scholarly journals Structure-Function Analysis of IntDOT

2009 ◽  
Vol 192 (2) ◽  
pp. 575-586 ◽  
Author(s):  
Seyeun Kim ◽  
Brian M. Swalla ◽  
Jeffrey F. Gardner
Keyword(s):  
Homology Modeling ◽  
Protein Interactions ◽  
Active Site ◽  
Dna Cleavage ◽  
Function Analysis ◽  
Indicator Strain ◽  
Amino Terminal ◽  
Dna Ligation ◽  
Polypeptide Backbone

ABSTRACT CTnDOT integrase (IntDOT) is a member of the tyrosine family of site-specific DNA recombinases. IntDOT is unusual in that it catalyzes recombination between nonidentical sequences. Previous mutational analyses centered on mutants with substitutions of conserved residues in the catalytic (CAT) domain or residues predicted by homology modeling to be close to DNA in the core-binding (CB) domain. That work suggested that a conserved active-site residue (Arg I) of the CAT domain is missing and that some residues in the CB domain are involved in catalysis. Here we used a genetic approach and constructed an Escherichia coli indicator strain to screen for random mutations in IntDOT that disrupt integrative recombination in vivo. Twenty-five IntDOT mutants were isolated and characterized for DNA binding, DNA cleavage, and DNA ligation activities. We found that mutants with substitutions in the amino-terminal (N) domain were catalytically active but defective in forming nucleoprotein complexes, suggesting that they have altered protein-protein interactions or altered interactions with DNA. Replacement of Ala-352 of the CAT domain disrupted DNA cleavage but not DNA ligation, suggesting that Ala-352 may be important for positioning the catalytic tyrosine (Tyr-381) during cleavage. Interestingly, our biochemical data and homology modeling of the CAT domain suggest that Arg-285 is the missing Arg I residue of IntDOT. The predicted position of Arg-285 shows it entering the active site from a position on the polypeptide backbone that is not utilized in other tyrosine recombinases. IntDOT may therefore employ a novel active-site architecture to catalyze recombination.

scholarly journals Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 21-29 ◽  
Author(s):  
David R H Evans ◽  
Brian A Hemmings
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Protein Function ◽  
Mutational Analysis ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Active Site Residues ◽  
Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

scholarly journals The toxofilin–actin–PP2C complex of Toxoplasma: identification of interacting domains

2007 ◽  
Vol 401 (3) ◽  
pp. 711-719 ◽  
Author(s):  
Gaelle Jan ◽  
Violaine Delorme ◽  
Violaine David ◽  
Celine Revenu ◽  
Angelita Rebollo ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Actin Filaments ◽  
Function Analysis ◽  
Protozoan Parasite ◽  
Actin Binding ◽  
Protein Protein Interactions ◽  
The Third ◽  
Parasite Motility

Toxofilin is a 27 kDa protein isolated from the human protozoan parasite Toxoplasma gondii, which causes toxoplasmosis. Toxofilin binds to G-actin, and in vitro studies have shown that it controls elongation of actin filaments by sequestering actin monomers. Toxofilin affinity for G-actin is controlled by the phosphorylation status of its Ser53, which depends on the activities of a casein kinase II and a type 2C serine/threonine phosphatase (PP2C). To get insights into the functional properties of toxofilin, we undertook a structure–function analysis of the protein using a combination of biochemical techniques. We identified a domain that was sufficient to sequester G-actin and that contains three peptide sequences selectively binding to G-actin. Two of these sequences are similar to sequences present in several G- and F-actin-binding proteins, while the third appears to be specific to toxofilin. Additionally, we identified two toxofilin domains that interact with PP2C, one of which contains the Ser53 substrate. In addition to characterizing the interacting domains of toxofilin with its partners, the present study also provides information on an in vivo-based approach to selectively and competitively disrupt the protein–protein interactions that are important to parasite motility.

