Structural Analysis of the Menangle Virus P Protein Reveals a Soft Boundary between Ordered and Disordered Regions

The paramyxoviral phosphoprotein (P protein) is the non-catalytic subunit of the viral RNA polymerase, and coordinates many of the molecular interactions required for RNA synthesis. All paramyxoviral P proteins oligomerize via a centrally located coiled-coil that is connected to a downstream binding domain by a dynamic linker. The C-terminal region of the P protein coordinates interactions between the catalytic subunit of the polymerase, and the viral nucleocapsid housing the genomic RNA. The inherent flexibility of the linker is believed to facilitate polymerase translocation. Here we report biophysical and structural characterization of the C-terminal region of the P protein from Menangle virus (MenV), a bat-borne paramyxovirus with zoonotic potential. The MenV P protein is tetrameric but can dissociate into dimers at sub-micromolar protein concentrations. The linker is globally disordered and can be modeled effectively as a worm-like chain. However, NMR analysis suggests very weak local preferences for alpha-helical and extended beta conformation exist within the linker. At the interface between the disordered linker and the structured C-terminal binding domain, a gradual disorder-to-order transition occurs, with X-ray crystallographic analysis revealing a dynamic interfacial structure that wraps the surface of the binding domain.

Download Full-text

Hice1, a Novel Microtubule-Associated Protein Required for Maintenance of Spindle Integrity and Chromosomal Stability in Human Cells

Molecular and Cellular Biology ◽

10.1128/mcb.01923-07 ◽

2008 ◽

Vol 28 (11) ◽

pp. 3652-3662 ◽

Cited By ~ 37

Author(s):

Guikai Wu ◽

Yi-Tzu Lin ◽

Randy Wei ◽

Yumay Chen ◽

Zhiyin Shan ◽

...

Keyword(s):

Cold Treatment ◽

Coiled Coil ◽

Spindle Pole ◽

Terminal Region ◽

Mitotic Spindles ◽

Mitotic Progression ◽

Physiological Concentration ◽

Chromosomal Stability ◽

Human Sarcoma

ABSTRACT Spindle integrity is critical for efficient mitotic progression and accurate chromosome segregation. Deregulation of spindles often leads to structural and functional aberrations, ultimately promoting segregation errors and aneuploidy, a hallmark of most human cancers. Here we report the characterization of a previously identified human sarcoma antigen (gene located at 19p13.11), Hice1, an evolutionarily nonconserved 46-kDa coiled-coil protein. Hice1 shows distinct cytoplasmic localization and associates with interphase centrosomes and mitotic spindles, preferentially at the spindle pole vicinity. Depletion of Hice1 by RNA interference resulted in abnormal and unstable spindle configurations, mitotic delay at prometaphase and metaphase, and elevated aneuploidy. Conversely, loss of Hice1 had minimal effects on interphase centrosome duplication. We also found that both full-length Hice1 and Hice1-N1, which is composed of 149 amino acids of the N-terminal region, but not the mutant lacking the N-terminal region, exhibited activities of microtubule bundling and stabilization at a near-physiological concentration. Consistently, overexpression of Hice1 rendered microtubule bundles in cells resistant to nocodazole- or cold-treatment-induced depolymerization. These results demonstrate that Hice1 is a novel microtubule-associated protein important for maintaining spindle integrity and chromosomal stability, in part by virtue of its ability to bind, bundle, and stabilize microtubules.

Download Full-text

Characterization of the P Protein-Binding Domain on the 10-Kilodalton Protein of Borna Disease Virus

Journal of Virology ◽

10.1128/jvi.74.7.3413-3417.2000 ◽

2000 ◽

Vol 74 (7) ◽

pp. 3413-3417 ◽

Cited By ~ 18

Author(s):

Tahir H. Malik ◽

Masahiko Kishi ◽

Patrick K. Lai

Keyword(s):

Disease Virus ◽

Nuclear Localization ◽

Cell Nucleus ◽

Binding Domain ◽

Borna Disease Virus ◽

Nuclear Localization Signals ◽

P Protein ◽

Infected Cells ◽

Borna Disease

ABSTRACT The Borna disease virus (BDV) is the prototype member of the Bornaviridae, and it replicates in the cell nucleus. The BDV p24P and p40N proteins carry nuclear localization signals (NLS) and are found in the nuclei of infected cells. The BDV p10 protein does not have an NLS, but it binds with P and/or N and is translocated to the nucleus. Hence, p10 may play a role in the replication of BDV in the cell nucleus. Here, we show that the P-binding domain is located in the N terminus of p10 and that S3 and L16 are important for the interaction.

