138 results on '"Boulanger, Martin J"'
Search Results
102. Structural Characterization of the Bradyzoite Surface Antigen (BSR4) from Toxoplasma gondii, a Unique Addition to the Surface Antigen Glycoprotein 1-related Superfamily
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Crawford, Joanna, primary, Grujic, Ognjen, additional, Bruic, Ekaterina, additional, Czjzek, Mirjam, additional, Grigg, Michael E., additional, and Boulanger, Martin J., additional
- Published
- 2009
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103. Structural and Biochemical Characterization of a Novel Aldehyde Dehydrogenase Encoded by the Benzoate Oxidation Pathway in Burkholderia xenovorans LB400
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Bains, Jasleen, primary and Boulanger, Martin J., additional
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- 2008
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104. Insect-cell expression, crystallization and X-ray data collection of the bradyzoite-specific antigen BSR4 fromToxoplasma gondii
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Grujic, Ognjen, primary, Grigg, Michael E., additional, and Boulanger, Martin J., additional
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- 2008
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105. Purification, crystallization and X-ray diffraction analysis of a novel ring-cleaving enzyme (BoxCC) fromBurkholderia xenovoransLB400
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Bains, Jasleen, primary and Boulanger, Martin J., additional
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- 2008
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106. Biochemical and Structural Characterization of the Paralogous Benzoate CoA Ligases from Burkholderia xenovorans LB400: Defining the Entry Point into the Novel Benzoate Oxidation (box) Pathway
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Bains, Jasleen, primary and Boulanger, Martin J., additional
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- 2007
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107. Signaling conformations of the tall cytokine receptor gp130 when in complex with IL-6 and IL-6 receptor
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Skiniotis, Georgios, primary, Boulanger, Martin J, additional, Garcia, K Christopher, additional, and Walz, Thomas, additional
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- 2005
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108. A Caged Lanthanide Complex as a Paramagnetic Shift Agent for Protein NMR
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Prudêncio, Miguel, primary, Rohovec, Jan, additional, Peters, Joop A., additional, Tocheva, Elitza, additional, Boulanger, Martin J., additional, Murphy, Michael E. P., additional, Hupkes, Hermen-Jan, additional, Kosters, Walter, additional, Impagliazzo, Antonietta, additional, and Ubbink, Marcellus, additional
- Published
- 2004
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109. Molecular Mechanisms for Viral Mimicry of a Human Cytokine: Activation of gp130 by HHV-8 Interleukin-6
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Boulanger, Martin J., primary, Chow, Dar-chone, additional, Brevnova, Elena, additional, Martick, Monika, additional, Sandford, Gordon, additional, Nicholas, John, additional, and Garcia, K.Christopher, additional
- Published
- 2004
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110. Convergent Mechanisms for Recognition of Divergent Cytokines by the Shared Signaling Receptor gp130
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Boulanger, Martin J, primary, Bankovich, Alexander J, additional, Kortemme, Tanja, additional, Baker, David, additional, and Garcia, K.Christopher, additional
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- 2003
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111. Crystal structure of the soluble domain of the major anaerobically induced outer membrane protein (AniA) from pathogenic Neisseria: a new class of copper-containing nitrite reductases
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Boulanger, Martin J, primary and Murphy, Michael E.P, additional
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- 2002
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112. Catalytic Roles for Two Water Bridged Residues (Asp-98 and His-255) in the Active Site of Copper-containing Nitrite Reductase
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Boulanger, Martin J., primary, Kukimoto, Mutsuko, additional, Nishiyama, Makoto, additional, Horinouchi, Sueharu, additional, and Murphy, Michael E.P., additional
- Published
- 2000
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113. Carbon Monoxide Binding to Copper-Containing Nitrite Reductase from Alcaligenes faecalis
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Zhang, Haoming, primary, Boulanger, Martin J., additional, Mauk, A. Grant, additional, and Murphy, Michael E. P., additional
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- 2000
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114. Chromodomain Antagonists ThatTarget the Polycomb-GroupMethyllysine Reader Protein Chromobox Homolog 7 (CBX7).
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Simhadri, Chakravarthi, Daze, Kevin D., Douglas, Sarah F., Quon, Taylor T. H., Dev, Amarjot, Gignac, Michael C., Peng, Fangni, Heller, Markus, Boulanger, Martin J., Wulff, Jeremy E., and Hof, Fraser
- Published
- 2014
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115. The multifunctional role of the pallilysin-associated T reponema pallidum protein, Tp0750, in promoting fibrinolysis and extracellular matrix component degradation.
