scholarly journals A metal ion orients SARS-CoV-2 mRNA to ensure accurate 2′-O methylation of its first nucleotide

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Thiruselvam Viswanathan ◽  
Anurag Misra ◽  
Siu-Hong Chan ◽  
Shan Qi ◽  
Nan Dai ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Metal Ion ◽  
Immune Surveillance ◽  
Binding Mode ◽  
Product Formation ◽  
Host Cells ◽  
Structural Basis ◽  
Outbreak Strain ◽  
The Status ◽  
Divalent Metal Ion

AbstractThe SARS-CoV-2 nsp16/nsp10 enzyme complex modifies the 2′-OH of the first transcribed nucleotide of the viral mRNA by covalently attaching a methyl group to it. The 2′-O methylation of the first nucleotide converts the status of mRNA cap from Cap-0 to Cap-1, and thus, helps the virus evade immune surveillance in host cells. Here, we report two structures of nsp16/nsp10 representing pre- and post-release states of the RNA product (Cap-1). We observe overall widening of the enzyme upon product formation, and an inward twisting motion in the substrate binding region upon product release. These conformational changes reset the enzyme for the next round of catalysis. The structures also identify a unique binding mode and the importance of a divalent metal ion for 2′-O methylation. We also describe underlying structural basis for the perturbed enzymatic activity of a clinical variant of SARS-CoV-2, and a previous SARS-CoV outbreak strain.

scholarly journals A metal ion orients mRNA to ensure accurate 2’-O ribosyl methylation of the first nucleotide of the SARS-CoV-2 genome

2021 ◽  
Author(s):  
Thiruselvam Viswanathan ◽  
Anurag Misra ◽  
Siu-Hong Chan ◽  
Shan Qi ◽  
Nan Dai ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Metal Ion ◽  
Immune Surveillance ◽  
Binding Mode ◽  
Product Formation ◽  
Structural Basis ◽  
Outbreak Strain ◽  
Product Release ◽  
The Status ◽  
Divalent Metal Ion

AbstractThe SARS-CoV-2 nsp16/nsp10 enzyme complex modifies the 2’-OH of the first transcribed nucleotide of the viral mRNA by covalently attaching a methyl group to it. The 2’-O methylation of the first nucleotide converts the status of mRNA cap from Cap-0 to Cap-1, and thus, helps the virus evade immune surveillance in the host cell. Here, we report two structures of nsp16/nsp10 representing pre- and post-release states of the RNA product (Cap-1). We observe overall widening of the enzyme upon product formation, and an inward twisting motion in the substrate binding region upon product release. These conformational changes reset the enzyme for the next round of catalysis. The structures also identify a unique binding mode and the importance of a divalent metal ion for 2’-O methylation. We also describe underlying structural basis for the perturbed enzymatic activity of a clinical variant of SARS-CoV-2, and a previous SARS-CoV outbreak strain.

scholarly journals Structural Basis of CD4 Downregulation by HIV-1 Nef

2020 ◽  
Author(s):  
Yonghwa Kwon ◽  
Robyn Kaake ◽  
Ignacia Echeverria ◽  
Marissa Suarez ◽  
Charlotte Stoneham ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Viral Entry ◽  
Neutralizing Antibodies ◽  
Immune Surveillance ◽  
Underlying Mechanism ◽  
Host Cells ◽  
Structural Basis ◽  
Viral Glycoprotein ◽  
Class I Mhc ◽  
Hiv 1

