scholarly journals Crystal Structure of the Japanese Encephalitis Virus Capsid Protein

Viruses ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 623 ◽  
Author(s):  
Thanalai Poonsiri ◽  
Gareth S. A. Wright ◽  
Tom Solomon ◽  
Svetlana V. Antonyuk
Keyword(s):  
Crystal Structure ◽  
Nucleic Acid ◽  
Capsid Protein ◽  
Japanese Encephalitis ◽  
Protein A ◽  
Coiled Coil ◽  
Specific Treatment ◽  
Capsid Proteins ◽  
Nucleic Acid Binding ◽  
Α Helix

Japanese encephalitis (JE) is inflammation and swelling of the brain caused by the JE virus (JEV), a mosquito-borne member of the Flavivirus family. There are around 68,000 JE cases worldwide each year, many of which result in permanent brain damage and death. There is no specific treatment for JE. Here we present the crystal structure of the JEV capsid protein, a potential drug target, at 1.98 Å, and compare it to other flavivirus capsid proteins. The JEV capsid has a helical secondary structure (α helixes 1–4) and a similar protein fold to the dengue virus (DENV), the West Nile virus (WNV), and the Zika virus (ZIKV) capsid proteins. It forms a homodimer by antiparallel pairing with another subunit (‘) through α-helix 1-1’, 2-2’, and 4-4’ interactions. This dimeric form is believed to be the building block of the nucleocapsid. The flexibility of the N-terminal α helix-1 allows the formation of closed and open conformations with possible functional importance. The basic C-terminal pairing of α4-4’ forms a coiled-coil-like structure, indicating possible nucleic acid binding functionality. However, a comparison with other nucleic acid interacting domains indicates that homodimerization would preclude binding. This is the first JEV capsid protein to be described and is an addition to the structural biology of the Flavivirus.

scholarly journals Crystal Structure of the YchF Protein Reveals Binding Sites for GTP and Nucleic Acid

2003 ◽  
Vol 185 (14) ◽  
pp. 4031-4037 ◽  
Author(s):  
Alexey Teplyakov ◽  
Galina Obmolova ◽  
Seung Y. Chu ◽  
John Toedt ◽  
Edward Eisenstein ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Nucleic Acid ◽  
Binding Site ◽  
Coiled Coil ◽  
Genomic Analysis ◽  
Protein Fluorescence ◽  
Terminal Domain ◽  
Fluorescence Measurements ◽  
Α Helix

ABSTRACT The bacterial protein encoded by the gene ychF is 1 of 11 universally conserved GTPases and the only one whose function is unknown. The crystal structure determination of YchF was sought to help with the functional assignment of the protein. The YchF protein from Haemophilus influenzae was cloned and expressed, and the crystal structure was determined at 2.4 Å resolution. The polypeptide chain is folded into three domains. The N-terminal domain has a mononucleotide binding fold typical for the P-loop NTPases. An 80-residue domain next to it has a pronounced α-helical coiled coil. The C-terminal domain features a six-stranded half-barrel that curves around an α-helix. The crablike three-domain structure of YchF suggests the binding site for a double-stranded nucleic acid in the cleft between the domains. The structure of the putative GTP-binding site is consistent with the postulated guanine specificity of the protein. Fluorescence measurements have demonstrated the ability of YchF to bind a double-stranded nucleic acid and GTP. Taken together with other experimental data and genomic analysis, these results suggest that YchF may be part of a nucleoprotein complex and may function as a GTP-dependent translation factor.

scholarly journals A new crystal structure and small-angle X-ray scattering analysis of the homodimer of human SFPQ

2019 ◽  
Vol 75 (6) ◽  
pp. 439-449 ◽  
Author(s):  
Thushara Welwelwela Hewage ◽  
Sofia Caria ◽  
Mihwa Lee
Keyword(s):  
Crystal Structure ◽  
Small Angle ◽  
Solution Structure ◽  
Rna Binding ◽  
Binding Protein ◽  
Coiled Coil ◽  
Scattering Data ◽  
Nucleic Acid Binding ◽  
Dimerization Domain ◽  
Orthorhombic Space Group