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Houston, Simon, Russell, Shannon, Hof, Rebecca, Roberts, Alanna K., Cullen, Paul, Irvine, Kyle, Smith, Derek S., Borchers, Christoph H., Tonkin, Michelle L., Boulanger, Martin J., and Cameron, Caroline E.
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FIBRINOLYSIS ,EXTRACELLULAR matrix proteins ,METALLOPROTEINASES ,TREPONEMA pallidum ,FIBRONECTINS ,VON Willebrand factor - Abstract
The mechanisms that facilitate dissemination of the highly invasive spirochaete, T reponema pallidum, are incompletely understood. Previous studies showed the treponemal metalloprotease pallilysin ( Tp0751) possesses fibrin clot degradation capability, suggesting a role in treponemal dissemination. In the current study we report characterization of the functionally linked protein Tp0750. Structural modelling predicts Tp0750 contains a von Willebrand factor type A ( vWFA) domain, a protein-protein interaction domain commonly observed in extracellular matrix ( ECM)-binding proteins. We report Tp0750 is a serine protease that degrades the major clot components fibrinogen and fibronectin. We also demonstrate Tp0750 cleaves a matrix metalloprotease ( MMP) peptide substrate that is targeted by several MMPs, enzymes central to ECM remodelling. Through proteomic analyses we show Tp0750 binds the endothelial fibrinolytic receptor, annexin A2, in a specific and dose-dependent manner. These results suggest Tp0750 constitutes a multifunctional protein that is able to (1) degrade infection-limiting clots by both inhibiting clot formation through degradation of host coagulation cascade proteins and promoting clot dissolution by complexing with host proteins involved in the fibrinolytic cascade and (2) facilitate ECM degradation via MMP-like proteolysis of host components. We propose that through these activities Tp0750 functions in concert with pallilysin to enable T . pallidum dissemination. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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116. Toxoplasma gondii Sporozoites Invade Host Cells Using Two Novel Paralogues of RON2 and AMA1.
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Poukchanski, Anna, Fritz, Heather M., Tonkin, Michelle L., Treeck, Moritz, Boulanger, Martin J., and Boothroyd, John C.
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TOXOPLASMA gondii ,SPOROZOITES ,IMMUNOLOGY ,CRYSTALLOGRAPHY ,PROTOZOOLOGY ,IMMUNOFLUORESCENCE - Abstract
Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa. The interaction of two well-studied proteins, Apical Membrane Antigen 1 (AMA1) and Rhoptry Neck protein 2 (RON2), has been shown to be critical for invasion by the asexual tachyzoite stage. Recently, two paralogues of these proteins, dubbed sporoAMA1 and sporoRON2 (or RON2L2), respectively, have been identified but not further characterized in proteomic and transcriptomic analyses of Toxoplasma sporozoites. Here, we show that sporoAMA1 and sporoRON2 localize to the apical region of sporozoites and that, in vitro, they interact specifically and exclusively, with no detectable interaction of sporoAMA1 with generic RON2 or sporoRON2 with generic AMA1. Structural studies of the interacting domains of sporoRON2 and sporoAMA1 indicate a novel pairing that is similar in overall form but distinct in detail from the previously described interaction of the generic pairing. Most notably, binding of sporoRON2 domain 3 to domains I/II of sporoAMA1 results in major alterations in the latter protein at the site of binding and allosterically in the membrane-proximal domain III of sporoAMA1 suggesting a possible role in signaling. Lastly, pretreatment of sporozoites with domain 3 of sporoRON2 substantially impedes their invasion into host cells while having no effect on tachyzoites, and vice versa for domain 3 of generic RON2 (which inhibits tachyzoite but not sporozoite invasion). These data indicate that sporozoites and tachyzoites each use a distinct pair of paralogous AMA1 and RON2 proteins for invasion into host cells, possibly due to the very different environment in which they each must function. [ABSTRACT FROM AUTHOR]
- Published
- 2013
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117. Purification, crystallization and X-ray diffraction analysis of Trypanosoma congolense insect-stage surface antigen (TcCISSA).
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Tonkin, Michelle L., Workman, Sean D., Eyford, Brett A., Loveless, Bianca C., Fudge, Jessica L., Pearson, Terry W., and Boulanger, Martin J.