The HIV-1 protein Nef suppresses multiple immune surveillance mechanisms to promote viral pathogenesis1. Individuals infected with HIV-1 encoding defective nef genes do not develop AIDS for decades2,3. A key target of Nef is the cellular receptor CD4. Although essential for viral entry into host cells, CD4 is problematic for the virus later in its replication cycle: CD4 disrupts processing of the viral glycoprotein, Env, inhibiting infectivity4; it interferes with the release of new virions5,6; and it causes vulnerability to superinfection, causing premature cell death and limiting viral productivity7. Furthermore, binding of CD4 to Env exposes otherwise-concealed Env epitopes, rendering infected cells more susceptible to antibody-dependent cellular cytotoxicity and virus particles more susceptible to neutralizing antibodies8-10. HIV-1 has evolved strategies to mitigate these problems. Newly synthesized CD4 is targeted in the endoplasmic reticulum by the viral Vpu protein for proteasomal degradation11. Surface-expressed CD4, in contrast, is targeted by Nef for endocytosis and lysosomal degradation12-15. Nef’s effect on CD4 involves hijacking of clathrin adaptor complex 2 (AP2)-dependent endocytosis16,17. Although how Nef associates with a part of the tetrameric AP2 is understood18, a complete understanding of the interaction, especially how CD4 is sequestered by Nef into a complex with AP2, has remained elusive. Here, we present a high-resolution crystal structure that describes the underlying mechanism. An intricate combination of conformational changes occurs in both Nef and AP2 to enable CD4 binding and downregulation. Strikingly, a pocket on Nef previously identified as crucial for recruiting class I MHC is also responsible for recruiting CD4, revealing a potential approach to inhibit two of Nef’s activities and sensitize the virus to immune clearance

Interaction of D-phenylalanine with Co(II)-substituted rabbit muscle pyruvate kinase: kinetic and optical properties

1982 ◽  
Vol 60 (9) ◽  
pp. 861-866
Author(s):  
Chiu-Yin Kwan ◽  
Robert C. Davis
Keyword(s):  
Amino Acids ◽  
Optical Properties ◽  
Pyruvate Kinase ◽  
Conformational Changes ◽  
Metal Ion ◽  
Inhibitory Effect ◽  
Rabbit Muscle ◽  
Divalent Metal Ion ◽  
Muscle Pyruvate Kinase ◽  
Difference Absorption

The kinetic and optical properties of Co(II)-substituted pyruvate kinase in the presence of D-phenylalanine (D-Phe) were investigated. The results are discussed in comparison with the effects of its optical isomer L-phenylalanine (L-Phe) on the same enzyme. The catalytic effect of D-Phe on rabbit muscle pyruvate kinase depended upon the nature of the activating divalent metal ion used. It has stimulatory effect on Mg(II)-activated enzyme, but inhibitory effect on Co(II)-activated enzyme. Unlike the inhibitory effect of L-Phe, the inhibition of Co(II)–enzyme by D-Phe was not sensitive to the changes of pH and temperature, could not be reversed by L-alanine (L-Ala), displayed hyperbolic kinetics, and was noncompetitive with respect to phosphoenolpyruvate saturation. D-Phe induced substantial visible circular dichroism (CD) spectral changes of Co(II)–enzyme similar to those induced by L-Phe. Although ultraviolet CD spectrum was not affected, D-Phe induced an ultraviolet difference absorption spectral change very similar to, but much smaller than, that induced by L-Phe. Our results support that D-Phe and other amino acids interact with the enzyme at two different sites: a common site, causing similar conformational changes which bear little direct kinetic relevance, and a kinetically relevant site, which is sterically dependent upon the side chain of the amino acids.

scholarly journals Structural basis for inhibition of complement C5a by an L-RNA aptamer

2014 ◽  
Vol 70 (a1) ◽  
pp. C202-C202
Author(s):  
Laure Yatime ◽  
Christian Maasch ◽  
Kai Hoehlig ◽  
Sven Klussmann ◽  
Axel Vater ◽  
...  
Keyword(s):  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Healing Process ◽  
Immune Surveillance ◽  
Binding Mode ◽  
Mirror Image ◽  
Structural Basis ◽  
Central Component ◽  
Crystallographic Structure ◽  
C5a Anaphylatoxin

