions contribute to the formation of non-covalent chainmail in BPP-1, unlike covalent inter-protein linkages of the HK97 chainmail. Despite these complex interactions, the termini of both CP and MCP are ideally positioned for DGR-based phage-display engineering.

scholarly journals Initiation of Attachment and Generation of Mature Focal Adhesions by Integrin-containing Filopodia in Cell Spreading

2006 ◽  
Vol 17 (10) ◽  
pp. 4237-4248 ◽  
Author(s):  
Michael A. Partridge ◽  
Eugene E. Marcantonio
Keyword(s):  
Total Internal Reflection ◽  
Fluorescent Protein ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Integrin Receptors ◽  
Cortical Actin ◽  
Cell Matrix ◽  
Cytoplasmic Proteins ◽  
Radial Projection ◽  
Anchor Points

Integrin receptors, and associated cytoplasmic proteins mediate adhesion, cell signaling and connections to the cytoskeleton. Using fluorescent protein chimeras, we analyzed initial integrin adhesion in spreading fibroblasts with Total Internal Reflection Fluorescence (TIRF) microscopy. Surprisingly, sequential radial projection of integrin and actin containing filopodia formed the initial cell-matrix contacts. These Cdc42-dependent, integrin-containing projections recruited cytoplasmic focal adhesion (FA) proteins in a hierarchical manner; initially talin with integrin and subsequently FAK and paxillin. Radial FA structures then anchored cortical actin bridges between them and subsequently cells reorganized their actin, a process promoted by Src, and characterized by lateral FA reorientation to provide anchor points for actin stress fibers. Finally, the nascent adhesions coalesced until they formed mature FAs.

Interaction of neutrophil elastase with hydrophobic polyanionic chelators

1991 ◽  
Vol 69 (9) ◽  
pp. 624-629 ◽  
Author(s):  
Suresh C. Tyagi ◽  
Sanford R. Simon
Keyword(s):  
Calcium Ions ◽  
Neutrophil Elastase ◽  
Metal Ion ◽  
Electrostatic Interactions ◽  
Enzyme Inhibitor ◽  
Human Neutrophil Elastase ◽  
Tetraacetic Acid ◽  
Fura 2 ◽  
A Site ◽  
Substrate Binding Domain

The polyanionic calcium chelators, ethylenediamine-tetraacetic acid (EDTA), ethylene-bis-(oxyethylenenitrilo)-tetraacetic acid (EGTA), [bis-(O-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid (BAPTA), 1-[2-amino-5-(6-carboxy-indol-2-yl)phenoxyl]-2-(2′-amino-5′-methylphenoxy)ethane- N,N,N′,N′-tetraacetic acid (INDO-1), 1-[2-(5-carboxyoxazol-2yl)-6-phenoxyl]-2-(2′-amino-5′-methylphenoxy)ethane-N,N,N′,N′-tetraacetic acid (FURA-2), and 2-{[2-bis-(carboxymethyl)-amino-5-methylphenoxy]-methyl}-6-methyl-8-bis-(bis-(carboxymethyl)-aminoquinoline (QUIN-2), are all inhibitors of amidolytic activity of human neutrophil elastase (HNE). With MeOSuc-Ala-Ala-Pro-Val-pNA as substrate, these chelators all display mixed partial competitive and partial noncompetitive inhibition, but with the smaller substrate, pGlu-Pro-Val-pNA, only the noncompetitive component persists. The most effective inhibitor is FURA-2, with an apparent Ki of 0.5–0.7 mM. QUIN-2 is somewhat less effective, with a Ki of 2 mM, while EDTA is much less effective, with a Ki of 7 mM. In general, the more hydrophobic chelators are the best inhibitors, although INDO-1, which is about the same size as FURA-2, is surprisingly ineffective as an inhibitor. The chelators no longer function as effective inhibitors if their carboxyl groups are blocked by esterification with acetoxymethyl groups or by complexation with calcium ions, indicating that their binding to HNE is mediated in part through electrostatic interactions with a center of positive charge on the protein. The excitation spectrum of the complex of FURA-2 with HNE differs from that of the chelator with calcium ions, indicating that the structure of the enzyme-inhibitor complex is not like that of the coordination complex of the chelator with the metal ion. The inhibitory capacity of FURA-2 apparently arises from binding to a site that is in the vicinity of the S4 and S5 subsites of the extended substrate binding domain on HNE through a combination of hydrophobic and electrostatic interactions with the enzyme.Key words: elastase, protease inhibitors, chelators, poly anions, calcium.