Download Full-text

A Role for the Sendai Virus P Protein Trimer in RNA Synthesis

Journal of Virology ◽

10.1128/jvi.72.5.4274-4280.1998 ◽

1998 ◽

Vol 72 (5) ◽

pp. 4274-4280 ◽

Cited By ~ 47

Author(s):

Joseph Curran

Keyword(s):

Amino Acids ◽

Mammalian Cells ◽

Essential Role ◽

Rna Synthesis ◽

Sendai Virus ◽

Coiled Coil ◽

The Third ◽

P Protein ◽

P Proteins

ABSTRACT The SeV P protein is found as a homotrimer (P3) when it is expressed in mammalian cells, and trimerization is mediated by a predicted coiled-coil motif which maps within amino acids (aa) 344 to 411 (the BoxA region). The bacterially expressed protein also appears to be trimeric, apparently precluding a role for phosphorylation in the association of the P monomers. I have examined the role of P trimerization both in the protein’s interaction with the nucleocapsid (N:RNA) template and in the protein’s function on the template during RNA synthesis. As with the results of earlier experiments (32), I found that both the BoxA and BoxC (aa 479 to 568) regions were required for stable binding of P to the N:RNA. Binding was also observed with P proteins containing less than three BoxC regions, suggesting that trimerization may be required to permit contacts between multiple BoxC regions and the N:RNA. However, these heterologous trimers failed to function in viral RNA synthesis, indicating that the third C-terminal leg of the trimer plays an essential role in P function on the template. We speculate that this function may involve the movement of P (and possibly the polymerase complex) on the template and the maintenance of processivity.

Download Full-text

Nipah Virus V and W Proteins Have a Common STAT1-Binding Domain yet Inhibit STAT1 Activation from the Cytoplasmic and Nuclear Compartments, Respectively

Journal of Virology ◽

10.1128/jvi.78.11.5633-5641.2004 ◽

2004 ◽

Vol 78 (11) ◽

pp. 5633-5641 ◽

Cited By ~ 153

Author(s):

Megan L. Shaw ◽

Adolfo García-Sastre ◽

Peter Palese ◽

Christopher F. Basler

Keyword(s):

Nipah Virus ◽

Binding Domain ◽

Viral Gene ◽

Virus Protein ◽

Antagonist Activity ◽

Terminal Domain ◽

P Protein ◽

The Common ◽

Β Receptor ◽

P Proteins

ABSTRACT In previous reports it was demonstrated that the Nipah virus V and W proteins have interferon (IFN) antagonist activity due to their ability to block signaling from the IFN-α/β receptor (J. J. Rodriguez, J. P. Parisien, and C. M. Horvath, J. Virol. 76:11476-11483, 2002; M. S. Park et al., J. Virol. 77:1501-1511, 2003). The V, W, and P proteins are all encoded by the same viral gene and share an identical 407-amino-acid N-terminal region but have distinct C-terminal sequences. We now show that the P protein also has anti-IFN function, confirming that the common N-terminal domain is responsible for the antagonist activity. Truncation of this N-terminal domain revealed that amino acids 50 to 150 retain the ability to block IFN and to bind STAT1, a key component of the IFN signaling pathway. Subcellular localization studies demonstrate that the V and P proteins are predominantly cytoplasmic whereas the W protein is localized to the nucleus. In all cases, STAT1 colocalizes with the corresponding Nipah virus protein. These interactions are sufficient to inhibit STAT1 activation, as demonstrated by the lack of STAT1 phosphorylation on tyrosine 701 in IFN-stimulated cells expressing P, V, or W. Therefore, despite their common STAT1-binding domain, the Nipah virus V and P proteins act by retaining STAT1 in the cytoplasm while the W protein sequesters STAT1 in the nucleus, creating both a cytoplasmic and a nuclear block for STAT1. We also show that the IFN antagonist activity of the P protein is not as strong as that of V or W, perhaps explaining why Nipah virus has evolved to express these two edited products.

Download Full-text

Identification and characterization of photomedins: novel olfactomedin-domain-containing proteins with chondroitin sulphate-E-binding activity

Biochemical Journal ◽

10.1042/bj20050120 ◽

2005 ◽

Vol 389 (3) ◽

pp. 675-684 ◽

Cited By ~ 32

Author(s):

Yutaka Furutani ◽

Ri-ichiroh Manabe ◽

Ko Tsutsui ◽

Tomiko Yamada ◽

Nagisa Sugimoto ◽

...