Complement is a central component of innate immunity providing a first line of defense against invading pathogens. It also bridges the innate and adaptive immunity, initiates the inflammatory response, and participates in immune surveillance. The anaphylatoxin C5a, generated during complement activation, is a potent inflammatory mediator which induces chemotaxis, oxidative burst, histamine release and increased vasodilatation, through G-protein coupled receptor signaling. Although inflammation is an integral part of the healing process following tissue damage and infection, excessive levels of C5a correlate with the onset of various inflammatory disorders including sepsis, rheumatoid arthritis, acute lung injury, ischemia-reperfusion injury, allergy, transplantation and asthma. Therapeutical targeting of the C5a:receptor axis is considered a promising strategy to down-regulate complement-mediated inflammation. The L-aptamer NOX-D20, fully composed of non-natural mirror-image nucleotides (a so called Spiegelmer), has been identified as a potent C5a inhibitor. NOX-D20 has already shown encouraging efficacy in an experimental model of sepsis [1]. Here, we present the first crystallographic structure of an active Spiegelmer®, NOX-D20, bound to its physiological target, the mouse C5a anaphylatoxin, determined at 1.8 Å resolution. The structure reveals a complex 3D-architecture for the L-RNA molecule that wraps around C5a, including an intramolecular G-quadruplex stabilized by a Ca2+ ion as validated through anomalous diffraction data. The aptamer:C5a binding mode observed in the structure was validated through mutational studies using SPR. Our structure provides a molecular basis for NOX-D20 inhibitory properties and allows us to rationalize NOX-D20 selectivity towards human and mouse C5a

scholarly journals Structural basis for oligomerization and glycosaminoglycan binding of CCL5 and CCL3

2016 ◽  
Vol 113 (18) ◽  
pp. 5000-5005 ◽  
Author(s):  
Wenguang G. Liang ◽  
Catherine G. Triandafillou ◽  
Teng-Yi Huang ◽  
Medel Manuel L. Zulueta ◽  
Shiladitya Banerjee ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Immune Surveillance ◽  
Polymerization Process ◽  
Structural Basis ◽  
Cc Chemokine ◽  
Inflammatory Conditions ◽  
Chemokine Ligand ◽  
Cc Chemokines ◽  
Relevant Regulation ◽  
Positively Charged

CC chemokine ligand 5 (CCL5) and CCL3 are critical for immune surveillance and inflammation. Consequently, they are linked to the pathogenesis of many inflammatory conditions and are therapeutic targets. Oligomerization and glycosaminoglycan (GAG) binding of CCL5 and CCL3 are vital for the functions of these chemokines. Our structural and biophysical analyses of human CCL5 reveal that CCL5 oligomerization is a polymerization process in which CCL5 forms rod-shaped, double-helical oligomers. This CCL5 structure explains mutational data and offers a unified mechanism for CCL3, CCL4, and CCL5 assembly into high-molecular-weight, polydisperse oligomers. A conserved, positively charged BBXB motif is key for the binding of CC chemokines to GAG. However, this motif is partially buried when CCL3, CCL4, and CCL5 are oligomerized; thus, the mechanism by which GAG binds these chemokine oligomers has been elusive. Our structures of GAG-bound CCL5 and CCL3 oligomers reveal that these chemokine oligomers have distinct GAG-binding mechanisms. The CCL5 oligomer uses another positively charged and fully exposed motif, KKWVR, in GAG binding. However, residues from two partially buried BBXB motifs along with other residues combine to form a GAG-binding groove in the CCL3 oligomer. The N termini of CC chemokines are shown to be involved in receptor binding and oligomerization. We also report an alternative CCL3 oligomer structure that reveals how conformational changes in CCL3 N termini profoundly alter its surface properties and dimer–dimer interactions to affect GAG binding and oligomerization. Such complexity in oligomerization and GAG binding enables intricate, physiologically relevant regulation of CC chemokine functions.

scholarly journals CD94-NKG2A recognition of human leukocyte antigen (HLA)-E bound to an HLA class I leader sequence