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TRYPANOSOMA ,CRYSTALLIZATION ,X-ray diffraction ,CELL surface antigens ,RECOMBINANT DNA - Abstract
Trypanosoma congolense is a major contributor to the vast socioeconomic devastation in sub-Saharan Africa caused by animal African trypanosomiasis. These protozoan parasites are transmitted between mammalian hosts by tsetse-fly vectors. A lack of understanding of the molecular basis of tsetse-trypanosome interactions stands as a barrier to the development of improved control strategies. Recently, a stage-specific T. congolense protein, T. congolense insect-stage surface antigen (TcCISSA), was identified that shows considerable sequence identity (>60%) to a previously identified T. brucei insect-stage surface molecule that plays a role in the maturation of infections. TcCISSA has multiple di-amino-acid and tri-amino-acid repeats in its extracellular domain, making it an especially interesting structure-function target. The predicted mature extracellular domain of TcCISSA was produced by recombinant DNA techniques, purified from Escherichia coli, crystallized and subjected to X-ray diffraction analysis; the data were processed to 2.7 Å resolution. [ABSTRACT FROM AUTHOR]
- Published
- 2012
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118. Defining a Structural and Kinetic Rationale for Paralogous qopies of Phenylacetate-CoA Ligases from the Cystic Fibrosis Pathogen Burkholderia cenocepacia J231 5.
- Author
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Law, Adrienne and Boulanger, Martin J.
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PHENYLACETIC acid , *BURKHOLDERIA infections , *CYSTIC fibrosis , *LIGASES , *ADENOSINE triphosphate , *PATIENTS - Abstract
he phenylacetic acid (PAA) degradation pathway is the sole ae obic route for phenylacetic acid metabolism in bacteria and facilitates degradation of environmental pollutants such as styrene and ethylbenzene. The PAA pathway also is implicated in promoting Burkholderia cenocepacia infections in cystic fibrosis patients. Intriguingly, the first enzyme in the PAA pathway is present in two copies (paaKl and paaK2), yet each subsequent enzyme is present in only a single copy. Furthermore, sequence divergence indicates that PaaK1 and PaaK2 form a unique subgroup within the adenylate-forming enzyme (AFE) superfamily. To establish a biochemical rationale for the existence of the PaaK paralogs in B. cenocepacia, we present high resolution x-ray crystal structures of a selenomethionine derivative of PaaK1 in complex with ATP and adenylated phenylacetate intermediate complexes of PaaK1 and PaaK2 in distinct co formations. Structural analysis reveals a novel N-terminal microdomain that may serve to recruit subsequent PAA enzymes, whereas a bifunctional role is proposed for the P-loop in stabilizing the C-terminal domain in conformation 2. The potential for different kinetic profiles was suggested by a structurally divergent extension of the aryl substrate pocket in PaaK1 relative to PaaK2. Functional characterization confirmed this production, with PaaK1 possessing a lower Km for phenylacetic acid and better able to accommodate 3' and 4' substitutions on th phenyl ring. Collectively, these results offer detailed insight in the reaction mechanism of a novel subgroup of the AFE superfamily and provide a clear biochemical rationale for the presence of paralogous copies of PaaK of B. cenocepacia. [ABSTRACT FROM AUTHOR]
- Published
- 2011
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119. Structure of the micronemal protein 2 A/I domain from Toxoplasma gondii.
- Author
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Tonkin, Michelle L., Grujic, Ognjen, Pearce, Mark, Crawford, Joanna, and Boulanger, Martin J.
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- 2010
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120. Structural elucidation of the ml 57 mouse cytomegalovirus ligand for Ly49 natural killer cell receptors.
- Author
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Adams, Erin J., Juo, Z. Sean, Venook, Rayna Takaki, Boulanger, Martin J., Arase, Hisashi, Lanier, Lewis L., and Garcia, K. Christopher
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CYTOMEGALOVIRUSES ,KILLER cells ,VIRAL antigens ,MAJOR histocompatibility complex ,CELL communication ,IMMUNE system ,MOLECULAR recognition - Abstract
Natural killer (NK) cells express activating and inhibitory receptors that, in concert, survey cells for proper expression of cell surface major histocompatibility complex (MHC) class I molecules. The mouse cytomegalovirus encodes an MHC-like protein, m157, which is the only known viral antigen to date capable of engaging both activating (Ly49H) and inhibitory (Ly491) NK cell receptors. We have determined the 3D structure of m157 and studied its biochemical and cellular interactions with the Ly49H and Ly491 receptors, m157 has a characteristic MHC-fold, yet possesses several unique structural features not found in other MHC class I-like molecules, m157 does not bind peptides or other small ligands, nor does it associate with β
2 -microglobulin. Instead, m157 engages in extensive intra- and intermolecular interactions within and between its domains to generate a compact minimal MHC-like molecule, m157's binding affinity for Ly49I (Kd ≈ 0.2 μM) is significantly higher than that of classical inhibitory Ly49-MHC interactions. Analysis of viral escape mutations on m157 that render it resistant to NK killing reveals that it is likely to be recognized by Ly49H in a binding mode that differs from Ly49/MHC-I. In addition, Ly49H+ NK cells can efficiently lyse RMA cells expressing m157, despite the presence of native MHC class I. Collectively, our results show that m157 represents a structurally divergent form of MHC class I-like proteins that directly engage Ly49 receptors with appreciable affinity in a noncanonical fashion. [ABSTRACT FROM AUTHOR]- Published