Splicing factor proline/glutamine-rich (SFPQ) is an essential RNA-binding protein that is implicated in many aspects of nuclear function. The structures of SFPQ and two paralogs, non-POU domain-containing octamer-binding protein and paraspeckle component 1, from theDrosophilabehavior human splicing protein family have previously been characterized. The unusual arrangement of the four domains, two RNA-recognition motifs (RRMs), a conserved region termed the NonA/paraspeckle (NOPS) domain and a C-terminal coiled coil, in the intertwined dimer provides a potentially unique RNA-binding surface. However, the molecular details of how the four RRMs in the dimeric SFPQ interact with RNA remain to be characterized. Here, a new crystal structure of the dimerization domain of human SFPQ in theC-centered orthorhombic space groupC2221with one monomer in the asymmetric unit is presented. Comparison of the new crystal structure with the previously reported structure of SFPQ and analysis of the solution small-angle X-scattering data revealed subtle domain movements in the dimerization domain of SFPQ, supporting the concept of multiple conformations of SFPQ in equilibrium in solution. The domain movement of RRM1, in particular, may reflect the complexity of the RNA substrates of SFPQ. Taken together, the crystal and solution structure analyses provide a molecular basis for further investigation into the plasticity of nucleic acid binding by SFPQ in the absence of the structure in complex with its cognate RNA-binding partners.

scholarly journals Structural basis of nucleic-acid recognition and double-strand unwinding by the essential neuronal protein Pur-alpha

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Janine Weber ◽  
Han Bao ◽  
Christoph Hartlmüller ◽  
Zhiqin Wang ◽  
Almut Windhager ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Nucleic Acid ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Point Mutations ◽  
Structural Basis ◽  
Binding Modes ◽  
Nucleic Acid Binding

The neuronal DNA-/RNA-binding protein Pur-alpha is a transcription regulator and core factor for mRNA localization. Pur-alpha-deficient mice die after birth with pleiotropic neuronal defects. Here, we report the crystal structure of the DNA-/RNA-binding domain of Pur-alpha in complex with ssDNA. It reveals base-specific recognition and offers a molecular explanation for the effect of point mutations in the 5q31.3 microdeletion syndrome. Consistent with the crystal structure, biochemical and NMR data indicate that Pur-alpha binds DNA and RNA in the same way, suggesting binding modes for tri- and hexanucleotide-repeat RNAs in two neurodegenerative RNAopathies. Additionally, structure-based in vitro experiments resolved the molecular mechanism of Pur-alpha's unwindase activity. Complementing in vivo analyses in Drosophila demonstrated the importance of a highly conserved phenylalanine for Pur-alpha's unwinding and neuroprotective function. By uncovering the molecular mechanisms of nucleic-acid binding, this study contributes to understanding the cellular role of Pur-alpha and its implications in neurodegenerative diseases.

scholarly journals A noncanonical PWI domain in the N-terminal helicase-associated region of the spliceosomal Brr2 protein

2015 ◽  
Vol 71 (4) ◽  
pp. 762-771 ◽  
Author(s):  
Eva Absmeier ◽  
Leonie Rosenberger ◽  
Luise Apelt ◽  
Christian Becke ◽  
Karine F. Santos ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Nucleic Acid ◽  
Messenger Rna ◽  
Spectroscopic Studies ◽  
Terminal Region ◽  
Surface Patch ◽  
Molecular Architecture ◽  
Nucleic Acid Binding ◽  
Band Shift ◽  
Binding Domains

The spliceosomal RNA helicase Brr2 is required for the assembly of a catalytically active spliceosome on a messenger RNA precursor. Brr2 exhibits an unusual organization with tandem helicase units, each comprising dual RecA-like domains and a Sec63 homology unit, preceded by a more than 400-residue N-terminal helicase-associated region. Whereas recent crystal structures have provided insights into the molecular architecture and regulation of the Brr2 helicase region, little is known about the structural organization and function of its N-terminal part. Here, a near-atomic resolution crystal structure of a PWI-like domain that resides in the N-terminal region ofChaetomium thermophilumBrr2 is presented. CD spectroscopic studies suggested that this domain is conserved in the yeast and human Brr2 orthologues. Although canonical PWI domains act as low-specificity nucleic acid-binding domains, no significant affinity of the unusual PWI domain of Brr2 for a broad spectrum of DNAs and RNAs was detected in band-shift assays. Consistently, theC. thermophilumBrr2 PWI-like domain, in the conformation seen in the present crystal structure, lacks an expanded positively charged surface patch as observed in at least one canonical, nucleic acid-binding PWI domain. Instead, in a comprehensive yeast two-hybrid screen against human spliceosomal proteins, fragments of the N-terminal region of human Brr2 were found to interact with several other spliceosomal proteins. At least one of these interactions, with the Prp19 complex protein SPF27, depended on the presence of the PWI-like domain. The results suggest that the N-terminal region of Brr2 serves as a versatile protein–protein interaction platform in the spliceosome and that some interactions require or are reinforced by the PWI-like domain.

