scholarly journals Probing the Structure of Rotavirus NSP4: a Short Sequence at the Extreme C Terminus Mediates Binding to the Inner Capsid Particle

2000 ◽  
Vol 74 (11) ◽  
pp. 5388-5394 ◽  
Author(s):  
Judith A. O'Brien ◽  
John A. Taylor ◽  
A. R. Bellamy
Keyword(s):  
Amino Acids ◽  
Binding Sites ◽  
Limited Proteolysis ◽  
Coiled Coil ◽  
Surface Binding ◽  
Virus Maturation ◽  
C Terminus ◽  
Tetramerization Domain ◽  
Necessary And Sufficient ◽  
Sequence Requirements

ABSTRACT The rotavirus nonstructural glycoprotein NSP4 functions as the receptor for the inner capsid particle (ICP) which buds into the lumen of the endoplasmic reticulum during virus maturation. The structure of the cytoplasmic domain of NSP4 from rotavirus strain SA11 has been investigated by using limited proteolysis and mass spectrometry. Digestion with trypsin and V8 protease reveals a C-terminal protease-sensitive region that is 28 amino acids long. The minimal sequence requirements for receptor function have been defined by constructing fusions with glutathione S-transferase and assessing their ability to bind ICPs. These experiments demonstrate that 17 to 20 amino acids from the extreme C terminus are necessary and sufficient for ICP binding and that this binding is cooperative. These observations are consistent with a model for the structure of the NSP4 cytoplasmic region in which four flexible regions of 28 amino acids are presented by a protease-resistant coiled-coil tetramerization domain, with only the last ∼20 amino acids of each peptide interacting with the surface binding sites on the ICP.

scholarly journals Sir3p Domains Involved in the Initiation of Telomeric Silencing in Saccharomyces cerevisiae

Genetics ◽  
1998 ◽  
Vol 150 (3) ◽  
pp. 977-986 ◽  
Author(s):  
Yangsuk Park ◽  
John Hanish ◽  
Arthur J Lustig
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
Fusion Proteins ◽  
Initiation Site ◽  
Negative Control ◽  
Model System ◽  
Mutant Protein ◽  
Regulatory Domain ◽  
C Terminus ◽  
Necessary And Sufficient

Abstract Previous studies from our laboratory have demonstrated that tethering of Sir3p at the subtelomeric/telomeric junction restores silencing in strains containing Rap1-17p, a mutant protein unable to recruit Sir3p. This tethered silencing assay serves as a model system for the early events that follow recruitment of silencing factors, a process we term initiation. A series of LexA fusion proteins in-frame with various Sir3p fragments were constructed and tested for their ability to support tethered silencing. Interestingly, a region comprising only the C-terminal 144 amino acids, termed the C-terminal domain (CTD), is both necessary and sufficient for restoration of silencing. Curiously, the LexA-Sir3N205 mutant protein overcomes the requirement for the CTD, possibly by unmasking a cryptic initiation site. A second domain spanning amino acids 481-835, termed the nonessential for initiation domain (NID), is dispensable for the Sir3p function in initiation, but is required for the recruitment of the Sir4p C terminus. In addition, in the absence of the N-terminal 481 amino acids, the NID negatively influences CTD activity. This suggests the presence of a third region, consisting of the N-terminal half (1-481) of Sir3p, termed the positive regulatory domain (PRD), which is required to initiate silencing in the presence of the NID. These data suggest that the CTD “active” site is under both positive and negative control mediated by multiple Sir3p domains.

Dissecting interactions between EB1, microtubules and APC in cortical clusters at the plasma membrane

2002 ◽  
Vol 115 (8) ◽  
pp. 1583-1590 ◽  
Author(s):  
Angela I. M. Barth ◽  
Kathleen A. Siemers ◽  
W. James Nelson
Keyword(s):  
Amino Acids ◽  
Cluster Formation ◽  
Adenomatous Polyposis ◽  
Polyposis Coli ◽  
Binding Domains ◽  
C Terminus ◽  
Endogenous Proteins ◽  
Apc Protein ◽  
Necessary And Sufficient ◽  
Armadillo Repeats

End-binding protein (EB) 1 binds to the C-terminus of adenomatous polyposis coli (APC) protein and to the plus ends of microtubules (MT) and has been implicated in the regulation of APC accumulation in cortical clusters at the tip of extending membranes. We investigated which APC domains are involved in cluster localization and whether binding to EB1 or MTs is essential for APC cluster localization. Armadillo repeats of APC that lack EB1- and MT-binding domains are necessary and sufficient for APC localization in cortical clusters; an APC fragment lacking the armadillo repeats, but containing MT-and EB1-binding domains, does not localize to the cortical clusters but instead co-aligns with MTs throughout the cell. Significantly, analysis of endogenous proteins reveals that EB1 does not accumulate in the APC clusters. However, overexpressed EB1 does accumulate in APC clusters; the APC-binding domain in EB1 is located in the C-terminal region of EB1 between amino acids 134 and 268. Overexpressed APC- or MT-binding domains of EB1 localize to APC cortical clusters and MT, respectively, without affecting APC cluster formation itself. These results show that localization of APC in cortical clusters is different from that of EB1 at MT plus ends and appears to be independent of EB1.