- 2007
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121. Compensatory Energetic Mechanisms Mediating the Assembly of Signaling Complexes Between Interleukin-2 and its α, β, and γc Receptors
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Rickert, Mathias, Boulanger, Martin J., Goriatcheva, Natalia, and Garcia, K. Christopher
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INTERLEUKIN-2 , *CELL receptors , *THERMODYNAMICS , *BIOLOGICAL interfaces - Abstract
Interleukin-2 is a key immuno-regulatory cytokine whose actions are mediated by three different cell surface receptors: the α, β and the “common γ” (γc) chains. We have undertaken a complete thermodynamic characterization of the stepwise assembly cycle for multiple possible combinations of the receptor–ligand, and receptor–receptor interactions that are necessary for formation of the high-affinity IL-2/αβγc signaling complex. We find an entropically favorable high affinity interaction between IL-2 and its α receptor, a moderately entropically favorable low affinity interaction between IL-2 and its β receptor, and no interaction between IL-2 and the shared receptor, γc. Formation of the stable intermediate trimolecular complexes of IL-2 with α and β receptors, as well as IL-2 with β and γc receptors proceeds through enthalpy–entropy compensation mechanisms. Surprisingly, we see a moderate affinity interaction between the unliganded receptor α and β chains, suggesting that a preformed αβ complex may serve as the initial interaction complex for IL-2. Reconstitution of the IL-2/Rαβγc high-affinity quaternary signaling complex shows it to be assembled through cooperative energetics to form a 1:1:1:1 assembly. Collectively, the favorable entropy of the bimolecular interactions appears to be offset by the loss in rigid body entropy of the receptor components in the higher-order complexes, but overcome by the formation of increasingly enthalpically favorable composite interfaces. This enthalpic mechanism utilized by γc contrasts with the favorable entropic mechanism utilized by gp130 for degenerate cytokine interaction. In conclusion, we find that several energetically redundant pathways exist for formation of IL-2 receptor signaling complexes, suggesting a more complex equilibrium on the cell surface than has been previously appreciated. [Copyright &y& Elsevier]
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- 2004
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122. Hexameric Structure and Assembly of the Interleukin-6/IL-6 α-Receptor/gp 130 Complex.
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Boulanger, Martin J., Chow, Dar-chone, Brevnova, Elena E., and Garcia, K. Christopher
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INTERLEUKIN-6 , *THERMODYNAMICS , *ELECTRON distribution , *CYTOKINES - Abstract
Interleukin-6 (IL-6) is an immunoregulatory cytokine that activates a cell-surface signaling assembly composed of IL-6, the IL-6 a-receptor (IL-6Ra), and the shared signaling receptor gp130. The 3.65 angstrom-resolution structure of the extracellular signaling complex reveals a hexameric, interlocking assembly mediated by a total of 10 symmetry-related, thermodynamically coupled interfaces. Assembly of the hexameric complex occurs sequentially: IL-6 is first engaged by IL-6Ra and then presented to gp130 in the proper geometry to facilitate a cooperative transition into the high-affinity, signaling-competent hexamer. The quaternary structures of other IL-6/IL-12 family signaling complexes are likely constructed by means of a similar topological blueprint. [ABSTRACT FROM AUTHOR]
- Published
- 2003
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123. Directing the mode of nitrite binding to a copper-containing nitrite reductase from Alcaligenes faecalis S-6: Characterization of an active site isoleucine.
- Author
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Boulanger, Martin J. and Murphy, Michael E.P.
- Abstract
Unlike the heme cd
1 -based nitrite reductase enzymes, the molecular mechanism of copper-containing nitrite reductases remains controversial. A key source of controversy is the productive binding mode of nitrite in the active site. To identify and characterize the molecular determinants associated with nitrite binding, we applied a combinatorial mutagenesis approach to generate a small library of six variants at position 257 in nitrite reductase from Alcaligenes faecalis S-6. The activities of these six variants span nearly two orders of magnitude with one variant, I257V, the only observed natural substitution for Ile257, showing greater activity than the native enzyme. High-resolution (> 1.8 Å) nitrite-soaked crystal structures of these variants display different modes of nitrite binding that correlate well with the altered activities. These studies identify for the first time that the highly conserved Ile257 in the native enzyme is a key molecular determinant in directing a catalytically competent mode of nitrite binding in the active site. The O-coordinate bidentate binding mode of nitrite observed in native and mutant forms with high activity supports a catalytic model distinct from the heme cd1 NiRs. (The atomic coordinates for I257V[NO2 − ], I257L[NO2 − ], I257A[NO2 − ], I257T[NO2 − ], I257M[NO2 − ] and I257G[NO2 − ] AfNiR have been deposited in the Protein Data Bank [PDB identification codes are listed in Table 2].) [ABSTRACT FROM AUTHOR]- Published
- 2003
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124. A Conserved Apicomplexan Microneme Protein Contributes to Toxoplasma gondiiInvasion and Virulence
- Author
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Huynh, My-Hang, Boulanger, Martin J., and Carruthers, Vern B.