scholarly journals Structure and assembly of the mouse ASC inflammasome by combined NMR spectroscopy and cryo-electron microscopy

2015 ◽  
Vol 112 (43) ◽  
pp. 13237-13242 ◽  
Author(s):  
Lorenzo Sborgi ◽  
Francesco Ravotti ◽  
Venkata P. Dandey ◽  
Mathias S. Dick ◽  
Adam Mazur ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Nmr Spectroscopy ◽  
Specific Binding ◽  
3D Structure ◽  
High Mobility ◽  
Atomic Resolution ◽  
Receptor Protein ◽  
Filament Formation ◽  
Cryo Electron Microscopy

Inflammasomes are multiprotein complexes that control the innate immune response by activating caspase-1, thus promoting the secretion of cytokines in response to invading pathogens and endogenous triggers. Assembly of inflammasomes is induced by activation of a receptor protein. Many inflammasome receptors require the adapter protein ASC [apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD)], which consists of two domains, the N-terminal pyrin domain (PYD) and the C-terminal CARD. Upon activation, ASC forms large oligomeric filaments, which facilitate procaspase-1 recruitment. Here, we characterize the structure and filament formation of mouse ASC in vitro at atomic resolution. Information from cryo-electron microscopy and solid-state NMR spectroscopy is combined in a single structure calculation to obtain the atomic-resolution structure of the ASC filament. Perturbations of NMR resonances upon filament formation monitor the specific binding interfaces of ASC-PYD association. Importantly, NMR experiments show the rigidity of the PYD forming the core of the filament as well as the high mobility of the CARD relative to this core. The findings are validated by structure-based mutagenesis experiments in cultured macrophages. The 3D structure of the mouse ASC-PYD filament is highly similar to the recently determined human ASC-PYD filament, suggesting evolutionary conservation of ASC-dependent inflammasome mechanisms.

scholarly journals Elucidation of AMPA receptor–stargazin complexes by cryo–electron microscopy

Science ◽  
2016 ◽  
Vol 353 (6294) ◽  
pp. 83-86 ◽  
Author(s):  
Edward C. Twomey ◽  
Maria V. Yelshanskaya ◽  
Robert A. Grassucci ◽  
Joachim Frank ◽  
Alexander I. Sobolevsky
Keyword(s):  
Electron Microscopy ◽  
Cognitive Processes ◽  
Electrostatic Interactions ◽  
Structural Basis ◽  
Cryo Electron Microscopy ◽  
Activated State ◽  
Ampar Trafficking ◽  
Excitatory Neurotransmission ◽  
Binding Interfaces ◽  
The Brain

AMPA-subtype ionotropic glutamate receptors (AMPARs) mediate fast excitatory neurotransmission and contribute to high cognitive processes such as learning and memory. In the brain, AMPAR trafficking, gating, and pharmacology is tightly controlled by transmembrane AMPAR regulatory proteins (TARPs). Here, we used cryo–electron microscopy to elucidate the structural basis of AMPAR regulation by one of these auxiliary proteins, TARP γ2, or stargazin (STZ). Our structures illuminate the variable interaction stoichiometry of the AMPAR-TARP complex, with one or two TARP molecules binding one tetrameric AMPAR. Analysis of the AMPAR-STZ binding interfaces suggests that electrostatic interactions between the extracellular domains of AMPAR and STZ play an important role in modulating AMPAR function through contact surfaces that are conserved across AMPARs and TARPs. We propose a model explaining how TARPs stabilize the activated state of AMPARs and how the interactions between AMPARs and their auxiliary proteins control fast excitatory synaptic transmission.