scholarly journals Crystal structure of an Escherichia coli Hfq Core (residues 2–69)–DNA complex reveals multifunctional nucleic acid binding sites

2020 ◽  
Vol 48 (7) ◽  
pp. 3987-3997 ◽  
Author(s):  
Jillian Orans ◽  
Alexander R Kovach ◽  
Kirsten E Hoff ◽  
Nicola M Horstmann ◽  
Richard G Brennan
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Nucleic Acid ◽  
Dna Interaction ◽  
Dna Duplexes ◽  
Nucleic Acid Binding ◽  
Binding Studies ◽  
Bacterial Gene ◽  
Bacterial Gene Expression ◽  
Microscopy Data

Abstract Hfq regulates bacterial gene expression post-transcriptionally by binding small RNAs and their target mRNAs, facilitating sRNA-mRNA annealing, typically resulting in translation inhibition and RNA turnover. Hfq is also found in the nucleoid and binds double-stranded (ds) DNA with a slight preference for A-tracts. Here, we present the crystal structure of the Escherichia coli Hfq Core bound to a 30 bp DNA, containing three 6 bp A-tracts. Although previously postulated to bind to the ‘distal’ face, three statistically disordered double stranded DNA molecules bind across the proximal face of the Hfq hexamer as parallel, straight rods with B-DNA like conformational properties. One DNA duplex spans the diameter of the hexamer and passes over the uridine-binding proximal-face pore, whereas the remaining DNA duplexes interact with the rims and serve as bridges between adjacent hexamers. Binding is sequence-independent with residues N13, R16, R17 and Q41 interacting exclusively with the DNA backbone. Atomic force microscopy data support the sequence-independent nature of the Hfq-DNA interaction and a role for Hfq in DNA compaction and nucleoid architecture. Our structure and nucleic acid-binding studies also provide insight into the mechanism of sequence-independent binding of Hfq to dsRNA stems, a function that is critical for proper riboregulation.

scholarly journals Alphavirus Nucleocapsid Protein Contains a Putative Coiled Coil α-Helix Important for Core Assembly

2001 ◽  
Vol 75 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Rushika Perera ◽  
Katherine E. Owen ◽  
Timothy L. Tellinghuisen ◽  
Alexander E. Gorbalenya ◽  
Richard J. Kuhn
Keyword(s):  
Amino Acid ◽  
Capsid Protein ◽  
Coiled Coil ◽  
Terminal Region ◽  
Wild Type Virus ◽  
Wild Type ◽  
Particle Assembly ◽  
In Vitro Assembly ◽  
Α Helix

ABSTRACT The alphavirus nucleocapsid core is formed through the energetic contributions of multiple noncovalent interactions mediated by the capsid protein. This protein consists of a poorly conserved N-terminal region of unknown function and a C-terminal conserved autoprotease domain with a major role in virion formation. In this study, an 18-amino-acid conserved region, predicted to fold into an α-helix (helix I) and embedded in a low-complexity sequence enriched with basic and Pro residues, has been identified in the N-terminal region of the alphavirus capsid proteins. In Sindbis virus, helix I spans residues 38 to 55 and contains three conserved leucine residues, L38, L45, and L52, conforming to the heptad amino acid organization evident in leucine zipper proteins. Helix I consists of an N-terminally truncated heptad and two complete heptad repeats with β-branched residues and conserved leucine residues occupying the a andd positions of the helix, respectively. Complete or partial deletion of helix I, or single-site substitutions at the conserved leucine residues (L45 and L52), caused a significant decrease in virus replication. The mutant viruses were more sensitive to elevated temperature than wild-type virus. These mutant viruses also failed to accumulate cores in the cytoplasm of infected cells, although they did not have defects in protein translation or processing. Analysis of these mutants using an in vitro assembly system indicated that the majority were defective in core particle assembly. Furthermore, mutant proteins showed a trans-dominant negative phenotype in in vitro assembly reactions involving mutant and wild-type proteins. We propose that helix I plays a central role in the assembly of nucleocapsid cores through coiled coil interactions. These interactions may stabilize subviral intermediates formed through the interactions of the C-terminal domain of the capsid protein and the genomic RNA and contribute to the stability of the virion.

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