scholarly journals Pentameric assembly of the Kv2.1 tetramerization domain.

2021 ◽  
Author(s):  
Zhen Xu ◽  
Saif Khan ◽  
Nicholas Schnicker ◽  
Sheila A Baker
Keyword(s):  
Electrostatic Interactions ◽  
Neuronal Excitability ◽  
Salt Bridge ◽  
X Ray ◽  
X Ray Crystallography ◽  
Kv Channels ◽  
Negative Stain ◽  
Tetramerization Domain ◽  
Membrane Contacts ◽  
T1 Domain

The Kv family of voltage-gated potassium channels regulate neuronal excitability. The biophysical characteristic of Kv channels can be matched to the needs of different neurons by forming homotetrameric or heterotetrameric channels within one of four subfamilies. The cytoplasmic tetramerization (T1) domain plays a major role in dictating the compatibility of different Kv subunits. The only Kv subfamily missing a representative structure of the T1 domain is the Kv2 family. We used X-ray crystallography to solve the structure of the human Kv2.1 T1 domain. The structure is similar to other T1 domains but surprisingly formed a pentamer instead of a tetramer. In solution the Kv2.1 T1 domain also formed a pentamer as determined with in-line SEC-MALS-SAXS and negative stain EM. The Kv2.1 T1-T1 interface involves electrostatic interactions including a salt bridge formed by the negative charges in a previously described CDD motif, and inter-subunit coordination of zinc. We show that zinc binding is important for stability. In conclusion, the Kv2.1 T1 domain behaves differently from the other Kv T1 domains which may reflect the versatility of Kv2.1, the only Kv subfamily that can assemble with the regulatory KvS subunits and scaffold ER-plasma membrane contacts.

scholarly journals A natural mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody

2020 ◽  
Author(s):  
Nicholas C. Wu ◽  
Meng Yuan ◽  
Sandhya Bangaru ◽  
Deli Huang ◽  
Xueyong Zhu ◽  
...  
Keyword(s):  
Antigenic Variation ◽  
Neutralizing Antibody ◽  
Spike Protein ◽  
Single Mutation ◽  
X Ray ◽  
X Ray Crystallography ◽  
Negative Stain ◽  
Natural Mutation ◽  
Reactive Antibody ◽  
Conserved Epitope

ABSTRACTEpitopes that are conserved among SARS-like coronaviruses are attractive targets for design of cross-reactive vaccines and therapeutics. CR3022 is a SARS-CoV neutralizing antibody to a highly conserved epitope on the receptor binding domain (RBD) on the spike protein that can cross-react with SARS-CoV-2, but with lower affinity. Using x-ray crystallography, mutagenesis, and binding experiments, we illustrate that of four amino acid differences in the CR3022 epitope between SARS-CoV-2 and SARS-CoV, a single mutation P384A fully determines the affinity difference. CR3022 does not neutralize SARS-CoV-2, but the increased affinity to SARS-CoV-2 P384A mutant now enables neutralization with a similar potency to SARS-CoV. We further investigated CR3022 interaction with the SARS-CoV spike protein by negative-stain EM and cryo-EM. Three CR3022 Fabs bind per trimer with the RBD observed in different up-conformations due to considerable flexibility of the RBD. In one of these conformations, quaternary interactions are made by CR3022 to the N-terminal domain (NTD) of an adjacent subunit. Overall, this study provides insights into antigenic variation and potential for cross-neutralizing epitopes on SARS-like viruses.