The coupling mechanism of mammalian respiratory complex I

Science ◽  
2020 ◽  
Vol 370 (6516) ◽  
pp. eabc4209 ◽  
Author(s):  
Domen Kampjut ◽  
Leonid A. Sazanov
Keyword(s):  
Conformational Changes ◽  
Complex I ◽  
Electrostatic Interactions ◽  
Proton Translocation ◽  
Proton Pumping ◽  
State Transitions ◽  
Coupling Mechanism ◽  
Cryo Electron Microscopy ◽  
Open Conformation ◽  
Respiratory Complex I

Mitochondrial complex I couples NADH:ubiquinone oxidoreduction to proton pumping by an unknown mechanism. Here, we present cryo–electron microscopy structures of ovine complex I in five different conditions, including turnover, at resolutions up to 2.3 to 2.5 angstroms. Resolved water molecules allowed us to experimentally define the proton translocation pathways. Quinone binds at three positions along the quinone cavity, as does the inhibitor rotenone that also binds within subunit ND4. Dramatic conformational changes around the quinone cavity couple the redox reaction to proton translocation during open-to-closed state transitions of the enzyme. In the induced deactive state, the open conformation is arrested by the ND6 subunit. We propose a detailed molecular coupling mechanism of complex I, which is an unexpected combination of conformational changes and electrostatic interactions.

Altered expression of the alpha7beta1 integrin in human and murine muscular dystrophies

1997 ◽  
Vol 110 (22) ◽  
pp. 2873-2881 ◽  
Author(s):  
B.L. Hodges ◽  
Y.K. Hayashi ◽  
I. Nonaka ◽  
W. Wang ◽  
K. Arahata ◽  
...  
Keyword(s):  
Muscular Dystrophy ◽  
Gene Transcription ◽  
Muscle Development ◽  
Structural Integrity ◽  
Laminin Receptor ◽  
Muscular Dystrophies ◽  
Mdx Mice ◽  
Altered Expression ◽  
Alternatively Spliced ◽  
Alpha7beta1 Integrin

The alpha7beta1 integrin is the primary laminin receptor on skeletal myoblasts and adult myofibers. It has distinct functions during muscle development and contributes to muscle structural integrity. We have studied this integrin in cases where expression of dystrophin or laminin are compromised. Immunofluorescence demonstrates an increase in alpha7beta1 in patients with Duchenne muscular dystrophy and in mdx mice that lack dystrophin. Analysis of RNA from mdx mice and from patients with Duchenne and Becker muscular dystrophies indicates that the increase in the alpha7beta1 integrin is regulated at the level of alpha7 gene transcription. In contrast, the levels of alpha7beta1 integrin are severely diminished in patients with laminin alpha2 chain congenital dystrophy muscular dystrophy and in dy/dy mice that also do not make the alpha2 laminin chain. Analysis of RNA from the hindlimbs of dy/dy mice demonstrated that in the absence of laminin alpha7 gene transcription is inhibited and limited to specific alternatively spliced isoforms. We suggest that the increased expression of alpha7beta1 integrin in the absence of dystrophin compensates for the reduced dystrophin-mediated linkage of fibers with the basal lamina and modulates the development of pathology associated with these diseases. The decrease in alpha7beta1 integrin and its transcripts in the absence of laminin likely contributes to the severe myopathy that results from laminin alpha2 chain deficiency and suggests that laminin-2 regulates expression of the alpha7 integrin gene. The role of the alpha7beta1 integrin in muscle integrity also suggests that compromised expression of this receptor may underlie as yet undefined myopathies.

scholarly journals Fluorescence Resonance Energy Transfer Analysis of Recombination Signal Sequence Configuration in the RAG1/2 Synaptic Complex

2007 ◽  
Vol 27 (13) ◽  
pp. 4745-4758 ◽  
Author(s):  
Mihai Ciubotaru ◽  
Aleksei N. Kriatchko ◽  
Patrick C. Swanson ◽  
Frank V. Bright ◽  
David G. Schatz
Keyword(s):  
Energy Transfer ◽  
Metal Ion ◽  
Signal Sequence ◽  
Resonance Energy Transfer ◽  
High Mobility ◽  
Resonance Energy ◽  
High Mobility Group Protein ◽  
Synaptic Complex ◽  
Recombination Signal ◽  
Group Protein