- Abstract
ABSTRACTThe obligate intracellular parasite Toxoplasma gondiicritically relies on host cell invasion during infection. Proteins secreted from the apical micronemes are central components for host cell recognition, invasion, egress, and virulence. Although previous work established that the sporozoite protein with an altered thrombospondin repeat (SPATR) is a micronemal protein conserved in other apicomplexan parasites, including Plasmodium, Neospora, and Eimeria, no genetic evidence of its contribution to invasion has been reported. SPATR contains a predicted epidermal growth factor domain and two thrombospondin type 1 repeats, implying a role in host cell recognition. In this study, we assess the contribution of T. gondiiSPATR (TgSPATR) to T. gondiiinvasion by genetically ablating it and restoring its expression by genetic complementation. Δspatrparasites were ∼50% reduced in invasion compared to parental strains, a defect that was reversed in the complemented strain. In mouse virulence assays, Δspatrparasites were significantly attenuated, with ∼20% of mice surviving infection. Given the conservation of this protein among the Apicomplexa, we assessed whether the Plasmodium falciparumSPATR ortholog (PfSPATR) could complement the absence of the TgSPATR. Although PfSPATR showed correct micronemal localization, it did not reverse the invasion deficiency of Δspatrparasites, because of an apparent failure in secretion. Overall, the results suggest that TgSPATR contributes to invasion and virulence, findings that have implications for the many genera and life stages of apicomplexans that express SPATR.
- Published
- 2014
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125. Bifunctional Role of the Treponema pallidumExtracellular Matrix Binding Adhesin Tp0751
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Houston, Simon, Hof, Rebecca, Francescutti, Teresa, Hawkes, Aaron, Boulanger, Martin J., and Cameron, Caroline E.
- Abstract
ABSTRACTTreponema pallidum, the causative agent of syphilis, is a highly invasive pathogenic spirochete capable of attaching to host cells, invading the tissue barrier, and undergoing rapid widespread dissemination via the circulatory system. The T. pallidumadhesin Tp0751 was previously shown to bind laminin, the most abundant component of the basement membrane, suggesting a role for this adhesin in host tissue colonization and bacterial dissemination. We hypothesized that similar to that of other invasive pathogens, the interaction of T. pallidumwith host coagulation proteins, such as fibrinogen, may also be crucial for dissemination via the circulatory system. To test this prediction, we used enzyme-linked immunosorbent assay (ELISA) methodology to demonstrate specific binding of soluble recombinant Tp0751 to human fibrinogen. Click-chemistry-based palmitoylation profiling of heterologously expressed Tp0751 confirmed the presence of a lipid attachment site within this adhesin. Analysis of the Tp0751 primary sequence revealed the presence of a C-terminal putative HEXXH metalloprotease motif, and in vitrodegradation assays confirmed that recombinant Tp0751 purified from both insect and Escherichia coliexpression systems degrades human fibrinogen and laminin. The proteolytic activity of Tp0751 was abolished by the presence of the metalloprotease inhibitor 1,10-phenanthroline. Further, inductively coupled plasma-mass spectrometry showed that Tp0751 binds zinc and calcium. Collectively, these results indicate that Tp0751 is a zinc-dependent, membrane-associated protease that exhibits metalloprotease-like characteristics. However, site-directed mutagenesis of the HEXXH motif to HQXXH did not abolish the proteolytic activity of Tp0751, indicating that further mutagenesis studies are required to elucidate the critical active site residues associated with this protein. This study represents the first published description of a T. pallidumprotease capable of degrading host components and thus provides novel insight into the mechanism of T. pallidumdissemination.
- Published
- 2011
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126. Bifunctional Role of the Treponema pallidum Extracellular Matrix Binding Adhesin Tp0751
- Author
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Houston, Simon, Hof, Rebecca, Francescutti, Teresa, Hawkes, Aaron, Boulanger, Martin J., and Cameron, Caroline E.