Crystalline two-dimensional nanosheet composed of tetraanionic porphyrins and tetracationic metallomacrocycles

2016 ◽  
Vol 20 (06) ◽  
pp. 694-699
Author(s):  
Toshio Nishino ◽  
Yasuyuki Yamada ◽  
Kentaro Tanaka
Keyword(s):  
Electrostatic Interactions ◽  
Two Dimensional ◽  
X Ray ◽  
X Ray Crystallography ◽  
Ionic Lattice

A tetraanionic porphyrin and a tetracationic metallomacrocycle formed a layered two-dimensional nanosheet through multiple electrostatic interactions. X-ray crystallography revealed that the sheet is an ionic lattice composed of [Formula: see text]-conjugated components

scholarly journals Integrative structural Investigation on the architecture of human Importin4_histone H3/H4_Asf1a complex and its histone H3 tail binding

2017 ◽  
Author(s):  
Jungmin Yoon ◽  
Seung Joong Kim ◽  
Sojin An ◽  
Alexander Leitner ◽  
Taeyang Jung ◽  
...  
Keyword(s):  
Histone H3 ◽  
Terminal Region ◽  
Nuclear Localization Sequence ◽  
Molecular Architecture ◽  
X Ray ◽  
X Ray Crystallography ◽  
Integrative Modeling ◽  
Negative Stain ◽  
Localization Sequence ◽  
Chaperone Complex

AbstractImportin4 transports histone H3/H4 in complex with Asf1a to the nucleus for chromatin assembly. Importin4 recognizes the nuclear localization sequence located at the N-terminal tail of histones. Here, we analyzed the structures and interactions of human Importin4, histones and Asf1a by cross-linking mass spectrometry, X-ray crystallography, negative-stain electron microscopy, small-angle X-ray scattering and integrative modeling. The XL-MS data showed that the C-terminal region of Importin4 interacts extensively with the histone H3 tail. We determined the crystal structure of the C-terminal region of Importin4 bound to the histone H3 peptide, thus revealing that the acidic path in Importin4 accommodates the histone H3 tail and that histone H3 Lys14 is the primary residue interacting with Importin4. Furthermore, the molecular architecture of the Importin4_histone H3/H4_Asf1a complex was produced through an integrative modeling approach. Overall, this work provides structural insights into how Importin4 recognizes histones and their chaperone complex.

scholarly journals A natural mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody

2020 ◽  
Vol 16 (12) ◽  
pp. e1009089
Author(s):  
Nicholas C. Wu ◽  
Meng Yuan ◽  
Sandhya Bangaru ◽  
Deli Huang ◽  
Xueyong Zhu ◽  
...  
Keyword(s):  
Antigenic Variation ◽  
Neutralizing Antibody ◽  
Spike Protein ◽  
Single Mutation ◽  
X Ray ◽  
X Ray Crystallography ◽  
Negative Stain ◽  
Natural Mutation ◽  
Reactive Antibody ◽  
Conserved Epitope

Epitopes that are conserved among SARS-like coronaviruses are attractive targets for design of cross-reactive vaccines and therapeutics. CR3022 is a SARS-CoV neutralizing antibody to a highly conserved epitope on the receptor binding domain (RBD) on the spike protein that is able to cross-react with SARS-CoV-2, but with lower affinity. Using x-ray crystallography, mutagenesis, and binding experiments, we illustrate that of four amino acid differences in the CR3022 epitope between SARS-CoV-2 and SARS-CoV, a single mutation P384A fully determines the affinity difference. CR3022 does not neutralize SARS-CoV-2, but the increased affinity to SARS-CoV-2 P384A mutant now enables neutralization with a similar potency to SARS-CoV. We further investigated CR3022 interaction with the SARS-CoV spike protein by negative-stain EM and cryo-EM. Three CR3022 Fabs bind per trimer with the RBD observed in different up-conformations due to considerable flexibility of the RBD. In one of these conformations, quaternary interactions are made by CR3022 to the N-terminal domain (NTD) of an adjacent subunit. Overall, this study provides insights into antigenic variation and potential cross-neutralizing epitopes on SARS-like viruses.

scholarly journals A Chemical Probe for Dark Kinase STK17B Derives its Potency and High Selectivity Through a Unique P-loop Conformation