scholarly journals Structure and Analysis of R1 and R2 Pyocin Receptor-Binding Fibers

Viruses ◽  
2018 ◽  
Vol 10 (8) ◽  
pp. 427 ◽  
Author(s):  
Sergey Buth ◽  
Mikhail Shneider ◽  
Dean Scholl ◽  
Petr Leiman
Keyword(s):  
Cell Surface ◽  
Host Cell ◽  
Receptor Binding ◽  
Binding Sites ◽  
Cell Envelope ◽  
Surface Binding ◽  
Cell Surface Binding ◽  
C Terminus ◽  
Host Cell Surface ◽  
Substrate Binding Sites

The R-type pyocins are high-molecular weight bacteriocins produced by some strains of Pseudomonas aeruginosa to specifically kill other strains of the same species. Structurally, the R-type pyocins are similar to “simple” contractile tails, such as those of phage P2 and Mu. The pyocin recognizes and binds to its target with the help of fibers that emanate from the baseplate structure at one end of the particle. Subsequently, the pyocin contracts its sheath and drives the rigid tube through the host cell envelope. This causes depolarization of the cytoplasmic membrane and cell death. The host cell surface-binding fiber is ~340 Å-long and is attached to the baseplate with its N-terminal domain. Here, we report the crystal structures of C-terminal fragments of the R1 and R2 pyocin fibers that comprise the distal, receptor-binding part of the protein. Both proteins are ~240 Å-long homotrimers in which slender rod-like domains are interspersed with more globular domains—two tandem knob domains in the N-terminal part of the fragment and a lectin-like domain at its C-terminus. The putative substrate binding sites are separated by about 100 Å, suggesting that binding of the fiber to the cell surface causes the fiber to adopt a certain orientation relative to the baseplate and this then triggers sheath contraction.

scholarly journals The Vaccinia Virus 14-Kilodalton (A27L) Fusion Protein Forms a Triple Coiled-Coil Structure and Interacts with the 21-Kilodalton (A17L) Virus Membrane Protein through a C-Terminal α-Helix

1998 ◽  
Vol 72 (12) ◽  
pp. 10126-10137 ◽  
Author(s):  
María-Isabel Vázquez ◽  
German Rivas ◽  
David Cregut ◽  
Luis Serrano ◽  
Mariano Esteban
Keyword(s):  
Amino Acids ◽  
Vaccinia Virus ◽  
Structural Model ◽  
Analytical Ultracentrifugation ◽  
Transmembrane Domain ◽  
Coiled Coil ◽  
C Terminus ◽  
Α Helix ◽  
Mutant Forms ◽  
Helical Region

ABSTRACT The vaccinia virus 14-kDa protein (encoded by the A27L gene) plays an important role in the biology of the virus, acting in virus-to-cell and cell-to-cell fusions. The protein is located on the surface of the intracellular mature virus form and is essential for both the release of extracellular enveloped virus from the cells and virus spread. Sequence analysis predicts the existence of four regions in this protein: a structureless region from amino acids 1 to 28, a helical region from residues 29 to 37, a triple coiled-coil helical region from residues 44 to 72, and a Leu zipper motif at the C terminus. Circular dichroism spectroscopy, analytical ultracentrifugation, and chemical cross-linking studies of the purified wild-type protein and several mutant forms, lacking one or more of the above regions or with point mutations, support the above-described structural division of the 14-kDa protein. The two contiguous cysteine residues at positions 71 and 72 are not responsible for the formation of 14-kDa protein trimers. The location of hydrophobic residues at the a and d positions on a helical wheel and of charged amino acids in adjacent positions, e and g, suggests that the hydrophobic and ionic interactions in the triple coiled-coil helical region are involved in oligomer formation. This conjecture was supported by the construction of a three-helix bundle model and molecular dynamics. Binding assays with purified proteins expressed in Escherichia coli and cytoplasmic extracts from cells infected with a virus that does not produce the 14-kDa protein during infection (VVindA27L) show that the 21-kDa protein (encoded by the A17L gene) is the specific viral binding partner and identify the putative Leu zipper, the predicted third α-helix on the C terminus of the 14-kDa protein, as the region involved in protein binding. These findings were confirmed in vivo, following transfection of animal cells with plasmid vectors expressing mutant forms of the 14-kDa protein and infected with VVindA27L. We find the structural organization of 14kDa to be similar to that of other fusion proteins, such as hemagglutinin of influenza virus and gp41 of human immunodeficiency virus, except for the presence of a protein-anchoring domain instead of a transmembrane domain. Based on our observations, we have established a structural model of the 14-kDa protein.