2008 ◽  
Vol 205 (3) ◽  
pp. 725-735 ◽  
Author(s):  
Emma J. Petrie ◽  
Craig S. Clements ◽  
Jie Lin ◽  
Lucy C. Sullivan ◽  
Darryl Johnson ◽  
...  
Keyword(s):  
Human Leukocyte Antigen ◽  
Conformational Changes ◽  
Specific Interaction ◽  
Human Leukocyte ◽  
Hla Class I ◽  
Binding Mode ◽  
Leader Sequence ◽  
Structural Basis ◽  
Peptide Ligand ◽  
Leukocyte Antigen

The recognition of human leukocyte antigen (HLA)-E by the heterodimeric CD94-NKG2 natural killer (NK) receptor family is a central innate mechanism by which NK cells monitor the expression of other HLA molecules, yet the structural basis of this highly specific interaction is unclear. Here, we describe the crystal structure of CD94-NKG2A in complex with HLA-E bound to a peptide derived from the leader sequence of HLA-G. The CD94 subunit dominated the interaction with HLA-E, whereas the NKG2A subunit was more peripheral to the interface. Moreover, the invariant CD94 subunit dominated the peptide-mediated contacts, albeit with poor surface and chemical complementarity. This unusual binding mode was consistent with mutagenesis data at the CD94-NKG2A–HLA-E interface. There were few conformational changes in either CD94-NKG2A or HLA-E upon ligation, and such a “lock and key” interaction is typical of innate receptor–ligand interactions. Nevertheless, the structure also provided insight into how this interaction can be modulated by subtle changes in the peptide ligand or by the pairing of CD94 with other members of the NKG2 family. Differences in the docking strategies used by the NKG2D and CD94-NKG2A receptors provided a basis for understanding the promiscuous nature of ligand recognition by NKG2D compared with the fidelity of the CD94-NKG2 receptors.

scholarly journals Structural basis of catalysis and substrate recognition by the NAD(H)-dependent α-d-glucuronidase from the glycoside hydrolase family 4

2021 ◽  
Vol 478 (4) ◽  
pp. 943-959
Author(s):  
Samar Ballabha Mohapatra ◽  
Narayanan Manoj
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Glycoside Hydrolase ◽  
Metal Ion ◽  
Substrate Recognition ◽  
Structural Basis ◽  
Glycoside Hydrolase Family ◽  
Diverse Range ◽  
Molecular Features ◽  
Hydrolase Family

Members of the glycoside hydrolase family 4 (GH4) employ an unusual glycosidic bond cleavage mechanism utilizing NAD(H) and a divalent metal ion, under reducing conditions. These enzymes act upon a diverse range of glycosides, and unlike most other GH families, homologs here are known to accommodate both α- and β-anomeric specificities within the same active site. Here, we report the catalytic properties and the crystal structures of TmAgu4B, an α-d-glucuronidase from the hyperthermophile Thermotoga maritima. The structures in three different states include the apo form, the NADH bound holo form, and the ternary complex with NADH and the reaction product d-glucuronic acid, at 2.15, 1.97 and 1.85 Å resolutions, respectively. These structures reveal the step-wise route of conformational changes required in the active site to achieve the catalytically competent state, and illustrate the direct role of residues that determine the reaction mechanism. Furthermore, a structural transition of a helical region in the active site to a turn geometry resulting in the rearrangement of a unique arginine residue governs the exclusive glucopyranosiduronic acid recognition in TmAgu4B. Mutational studies show that modifications of the glycone binding site geometry lead to catalytic failure and indicate overlapping roles of specific residues in catalysis and substrate recognition. The data highlight hitherto unreported molecular features and associated active site dynamics that determine the structure–function relationships within the unique GH4 family.