- Abstract
Treponema pallidum, the causative agent of syphilis, is a highly invasive pathogenic spirochete capable of attaching to host cells, invading the tissue barrier, and undergoing rapid widespread dissemination via the circulatory system. The T. pallidum adhesin Tp0751 was previously shown to bind laminin, the most abundant component of the basement membrane, suggesting a role for this adhesin in host tissue colonization and bacterial dissemination. We hypothesized that similar to that of other invasive pathogens, the interaction of T. pallidum with host coagulation proteins, such as fibrinogen, may also be crucial for dissemination via the circulatory system. To test this prediction, we used enzyme-linked immunosorbent assay (ELISA) methodology to demonstrate specific binding of soluble recombinant Tp0751 to human fibrinogen. Click-chemistry-based palmitoylation profiling of heterologously expressed Tp0751 confirmed the presence of a lipid attachment site within this adhesin. Analysis of the Tp0751 primary sequence revealed the presence of a C-terminal putative HEXXH metalloprotease motif, and in vitro degradation assays confirmed that recombinant Tp0751 purified from both insect and Escherichia coli expression systems degrades human fibrinogen and laminin. The proteolytic activity of Tp0751 was abolished by the presence of the metalloprotease inhibitor 1,10-phenanthroline. Further, inductively coupled plasma-mass spectrometry showed that Tp0751 binds zinc and calcium. Collectively, these results indicate that Tp0751 is a zinc-dependent, membrane-associated protease that exhibits metalloprotease-like characteristics. However, site-directed mutagenesis of the HEXXH motif to HQXXH did not abolish the proteolytic activity of Tp0751, indicating that further mutagenesis studies are required to elucidate the critical active site residues associated with this protein. This study represents the first published description of a T. pallidum protease capable of degrading host components and thus provides novel insight into the mechanism of T. pallidum dissemination.
- Published
- 2010
127. Purification, crystallization and preliminary X-ray diffraction analysis of inner membrane complex (IMC) subcompartment protein 1 (ISP1) from Toxoplasma gondii.
- Author
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Tonkin, Michelle L., Brown, Shannon, Beck, Josh R., Bradley, Peter J., and Boulanger, Martin J.
- Published
- 2012
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128. Purification and crystallization of a putative transcriptional regulator of the benzoate oxidation pathway in Burkholderia xenovorans LB400.
- Author
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Law, Adrienne M., Bains, Jasleen, and Boulanger, Martin J.
- Subjects
CRYSTALLIZATION ,X-ray diffraction ,ESCHERICHIA coli ,BENZOATES ,OXIDATION - Abstract
Burkholderia xenovorans LB400 harbours two paralogous copies of the recently discovered benzoate oxidation ( box) pathway. While both copies are functional, the paralogues are differentially regulated and flanked by putative transcriptional regulators from distinct families. The putative LysR-type transcriptional regulator (LTTR) adjacent to the megaplasmid-encoded box enzymes, Bxe_C0898, has been produced recombinantly in Escherichia coli and purified to homogeneity. Gel-filtration studies show that Bxe_C0898 is a tetramer in solution, consistent with previously characterized LTTRs. Bxe_C0898 crystallized with four molecules in the asymmetric unit of the P4
3 21 2/ P41 21 2 unit cell with a solvent content of 61.19%, as indicated by processing of the X-ray diffraction data. DNA-protection assays are currently under way in order to identify potential operator regions for this LTTR and to define its role in regulation of the box pathway. [ABSTRACT FROM AUTHOR]- Published
- 2009
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129. P1.29 Attachment of the syphilis spirochete, treponema pallidum, to the vascular endothelium
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Lithgow, Karen V, Kao, Wei-Chen, Hof, Rebecca, Petrosova, Helena, Lin, Yi-Pin, Boulanger, Martin J, Leong, John, Burke, John E, Moriarty, Tara J, and Cameron, Caroline E
- Abstract
Introduction:Treponema pallidumis the causative agent of venereal syphilis, a human-specific sexually transmitted infection characterised by multi-stage disease and diverse clinical manifestations. T. pallidumundergoes rapid hematogenous dissemination, accessing distant organ sites and penetrating tissue, placental, and blood-brain barriers. Tp0751 is an adhesin that interacts with the host vasculature and mediates bacterial adherence to endothelial cells under shear flow conditions. This study explores Tp0751-mediated adhesion to the vascular endothelium.MethodsTp0751, expressed in a non-infectious model spirochete [Borrelia burgdorferi(Bb-Tp0751)], was assessed for a gain-of-function adhesion phenotype using attachment assays. Interaction specificity was probed with competitive inhibition studies using synthetic peptides of Tp0751 host-binding regions. Affinity chromatography coupled with mass spectrometry was used to identify endothelial receptors for Tp0751. Membrane receptors isolated from human umbilical vein endothelial cells (HUVECs) were incubated with Tp0751-affinity columns and interacting proteins were identified with mass spectrometry. ResultsHere we demonstrate that Bb-Tp0751 adheres to HUVECs under stationary conditions. The laminin receptor (LamR) was identified as an endothelial receptor for Tp0751. LamR is a brain endothelial receptor for other neurotropic invasive pathogens, including Neisseria meningitidis. Current investigations will validate the Tp0751-LamR interaction and characterise the functional outcomes of Tp0751 adhesion to endothelial cells.ConclusionThese investigations reveal the mechanics of T. palliumattachment to endothelial cells, the fundamental step in the process of T. pallidumvascular dissemination. A complete understanding of this process will provide opportunities to prevent T. pallidumattachment to the host vasculature to facilitate syphilis vaccine development.