Keyword(s):

Chondroitin Sulphate ◽

Recombinant Expression ◽

Coiled Coil ◽

Photoreceptor Cells ◽

Binding Activity ◽

Extracellular Proteins ◽

Terminal Region ◽

Computational Screening ◽

Ecm Extracellular Matrix

We screened more than 60000 RIKEN mouse cDNAs for novel ECM (extracellular matrix) proteins by extensive computational screening followed by recombinant expression and immunohistochemical characterization. We identified two novel olfactomedin-family proteins characterized by the presence of tandem CXCXCX9C motifs in the N-terminal region, a coiled-coil domain and an olfactomedin domain in the C-terminal region. These proteins, named photomedin-1 and photomedin-2, were secreted as disulphide-bonded dimers (photomedin-1) or oligomers/multimers (photomedin-2) with O-linked carbohydrate chains, although photomedin-1 was proteolytically processed in the middle of the molecule after secretion. In the retina, photomedin-1 was selectively expressed in the outer segment of photoreceptor cells and photomedin-2 was expressed in all retinal neurons. Among a panel of ECM components, including glycosaminoglycans, photomedins preferentially bound to chondroitin sulphate-E and heparin. These results, together, indicate that photomedins are novel olfactomedin-domain-containing extracellular proteins capable of binding to proteoglycans containing these glycosaminoglycan chains.

Download Full-text

Characterization of the Human Sigma-1 Receptor Chaperone Domain Structure and Binding Immunoglobulin Protein (BiP) Interactions

Journal of Biological Chemistry ◽

10.1074/jbc.m113.450379 ◽

2013 ◽

Vol 288 (29) ◽

pp. 21448-21457 ◽

Cited By ~ 51

Author(s):

Jose Luis Ortega-Roldan ◽

Felipe Ossa ◽

Jason R. Schnell

Keyword(s):

Nucleotide Binding ◽

Binding Domain ◽

Terminal Region ◽

Nucleotide Binding Domain ◽

Sigma 1 Receptor ◽

Er Stress Response ◽

Sigma 1 ◽

The Central Nervous System ◽

Substrate Binding Domain

The sigma-1 receptor (S1R) is a ligand-regulated membrane protein chaperone involved in the ER stress response. S1R activity is implicated in diseases of the central nervous system including amnesia, schizophrenia, depression, Alzheimer disease, and addiction. S1R has been shown previously to regulate the Hsp70 binding immunoglobulin protein (BiP) and the inositol triphosphate receptor calcium channel through a C-terminal domain. We have developed methods for bacterial expression and reconstitution of the chaperone domain of human S1R into detergent micelles that enable its study by solution NMR spectroscopy. The chaperone domain is found to contain a helix at the N terminus followed by a largely dynamic region and a structured, helical C-terminal region that encompasses a membrane associated domain containing four helices. The helical region at residues ∼198–206 is strongly amphipathic and proposed to anchor the chaperone domain to micelles and membranes. Three of the helices in the C-terminal region closely correspond to previously identified cholesterol and drug recognition sites. In addition, it is shown that the chaperone domain interacts with full-length BiP or the isolated nucleotide binding domain of BiP, but not the substrate binding domain, suggesting that the nucleotide binding domain is sufficient for S1R interactions.

Download Full-text

Characterization of a novel DNA binding domain within the amino-terminal region of the RAG-1 protein

IUBMB Life ◽

10.1080/15216549800202922 ◽

1998 ◽

Vol 45 (3) ◽

pp. 535-544

Author(s):

Jackelyn Alva ◽

Albert Lin ◽

Christopher Lyon ◽

Renato Aguilera ◽

Zoran Galic

Keyword(s):

Dna Binding ◽

Binding Domain ◽

Terminal Region ◽

Dna Binding Domain ◽

Amino Terminal ◽

Rag 1

Download Full-text

HRTEM simulation of interfacial structure in amorphous multilayers

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100154305 ◽

1989 ◽

Vol 47 ◽

pp. 466-467

Author(s):

Margaret L. Sattler ◽

Michael A. O'Keefe

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Cross Section ◽

High Resolution Electron Microscopy ◽

Interfacial Structure ◽

Multilayer Sample ◽

Resolution Electron ◽

Multilayered Materials ◽

Simulated Images

Multilayered materials have been fabricated with such high perfection that individual layers having two atoms deep are possible. Characterization of the interfaces between these multilayers is achieved by high resolution electron microscopy and Figure 1a shows the cross-section of one type of multilayer. The production of such an image with atomically smooth interfaces depends upon certain factors which are not always reliable. For example, diffusion at the interface may produce complex interlayers which are important to the properties of the multilayers but which are difficult to observe. Similarly, anomalous conditions of imaging or of fabrication may occur which produce images having similar traits as the diffusion case above, e.g., imaging on a tilted/bent multilayer sample (Figure 1b) or deposition upon an unaligned substrate (Figure 1c). It is the purpose of this study to simulate the image of the perfect multilayer interface and to compare with simulated images having these anomalies.

Download Full-text