scholarly journals Cross-species recognition of SARS-CoV-2 to bat ACE2

2020 ◽  
Vol 118 (1) ◽  
pp. e2020216118
Author(s):  
Kefang Liu ◽  
Shuguang Tan ◽  
Sheng Niu ◽  
Jia Wang ◽  
Lili Wu ◽  
...  
Keyword(s):  
Binding Capacity ◽  
Species Recognition ◽  
Interaction Network ◽  
Binding Mode ◽  
Host Cells ◽  
Structural Basis ◽  
Receptor Binding Domain ◽  
Angiotensin Converting Enzyme 2 ◽  
Protein Receptor ◽  
Human Receptor

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emerged as a major threat to global health. Although varied SARS-CoV-2–related coronaviruses have been isolated from bats and SARS-CoV-2 may infect bat, the structural basis for SARS-CoV-2 to utilize the human receptor counterpart bat angiotensin-converting enzyme 2 (bACE2) for virus infection remains less understood. Here, we report that the SARS-CoV-2 spike protein receptor binding domain (RBD) could bind to bACE2 fromRhinolophus macrotis(bACE2-Rm) with substantially lower affinity compared with that to the human ACE2 (hACE2), and its infectivity to host cells expressing bACE2-Rm was confirmed with pseudotyped SARS-CoV-2 virus and SARS-CoV-2 wild virus. The structure of the SARS-CoV-2 RBD with the bACE2-Rm complex was determined, revealing a binding mode similar to that of hACE2. The analysis of binding details between SARS-CoV-2 RBD and bACE2-Rm revealed that the interacting network involving Y41 and E42 of bACE2-Rm showed substantial differences with that to hACE2. Bats have extensive species diversity and the residues for RBD binding in bACE2 receptor varied substantially among different bat species. Notably, the Y41H mutant, which exists in many bats, attenuates the binding capacity of bACE2-Rm, indicating the central roles of Y41 in the interaction network. These findings would benefit our understanding of the potential infection of SARS-CoV-2 in varied species of bats.

scholarly journals Structural basis of SARS-CoV-2 spike protein induced by ACE2

2020 ◽  
Author(s):  
Tomer Meirson ◽  
David Bomze ◽  
Gal Markel
Keyword(s):  
Conformational Changes ◽  
Viral Entry ◽  
Active Form ◽  
Intramolecular Interactions ◽  
Host Cells ◽  
Supplementary Information ◽  
Structural Basis ◽  
S Protein ◽  
Binding Interface ◽  
Recent Emergence

Abstract Motivation The recent emergence of the novel SARS-coronavirus 2 (SARS-CoV-2) and its international spread pose a global health emergency. The spike (S) glycoprotein binds ACE2 and promotes SARS-CoV-2 entry into host cells. The trimeric S protein binds the receptor using the receptor-binding domain (RBD) causing conformational changes in S protein that allow priming by host cell proteases. Unraveling the dynamic structural features used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal novel therapeutic targets. Using structures determined by X-ray crystallography and cryo-EM, we performed structural analysis and atomic comparisons of the different conformational states adopted by the SARS-CoV-2-RBD. Results Here, we determined the key structural components induced by the receptor and characterized their intramolecular interactions. We show that κ-helix (polyproline-II) is a predominant structure in the binding interface and in facilitating the conversion to the active form of the S protein. We demonstrate a series of conversions between switch-like κ-helix and β-strand, and conformational variations in a set of short α-helices which affect the hinge region. These conformational changes lead to an alternating pattern in conserved disulfide bond configurations positioned at the hinge, indicating a possible disulfide exchange, an important allosteric switch implicated in viral entry of various viruses, including HIV and murine coronavirus. The structural information presented herein enables to inspect and understand the important dynamic features of SARS-CoV-2-RBD and propose a novel potential therapeutic strategy to block viral entry. Overall, this study provides guidance for the design and optimization of structure-based intervention strategies that target SARS-CoV-2. Availability and implementation We have implemented the proposed methods in an R package freely available at https://github.com/Grantlab/bio3d. Supplementary information Supplementary data are available at Bioinformatics online.

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