- Published
- 2017
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130. Systems-Based Analysis of the Sarcocystis neuronaGenome Identifies Pathways That Contribute to a Heteroxenous Life Cycle
- Author
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Blazejewski, Tomasz, Nursimulu, Nirvana, Pszenny, Viviana, Dangoudoubiyam, Sriveny, Namasivayam, Sivaranjani, Chiasson, Melissa A., Chessman, Kyle, Tonkin, Michelle, Swapna, Lakshmipuram S., Hung, Stacy S., Bridgers, Joshua, Ricklefs, Stacy M., Boulanger, Martin J., Dubey, Jitender P., Porcella, Stephen F., Kissinger, Jessica C., Howe, Daniel K., Grigg, Michael E., and Parkinson, John
- Abstract
ABSTRACTSarcocystis neuronais a member of the coccidia, a clade of single-celled parasites of medical and veterinary importance including Eimeria, Sarcocystis, Neospora, and Toxoplasma. Unlike Eimeria, a single-host enteric pathogen, Sarcocystis, Neospora, and Toxoplasma are two-host parasites that infect and produce infectious tissue cysts in a wide range of intermediate hosts. As a genus, Sarcocystisis one of the most successful protozoan parasites; all vertebrates, including birds, reptiles, fish, and mammals are hosts to at least one Sarcocystisspecies. Here we sequenced Sarcocystis neurona, the causal agent of fatal equine protozoal myeloencephalitis. The S. neurona genome is 127 Mbp, more than twice the size of other sequenced coccidian genomes. Comparative analyses identified conservation of the invasion machinery among the coccidia. However, many dense-granule and rhoptry kinase genes, responsible for altering host effector pathways in Toxoplasma and Neospora, are absent from S. neurona. Further, S. neurona has a divergent repertoire of SRS proteins, previously implicated in tissue cyst formation in Toxoplasma. Systems-based analyses identified a series of metabolic innovations, including the ability to exploit alternative sources of energy. Finally, we present an S. neurona model detailing conserved molecular innovations that promote the transition from a purely enteric lifestyle (Eimeria) to a heteroxenous parasite capable of infecting a wide range of intermediate hosts.IMPORTANCESarcocystis neuronais a member of the coccidia, a clade of single-celled apicomplexan parasites responsible for major economic and health care burdens worldwide. A cousin of Plasmodium, Cryptosporidium, Theileria, and Eimeria, Sarcocystisis one of the most successful parasite genera; it is capable of infecting all vertebrates (fish, reptiles, birds, and mammals—including humans). The past decade has witnessed an increasing number of human outbreaks of clinical significance associated with acute sarcocystosis. Among Sarcocystisspecies, S. neurona has a wide host range and causes fatal encephalitis in horses, marine mammals, and several other mammals. To provide insights into the transition from a purely enteric parasite (e.g., Eimeria) to one that forms tissue cysts (Toxoplasma), we present the first genome sequence of S. neurona. Comparisons with other coccidian genomes highlight the molecular innovations that drive its distinct life cycle strategies.
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- 2015
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131. Structure of the micronemal protein 2 A/I domain from Toxoplasma gondii.