2020 ◽  
Author(s):  
Alfredo Picado ◽  
Apirat Chaikuad ◽  
Carrow Wells ◽  
Safal Shrestha ◽  
William Zuercher ◽  
...  
Keyword(s):  
Carboxylic Acid ◽  
High Selectivity ◽  
Salt Bridge ◽  
Competitive Inhibitor ◽  
Chemical Probe ◽  
High Quality ◽  
X Ray ◽  
X Ray Crystallography ◽  
Loop Conformation

We present the discovery of thieno[3,2-<i>d</i>]pyrimidine <b>SGC-STK17B-1</b> (<b>11s</b>), a high-quality chemical probe for this understudied “dark” kinase. <b>11s</b> is an ATP-competitive inhibitor that showed remarkable selectivity over other kinases including the closely related STK17A. X-ray crystallography of <b>11s</b> and related thieno[3,2-<i>d</i>]pyrimidines bound to STK17B revealed a unique P-loop conformation characterized by a salt bridge between R41 and the carboxylic acid of the inhibitor.

scholarly journals Conduction through a narrow inward-rectifier K+ channel pore

2019 ◽  
Vol 151 (10) ◽  
pp. 1231-1246 ◽  
Author(s):  
Harald Bernsteiner ◽  
Eva-Maria Zangerl-Plessl ◽  
Xingyu Chen ◽  
Anna Stary-Weinzinger
Keyword(s):  
Neuronal Excitability ◽  
Md Simulations ◽  
Inward Rectifier ◽  
K Channel ◽  
Resting Membrane Potential ◽  
Kir Channels ◽  
X Ray Crystallography ◽  
Kv Channels ◽  
Physiological Processes ◽  
Conduction Mechanisms

Inwardly rectifying potassium (Kir) channels play a key role in controlling membrane potentials in excitable and unexcitable cells, thereby regulating a plethora of physiological processes. G-protein–gated Kir channels control heart rate and neuronal excitability via small hyperpolarizing outward K+ currents near the resting membrane potential. Despite recent breakthroughs in x-ray crystallography and cryo-EM, the gating and conduction mechanisms of these channels are poorly understood. MD simulations have provided unprecedented details concerning the gating and conduction mechanisms of voltage-gated K+ and Na+ channels. Here, we use multi-microsecond–timescale MD simulations based on the crystal structures of GIRK2 (Kir3.2) bound to phosphatidylinositol-4,5-bisphosphate to provide detailed insights into the channel’s gating dynamics, including insights into the behavior of the G-loop gate. The simulations also elucidate the elementary steps that underlie the movement of K+ ions through an inward-rectifier K+ channel under an applied electric field. Our simulations suggest that K+ permeation might occur via direct knock-on, similar to the mechanism recently shown for Kv channels.

Crystal structure of the complex of DNA with the C-terminal domain of TYE7 from Saccharomyces cerevisiae

2021 ◽  
Vol 77 (10) ◽  
Author(s):  
Wei Gui ◽  
Lu Xue ◽  
Jian Yue ◽  
Zhiling Kuang ◽  
Yuping Jin ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Sulfur Metabolism ◽  
Salt Bridge ◽  
Mobility Shift ◽  
X Ray ◽  
X Ray Crystallography ◽  
Helix Loop Helix ◽  
Phosphate Backbone ◽  
Backbone Structure ◽  
Electrophoretic Mobility Shift Assays

TYE7, a bHLH (basic helix–loop–helix) transcription factor from Saccharomyces cerevisiae, is involved in the regulation of many genes, including glycolytic genes. Meanwhile, accumulating evidence indicates that TYE7 also functions as a cyclin and is linked to sulfur metabolism. Here, the structure of TYE7 (residues 165–291) complexed with its specific DNA was determined by X-ray crystallography. Structural analysis and comparison revealed that His185 and Glu189 are conserved in base recognition. However, Arg193 is also involved in base recognition in the structures that were compared. In the structure in this study, Arg193 in chain A has two conformations and makes a salt bridge with the phosphate backbone structure. In addition, a series of corresponding electrophoretic mobility shift assays were performed to better understand the DNA-binding mechanism of the bHLH domain of TYE7.