Cardiac hypertrophy and altered β-adrenergic signaling in transgenic mice that express the amino terminus of β-ARK1

2003 ◽  
Vol 285 (5) ◽  
pp. H2201-H2211 ◽  
Author(s):  
Janelle R. Keys ◽  
Emily A. Greene ◽  
Chris J. Cooper ◽  
Sathyamangla V. Naga Prasad ◽  
Howard A. Rockman ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Cardiac Hypertrophy ◽  
Protein Interactions ◽  
Amino Terminus ◽  
Amino Acid Residues ◽  
Protein Protein Interactions ◽  
Gpcr Signaling ◽  
Amino Terminal ◽  
G Protein Coupled

The G protein-coupled receptor (GPCR) kinase β-adrenergic receptor (β-AR) kinase-1 (β-ARK1) is elevated during heart failure; however, its role is not fully understood. β-ARK1 contains several domains that are capable of protein-protein interactions that may play critical roles in the regulation of GPCR signaling. In this study, we developed a novel line of transgenic mice that express an amino-terminal peptide of β-ARK1 that is comprised of amino acid residues 50–145 (β-ARKnt) in the heart to determine whether this domain has any functional significance in vivo. Surprisingly, the β-ARKnt transgenic mice presented with cardiac hypertrophy. Our data suggest that the phenotype was driven via an enhanced β-AR system, as β-ARKnt mice had elevated cardiac β-AR density. Moreover, administration of a β-AR antagonist reversed hypertrophy in these mice. Interestingly, signaling through the β-AR in response to agonist stimulation was not enhanced in these mice. Thus the amino terminus of β-ARK1 appears to be critical for normal β-AR regulation in vivo, which further supports the hypothesis that β-ARK1 plays a key role in normal and compromised cardiac GPCR signaling.

scholarly journals Structure/Function Analysis of the Vaccinia Virus F18 Phosphoprotein, an Abundant Core Component Required for Virion Maturation and Infectivity

2010 ◽  
Vol 84 (13) ◽  
pp. 6846-6860 ◽  
Author(s):  
Nadi T. Wickramasekera ◽  
Paula Traktman
Keyword(s):  
Amino Acids ◽  
Structure Function ◽  
Protein Interactions ◽  
Function Analysis ◽  
Aromatic Amino Acids ◽  
Protein Protein Interactions ◽  
Core Component ◽  
Virion Morphogenesis

ABSTRACT Poxvirus virions, whose outer membrane surrounds two lateral bodies and a core, contain at least 70 different proteins. The F18 phosphoprotein is one of the most abundant core components and is essential for the assembly of mature virions. We report here the results of a structure/function analysis in which the role of conserved cysteine residues, clusters of charged amino acids and clusters of hydrophobic/aromatic amino acids have been assessed. Taking advantage of a recombinant virus in which F18 expression is IPTG (isopropyl-β-d-thiogalactopyranoside) dependent, we developed a transient complementation assay to evaluate the ability of mutant alleles of F18 to support virion morphogenesis and/or to restore the production of infectious virus. We have also examined protein-protein interactions, comparing the ability of mutant and WT F18 proteins to interact with WT F18 and to interact with the viral A30 protein, another essential core component. We show that F18 associates with an A30-containing multiprotein complex in vivo in a manner that depends upon clusters of hydrophobic/aromatic residues in the N′ terminus of the F18 protein but that it is not required for the assembly of this complex. Finally, we confirmed that two PSSP motifs within F18 are the sites of phosphorylation by cellular proline-directed kinases in vitro and in vivo. Mutation of both of these phosphorylation sites has no apparent impact on virion morphogenesis but leads to the assembly of virions with significantly reduced infectivity.

scholarly journals Activation of brain calcineurin (Cn) by Cu-Zn superoxide dismutase (SOD1) depends on direct SOD1–Cn protein interactions occurring in vitro and in vivo

2007 ◽  
Vol 405 (1) ◽  
pp. 51-59 ◽  
Author(s):  
Abdulbaki Agbas ◽  
Dongwei Hui ◽  
Xinsheng Wang ◽  
Vekalet Tek ◽  
Asma Zaidi ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Protein Interactions ◽  
Active Site ◽  
Conformational State ◽  
Full Length ◽  
Poor Correlation ◽  
Calcineurin Activity ◽  
Human Sod1