ABSTRACT A critical step in V(D)J recombination is the synapsis of complementary (12/23) recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins to generate the paired complex (PC). Using a facilitated ligation assay and substrates that vary the helical phasing of the RSSs, we provide evidence that one particular geometric configuration of the RSSs is favored in the PC. To investigate this configuration further, we used fluorescent resonance energy transfer (FRET) to detect the synapsis of fluorescently labeled RSS oligonucleotides. FRET requires an appropriate 12/23 RSS pair, a divalent metal ion, and high-mobility-group protein HMGB1 or HMGB2. Energy transfer between the RSSs was detected with all 12/23 RSS end positions of the fluorescent probes but was not detected when probes were placed on the two ends of the same RSS. Energy transfer was confirmed to originate from the PC by using an in-gel FRET assay. The results argue against a unique planar configuration of the RSSs in the PC and are most easily accommodated by models in which synapsed 12- and 23-RSSs are bent and cross one another, with implications for the organization of the RAG proteins and the DNA substrates at the time of cleavage.

scholarly journals Differences in the subunit interface residues of alternatively spliced glutathione transferases affects catalytic and structural functions

2007 ◽  
Vol 401 (3) ◽  
pp. 635-644 ◽  
Author(s):  
Juthamart Piromjitpong ◽  
Jantana Wongsantichon ◽  
Albert J. Ketterman
Keyword(s):  
Active Site ◽  
Electrostatic Interactions ◽  
Hydrophobic Interactions ◽  
Glutathione Transferases ◽  
Active Site Residues ◽  
Interface Area ◽  
Subunit Interface ◽  
Alternatively Spliced ◽  
Active Site Conformation ◽  
Interface Residues

GSTs (glutathione transferases) are multifunctional widespread enzymes. Currently there are 13 identified classes within this family. Previously most structural characterization has been reported for mammalian Alpha, Mu and Pi class GSTs. In the present study we characterize two enzymes from the insect-specific Delta class, adGSTD3-3 and adGSTD4-4. These two proteins are alternatively spliced products from the same gene and have very similar tertiary structures. Several major contributions to the dimer interface area can be separated into three regions: conserved electrostatic interactions in region 1, hydrophobic interactions in region 2 and an ionic network in region 3. The four amino acid side chains studied in region 1 interact with each other as a planar rectangle. These interactions are highly conserved among the GST classes, Delta, Sigma and Theta. The hydrophobic residues in region 2 are not only subunit interface residues but also active site residues. Overall these three regions provide important contributions to stabilization and folding of the protein. In addition, decreases in yield as well as catalytic activity changes, suggest that the mutations in these regions can disrupt the active site conformation which decreases binding affinity, alters kinetic constants and alters substrate specificity. Several of these residues have only a slight effect on the initial folding of each subunit but have more influence on the dimerization process as well as impacting upon appropriate active site conformation. The results also suggest that even splicing products from the same gene may have specific features in the subunit interface area that would preclude heterodimerization.

scholarly journals Non-rigid image registration to reduce beam-induced blurring of cryo-electron microscopy images

2012 ◽  
Vol 20 (1) ◽  
pp. 58-66 ◽  
Author(s):  
Fatemeh Karimi Nejadasl ◽  
Manikandan Karuppasamy ◽  
Emily R. Newman ◽  
John E. McGeehan ◽  
Raimond B. G. Ravelli
Keyword(s):  
Electron Microscopy ◽  
Low Dose ◽  
Structural Alterations ◽  
Gold Particles ◽  
Correlated Motions ◽  
Cryo Electron Microscopy ◽  
Natural Neighbor ◽  
Radiation Induced ◽  
Anchor Points ◽  
Microscopy Images

The typical dose used to record cryo-electron microscopy images from vitrified biological specimens is so high that radiation-induced structural alterations are bound to occur during data acquisition. Integration of all scattered electrons into one image can lead to significant blurring, particularly if the data are collected from an unsupported thin layer of ice suspended over the holes of a support film. Here, the dose has been fractioned and exposure series have been acquired in order to study beam-induced specimen movements under low dose conditions, prior to bubbling. Gold particles were added to the protein sample as fiducial markers. These were automatically localized and tracked throughout the exposure series and showed correlated motions within small patches, with larger amplitudes of motion vectors at the start of a series compared with the end of each series. A non-rigid scheme was used to register all images within each exposure series, using natural neighbor interpolation with the gold particles as anchor points. The procedure increases the contrast and resolution of the examined macromolecules.

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