p53 domains: structure, oligomerization, and transformation

1994 ◽  
Vol 14 (8) ◽  
pp. 5182-5191
Author(s):  
P Wang ◽  
M Reed ◽  
Y Wang ◽  
G Mayr ◽  
J E Stenger ◽  
...  
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Gel Filtration ◽  
Dominant Negative ◽  
Wild Type ◽  
C Terminus ◽  
Tetramerization Domain ◽  
Wild Type P53 ◽  
Negative Phenotype

Wild-type p53 forms tetramers and multiples of tetramers. Friedman et al. (P. N. Friedman, X. B. Chen, J. Bargonetti, and C. Prives, Proc. Natl. Acad. Sci. USA 90:3319-3323, 1993) have reported that human p53 behaves as a larger molecule during gel filtration than it does during sucrose gradient sedimentation. These differences argue that wild-type p53 has a nonglobular shape. To identify structural and oligomerization domains in p53, we have investigated the physical properties of purified segments of p53. The central, specific DNA-binding domain within murine amino acids 80 to 320 and human amino acids 83 to 323 behaves predominantly as monomers during analysis by sedimentation, gel filtration, and gel electrophoresis. This consistent behavior argues that the central region of p53 is globular in shape. Under appropriate conditions, however, this segment can form transient oligomers without apparent preference for a single oligomeric structure. This region does not enhance transformation by other oncogenes. The biological implications of transient oligomerization by this central segment, therefore, remain to be demonstrated. Like wild-type p53, the C terminus, consisting of murine amino acids 280 to 390 and human amino acids 283 to 393, behaves anomalously during gel filtration and apparently has a nonglobular shape. Within this region, murine amino acids 315 to 350 and human amino acids 323 to 355 are sufficient for assembly of stable tetramers. The finding that murine amino acids 315 to 360 enhance transformation by other oncogenes strongly supports the role of p53 tetramerization in oncogenesis. Amino acids 330 to 390 of murine p53 and amino acids 340 to 393 of human p53, which have been implicated by Sturzbecher et al. in tetramerization (H.-W. Sturzbecher, R. Brain, C. Addison, K. Rudge, M. Remm, M. Grimaldi, E. Keenan, and J. R. Jenkins, Oncogene 7:1513-1523, 1992), do not form stable tetramers under our conditions. Our findings indicate that p53 has at least two autonomous oligomerization domains: a strong tetramerization domain in its C-terminal region and a weaker oligomerization domain in the central DNA binding region of p53. Together, these domains account for the formation of tetramers and multiples of tetramers by wild-type p53. The tetramerization domain is the major determinant of the dominant negative phenotype leading to transformation by mutant p53s.

scholarly journals Identification of fibronectin fragments that bind to carboxy-group-modified proteins

1983 ◽  
Vol 215 (3) ◽  
pp. 613-616 ◽  
Author(s):  
M Vuento ◽  
K Sekiguchi ◽  
M Korkolainen
Keyword(s):  
Binding Site ◽  
Human Plasma ◽  
Carboxy Group ◽  
Binding Sites ◽  
Limited Proteolysis ◽  
Plasma Fibronectin ◽  
C Terminus ◽  
Fibronectin Fragments ◽  
Modified Proteins ◽  
Polypeptide Chains

Limited proteolysis of human plasma fibronectin with chymotrypsin, trypsin or thermolysin has been used to localize binding sites responsible for binding [Vuento, Korkolainen & Stenman (1982) Biochem. J. 205, 303-311] of fibronectin to carboxy-group-modified proteins. These bindings sites are different from those mediating binding of fibronectin to gelatin or heparin. They are located close to the C-terminus of the polypeptide chains of fibronectin, and apparently overlap with the C-terminal fibrin binding site.

scholarly journals Multiple Rab GTPase Binding Sites in GCC185 Suggest a Model for Vesicle Tethering at the Trans-Golgi

2009 ◽  
Vol 20 (1) ◽  
pp. 209-217 ◽  
Author(s):  
Garret L. Hayes ◽  
Frank C. Brown ◽  
Alexander K. Haas ◽  
Ryan M. Nottingham ◽  
Francis A. Barr ◽  
...  
Keyword(s):  
Binding Sites ◽  
Cultured Cells ◽  
Coiled Coil ◽  
Cooperative Interaction ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Biochemical Tests ◽  
C Terminus ◽  
Golgi Network ◽  
Interfering Rna