Cn (calcineurin) activity is stabilized by SOD1 (Cu-Zn superoxide dismutase), a phenomenon attributed to protection from superoxide (O2•−). The effects of O2•− on Cn are still controversial. We found that O2•−, generated either in vitro or in vivo did not affect Cn activity. Yet native bovine, recombinant human or rat, and two chimaeras of human SOD1–rat SOD1, all activated Cn, but SOD2 (Mn-superoxide dismutase) did not affect Cn activity. There was also a poor correlation between SOD1 dismutase activity and Cn activation. A chimaera of human N-terminal SOD1 and rat C-terminal SOD1 had little detectable dismutase activity, yet stimulated Cn activity the same as full-length human or rat SOD1. Nevertheless, there was evidence that the active site of SOD1 was involved in Cn activation based on the loss of activation following chelation of Cu from the active site of SOD1. Also, SOD1 engaged in the catalysis of O2•− dismutation was ineffective in activating Cn. SOD1 activation of Cn resulted from a 90-fold decrease in phosphatase Km without a change in Vmax. A possible mechanism for the activation of Cn was identified in our studies as the prevention of Fe and Zn losses from the active site of Cn, suggesting a conformation-dependent SOD1–Cn interaction. In neurons, SOD1 and Cn were co-localized in cytoplasm and membranes, and SOD1 co-immunoprecipitated with Cn from homogenates of brain hippocampus and was present in immunoprecipitates as large multimers. Pre-incubation of pure SOD1 with Cn caused SOD1 multimer formation, an indication of an altered conformational state in SOD1 upon interaction with Cn.

scholarly journals Structure and Function Analysis of LIN-14, a Temporal Regulator of Postembryonic Developmental Events in Caenorhabditis elegans

2000 ◽  
Vol 20 (6) ◽  
pp. 2285-2295 ◽  
Author(s):  
Yang Hong ◽  
Rosalind C. Lee ◽  
Victor Ambros
Keyword(s):  
Caenorhabditis Elegans ◽  
Function Analysis ◽  
Cell Types ◽  
Terminal Region ◽  
Gene Products ◽  
Amino Terminal ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal ◽  
Terminal Domains

ABSTRACT During postembryonic development of Caenorhabditis elegans, the heterochronic gene lin-14 controls the timing of developmental events in diverse cell types. Three alternativelin-14 transcripts are predicted to encode isoforms of a novel nuclear protein that differ in their amino-terminal domains. In this paper, we report that the alternative amino-terminal domains of LIN-14 are dispensable and that a carboxy-terminal region within exons 9 to 13 is necessary and sufficient for in vivo LIN-14 function. A transgene capable of expressing only one of the three alternativelin-14 gene products rescues a lin-14 null mutation and is developmentally regulated by lin-4. This shows that the deployment of alternative lin-14 gene products is not critical for the ability of LIN-14 to regulate downstream genes in diverse cell types or for the in vivo regulation of LIN-14 level by lin-4. The carboxy-terminal region of LIN-14 contains an unusual expanded nuclear localization domain which is essential for LIN-14 function. These results support the view that LIN-14 controls developmental timing in C. elegans by regulating gene expression in the nucleus.

Molecular mechanisms of androgen receptor-mediated gene regulation: structure-function analysis of the AF-1 domain.

2004 ◽  
Vol 11 (2) ◽  
pp. 281-293 ◽  
Author(s):  
I J McEwan
Keyword(s):  
Androgen Receptor ◽  
Protein Interactions ◽  
Molecular Mechanisms ◽  
Function Analysis ◽  
Transactivation Domain ◽  
Protein Protein Interactions ◽  
Amino Terminal ◽  
Transcription Complexes ◽  
Amino Terminal Domain ◽  
General Transcription Machinery

The androgen receptor is a ligand-activated transcription factor that binds DNA response elements as a homodimer. Binding sites for the receptor have been identified both upstream and downstream of the transcription start site. Once bound to DNA, the receptor contacts chromatin remodelling complexes, coactivator proteins and components of the general transcription machinery in order to regulate target gene expression. The main transactivation domain, termed AF1, is located within the structurally distinct amino-terminal domain. This region is structurally flexible but adopts a more folded conformation in the presence of the binding partner TFIIF, and this in turn enhances subsequent protein-protein interactions. Thus, there is likely to be a dynamic interplay between protein-protein interactions and protein folding, involving AF1, that is proposed to lead to the assembly and/or disassembly of receptor-dependent transcription complexes.