Self-Assembly of Square-Planar Halide Complexes of Trimethylphosphine-Stabilized Diphenyl-Arsenium, -Stibenium, and -Bismuthenium Hexafluorophosphates

2016 ◽  
Vol 69 (5) ◽  
pp. 524 ◽  
Author(s):  
J. Wolfram Wielandt ◽  
Simon Petrie ◽  
Nathan L. Kilah ◽  
Anthony C. Willis ◽  
Rian D. Dewhurst ◽  
...  
Keyword(s):  
Self Assembly ◽  
Electrostatic Interactions ◽  
Main Group ◽  
Main Group Elements ◽  
X Ray ◽  
X Ray Crystallography ◽  
Square Planar ◽  
Halide Complexes ◽  
Positively Charged

Square-planar halide complexes in which four trimethylphosphine-stabilized diphenyl-stibenium or -bismuthenium ions surround a central halide ion in discrete centrosymmetrical structures of C4h symmetry have been isolated and their structures determined by X-ray crystallography. The structures are stabilized by electrostatic interactions between the halide ion and four positively charged trimethylphosphine-stabilized diphenyl-stibenium or -bismuthenium ions, as well as four edge-to-face phenyl–phenyl embraces above and below the plane containing the nine main group elements. The parent halide-free trimethylphosphine-stabilized diphenyl-arsenium, -stibenium, and -bismuthenium hexaflurophosphate complexes have also been prepared and structurally characterized.

scholarly journals A Chemical Probe for Dark Kinase STK17B Derives its Potency and High Selectivity Through a Unique P-loop Conformation

2020 ◽  
Author(s):  
Alfredo Picado ◽  
Apirat Chaikuad ◽  
Carrow Wells ◽  
Safal Shrestha ◽  
William Zuercher ◽  
...  
Keyword(s):  
Carboxylic Acid ◽  
High Selectivity ◽  
Salt Bridge ◽  
Competitive Inhibitor ◽  
Chemical Probe ◽  
High Quality ◽  
X Ray ◽  
X Ray Crystallography ◽  
Loop Conformation

We present the discovery of thieno[3,2-<i>d</i>]pyrimidine <b>SGC-STK17B-1</b> (<b>11s</b>), a high-quality chemical probe for this understudied “dark” kinase. <b>11s</b> is an ATP-competitive inhibitor that showed remarkable selectivity over other kinases including the closely related STK17A. X-ray crystallography of <b>11s</b> and related thieno[3,2-<i>d</i>]pyrimidines bound to STK17B revealed a unique P-loop conformation characterized by a salt bridge between R41 and the carboxylic acid of the inhibitor.

Difference Fourier Analysis of Glucose Embedded and Frozen Hydrated Purple Membrane

1982 ◽  
Vol 40 ◽  
pp. 74-75
Author(s):  
Jules S. Jaffe ◽  
Robert M. Glaeser
Keyword(s):  
Purple Membrane ◽  
Data Set ◽  
High Resolution Data ◽  
X Ray ◽  
X Ray Crystallography ◽  
Fourier Techniques ◽  
Versus Protein ◽  
The Difference ◽  
Difference Fourier ◽  
Ideal Method

Although difference Fourier techniques are standard in X-ray crystallography it has only been very recently that electron crystallographers have been able to take advantage of this method. We have combined a high resolution data set for frozen glucose embedded Purple Membrane (PM) with a data set collected from PM prepared in the frozen hydrated state in order to visualize any differences in structure due to the different methods of preparation. The increased contrast between protein-ice versus protein-glucose may prove to be an advantage of the frozen hydrated technique for visualizing those parts of bacteriorhodopsin that are embedded in glucose. In addition, surface groups of the protein may be disordered in glucose and ordered in the frozen state. The sensitivity of the difference Fourier technique to small changes in structure provides an ideal method for testing this hypothesis.

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