GCC185, a trans-Golgi network-localized protein predicted to assume a long, coiled-coil structure, is required for Rab9-dependent recycling of mannose 6-phosphate receptors (MPRs) to the Golgi and for microtubule nucleation at the Golgi via CLASP proteins. GCC185 localizes to the Golgi by cooperative interaction with Rab6 and Arl1 GTPases at adjacent sites near its C terminus. We show here by yeast two-hybrid and direct biochemical tests that GCC185 contains at least four additional binding sites for as many as 14 different Rab GTPases across its entire length. A central coiled-coil domain contains a specific Rab9 binding site, and functional assays indicate that this domain is important for MPR recycling to the Golgi complex. N-Terminal coiled-coils are also required for GCC185 function as determined by plasmid rescue after GCC185 depletion by using small interfering RNA in cultured cells. Golgi-Rab binding sites may permit GCC185 to contribute to stacking and lateral interactions of Golgi cisternae as well as help it function as a vesicle tether.

scholarly journals Domains of WNK1 kinase in the regulation of ROMK1

2008 ◽  
Vol 295 (2) ◽  
pp. F438-F445 ◽  
Author(s):  
Hao-Ran Wang ◽  
Zhen Liu ◽  
Chou-Long Huang
Keyword(s):  
Amino Acids ◽  
Potassium Channel ◽  
Protein Kinases ◽  
Kinase Activity ◽  
Coiled Coil ◽  
Kinase Domain ◽  
Autoinhibitory Domain ◽  
Rich Domain ◽  
Coiled Coil Domain ◽  
Necessary And Sufficient

WNK1 kinase belongs to a family of serine-threonine protein kinases with an atypical placement of the catalytic lysine. Increased expression of WNK1 causes hypertension and hyperkalemia in humans. WNK1 inhibits renal potassium channel ROMK1 by enhancing its endocytosis, likely contributing to hyperkalemia in affected patients. The domains of WNK1 involved in inhibition of ROMK1 have not been completely elucidated. Here, we reported that an NH2-terminal proline-rich domain (N-PRD; amino acids 1-119) is necessary and sufficient for WNK1 inhibition of ROMK1. A region (named “NL” for N-linker; amino acids 120-220) located between N-PRD and the kinase domain of WNK1 (amino acids 220-491) antagonized the inhibition of ROMK1 caused by N-PRD. The WNK1 kinase domain reversed the antagonism of NL on N-PRD. Mutagenesis studies revealed that charge-charge interactions between two conserved catalytic residues (Lys-233 and Asp-368) within the kinase domain (not the kinase activity) are critical for kinase domain to reverse the antagonism of NL domain. The WNK1 autoinhibitory domain (AID; amino acids 491-555) also affected ROMK, presumably by modulating the kinase domain conformation. Mutations of two conserved phenylalanine abolished the ability of AID to modulate ROMK1. Finally, the first coiled-coil domain (CC1; amino acids 555-640) of WNK1 alleviated the effect of AID domain toward kinase domain. Thus, multiple intra- and/or intermolecular interactions of WNK1 domains are at play for regulation of ROMK1 by WNK1.

scholarly journals Identification of HCN1 as a 14-3-3 client

2021 ◽  
Author(s):  
Colten K Lankford ◽  
Jon C Houtman ◽  
Sheila A Baker
Keyword(s):  
Protein Degradation ◽  
Binding Sites ◽  
Neuronal Excitability ◽  
Adaptive Modulation ◽  
Hek293 Cells ◽  
Titration Calorimetry ◽  
Intrinsically Disordered ◽  
C Terminus ◽  
Gated Channel ◽  
Necessary And Sufficient

Hyperpolarization activated cyclic nucleotide-gated channel 1 (HCN1) is expressed throughout the nervous system and is critical for regulating neuronal excitability, with mutations being associated with multiple forms of epilepsy. Adaptive modulation of HCN1 has been observed as has pathogenic dysregulation. While the mechanisms underlying this modulation remain incompletely understood, regulation of HCN1 has been shown to include phosphorylation. A candidate phosphorylation-dependent regulator of HCN1 channels is 14-3-3. We used bioinformatics to identify three potential 14-3-3 binding sites in HCN1. Isothermal titration calorimetry demonstrated that recombinant 14-3-3 binds all three phospho-peptides with low micromolar affinity. We confirmed that 14-3-3 could pull down HCN1 from multiple tissue sources and used HEK293 cells to detail the interaction. Two binding sites in the intrinsically disordered C-terminus of HCN1 were necessary and sufficient for a phosphorylation-dependent interaction with 14-3-3. The same region of HCN1 containing the 14-3-3 binding sites is required for phosphorylation-independent protein degradation. We propose a model in which phosphorylation of S810 and S867 (human S789 and S846) recruits 14-3-3 to inhibit a yet unidentified factor signaling for protein degradation, thus increasing the half-life of HCN1.

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