Abstract 365: Domain-specific Roles for GRK2 in Cardiac Hypertrophy and Heart Failure

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Sarah M Schumacher ◽  
Erhe Gao ◽  
J. Kurt Chuprun ◽  
Walter J Koch
Keyword(s):  
Heart Failure ◽  
G Protein ◽  
Protein Interactions ◽  
Heart Function ◽  
Pressure Overload ◽  
Left Ventricular ◽  
Amino Terminal ◽  
Domain Specific ◽  
Rgs Domain

During heart failure (HF), cardiac levels and activity of the G protein-coupled receptor (GPCR) kinase (GRK) GRK2 are elevated and contribute to adverse remodeling and contractile dysfunction, while inhibition via a carboxyl-terminal peptide, βARKct, enhances heart function and can prevent HF development. Mounting evidence supports the idea of a dynamic “interactome” in which GRK2 can uncouple GPCRs via novel protein-protein interactions. Several GRK2 interacting partners are important for adaptive and maladaptive myocyte growth; therefore, an understanding of domain-specific interactions with signaling and regulatory molecules could lead to novel targets for HF therapy. For instance, GRK2 contains a putative amino-terminal Regulator of G protein Signaling (RGS) domain (βARK-RGS) that directly interacts with Gq and appears to inhibit signaling without altering Gq enzymatic activity. Previously, our lab investigated cardiac-specific transgenic (Tg) expression of a fragment of this RGS domain (βARKnt). This fragment did not alter acute hypertrophy after pressure overload or demonstrate RGS activity in vivo against Gq-mediated signaling. In contrast, βARKnt induced hypertrophy and elevated β-adrenergic receptor (βAR) density without altering agonist-induced contractility or adenylyl cyclase activity, due to a compensatory increase in GRK2 activity. Importantly, though, βAR downregulation in response to chronic agonist administration was attenuated by βARKnt expression, indicating a novel regulation of βAR receptor density. Given these findings we have recently investigated the effect of βARKnt expression during chronic pressure overload post trans-aortic constriction (TAC). Echocardiographic analysis revealed increased posterior wall thickness and left-ventricular mass 4 weeks post-TAC compared to non-transgenic littermate controls (NLC). Importantly, despite enhanced hypertrophy, the progression to HF was inhibited in βARKnt mice 14 weeks post-TAC (%LV Ejection Fraction of 36.1 ± 0.2 in NLC versus 56.6 ± 0.9 in Tg mice). While mechanistic characterization is underway, these data indicate that βARKnt-mediated regulation of βAR density may provide a novel means of cardioprotection during pressure-overload induced HF.

scholarly journals Numb Independently Antagonizes Sanpodo Membrane Targeting and Notch Signaling in Drosophila Sensory Organ Precursor Cells

2010 ◽  
Vol 21 (5) ◽  
pp. 802-810 ◽  
Author(s):  
Xin Tong ◽  
Diana Zitserman ◽  
Ilya Serebriiskii ◽  
Mark Andrake ◽  
Roland Dunbrack ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Notch Signaling ◽  
Progenitor Cells ◽  
Cell Fate ◽  
Membrane Trafficking ◽  
Neural Progenitor Cells ◽  
Function Analysis ◽  
Neural Progenitor ◽  
Amino Terminal

In Drosophila , mitotic neural progenitor cells asymmetrically segregate the cell fate determinant Numb in order to block Notch signaling in only one of the two daughter cells. Sanpodo, a membrane protein required for Notch signaling in asymmetrically dividing cells, is sequestered from the plasma membrane to intracellular vesicles in a Numb-dependent way after neural progenitor cell mitosis. However, the significance of Numb-dependent Sanpodo regulation is unclear. In this study, we conducted a structure–function analysis to identify the determinants of Sanpodo targeting in vivo. We identified an NPAF motif in the amino-terminal cytoplasmic tail of Sanpodo, which is conserved among insect Sanpodo homologues. The Sanpodo NPAF motif is predicted to bind directly to the Numb phosphotyrosine-binding domain and is critical for Numb binding in vitro. Deletion or mutation of the NPAF motif results in accumulation of Sanpodo at the plasma membrane in Numb-positive cells in vivo. Genetic analysis of Sanpodo NPAF mutants shows that Numb-dependent Sanpodo endocytic targeting can be uncoupled from Notch signaling regulation. Our findings demonstrate that Sanpodo contains an evolutionarily conserved motif that has been linked to Numb-dependent regulation in vertebrates and further support the model that Numb regulates Notch signaling independently of Sanpodo membrane trafficking in neural progenitor cells.

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