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Tonkin ML, Grujic O, Pearce M, Crawford J, and Boulanger MJ
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- Amino Acid Sequence, Binding Sites genetics, Crystallography, X-Ray, Electrophoresis, Polyacrylamide Gel, Glycine chemistry, Glycine genetics, Glycine metabolism, Heparin chemistry, Heparin metabolism, Hydrophobic and Hydrophilic Interactions, Membrane Proteins genetics, Membrane Proteins metabolism, Molecular Sequence Data, Phenylalanine chemistry, Phenylalanine genetics, Phenylalanine metabolism, Protein Binding, Protein Folding, Protein Structure, Secondary, Protozoan Proteins genetics, Protozoan Proteins metabolism, Sequence Homology, Amino Acid, Static Electricity, Structure-Activity Relationship, Toxoplasma genetics, Toxoplasma metabolism, Membrane Proteins chemistry, Protein Structure, Tertiary, Protozoan Proteins chemistry
- Abstract
Toxoplasma gondii is a widespread zoonotic pathogen capable of causing serious disease in humans and animals. As an obligate intracellular parasite, T. gondii relies on the orchestrated secretion of proteins from its apical complex organelles including the multimodular, transmembrane micronemal protein 2 (MIC2) that couples recognition of the host cell with cytoskeletal reorganization of the parasite to drive invasion. To probe the basis by which the von Willebrand Factor A (vWA)-Integrin like module of TgMIC2 engages the host cell, we solved the crystal structure of a truncated form of TgMIC2A/I (TgMIC2A/Ic) phased by iodide SIRAS and refined to a resolution of 2.05 Å. The TgMIC2A/Ic core is organized into a central twisted beta sheet flanked by α-helices consistent with a canonical vWA fold. A restricted basic patch serves as the putative heparin binding site, but no heparin binding was detected in native gel shift assays. Furthermore, no metal was observed in the metal ion dependent adhesion site (MIDAS). Structural overlays with homologous A/I domains reveal a divergent organization of the MIDAS β4-α4 loop in TgMIC2A/Ic, which is stabilized through the burial of Phe195 into a deep pocket formed by Gly185. Intriguingly, Gly185 appears to be unique among A/I domains to TgMIC2A/I suggesting that the divergent loop conformation may also be unique to TgMIC2A/I. Although lacking the C-terminal extension, the TgMIC2A/Ic structure reported here is the first of an A/I domain from an apicomplexan parasite and provides valuable insight into defining the molecular recognition of host cells by these widespread pathogens.
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- 2010
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132. Structural and biophysical characterization of BoxC from Burkholderia xenovorans LB400: a novel ring-cleaving enzyme in the crotonase superfamily.
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Bains J, Leon R, and Boulanger MJ
- Subjects
- Binding Sites, Catalysis, Catalytic Domain, Crystallography, Dimerization, Evolution, Molecular, Protein Structure, Secondary, Protein Structure, Tertiary, Burkholderia enzymology, Enoyl-CoA Hydratase chemistry, Enoyl-CoA Hydratase metabolism, Hydrocarbons, Aromatic metabolism
- Abstract
The mineralization of aromatic compounds by microorganisms relies on a structurally and functionally diverse group of ring-cleaving enzymes. The recently discovered benzoate oxidation pathway in Burkholderia xenovorans LB400 encodes a novel such ring-cleaving enzyme, termed BoxC, that catalyzes the conversion of 2,3-dihydro-2,3-dihydroxybenzoyl-CoA to 3,4-dehydroadipyl-CoA without the requirement for molecular oxygen. Sequence analysis indicates that BoxC is a highly divergent member of the crotonase superfamily and nearly double the size of the average superfamily member. The structure of BoxC determined to 1.5 A resolution reveals an intriguing structural demarcation. A highly divergent region in the C terminus probably serves as a structural scaffold for the conserved N terminus that encompasses the active site and, in conjunction with a conserved C-terminal helix, mediates dimer formation. Isothermal titration calorimetry and molecular docking simulations contribute to a detailed view of the active site, resulting in a compelling mechanistic model where a pair of conserved glutamate residues (Glu146 and Glu168) work in tandem to deprotonate the dihydroxylated ring substrate, leading to cleavage. A final deformylation step incorporating a water molecule and Cys111 as a general base completes the formation of 3,4-dehydroadipyl-CoA product. Overall, this study establishes the basis for BoxC as one of the most divergent members of the crotonase superfamily and provides the first structural insight into the mechanism of this novel class of ring-cleaving enzymes.
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- 2009
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133. Purification, crystallization and X-ray diffraction analysis of a novel ring-cleaving enzyme (BoxC(C)) from Burkholderia xenovorans LB400.
- Author
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Bains J and Boulanger MJ
- Subjects
- Bacterial Proteins isolation & purification, Bacterial Proteins metabolism, Cloning, Molecular, Crystallization methods, Enoyl-CoA Hydratase isolation & purification, Enoyl-CoA Hydratase metabolism, X-Ray Diffraction, Bacterial Proteins chemistry, Burkholderia enzymology, Enoyl-CoA Hydratase chemistry
- Abstract
The assimilation of aromatic compounds by microbial species requires specialized enzymes to cleave the thermodynamically stable ring. In the recently discovered benzoate-oxidation (box) pathway in Burkholderia xenovorans LB400, this is accomplished by a novel dihydrodiol lyase (BoxC(C)). Sequence analysis suggests that BoxC(C) is part of the crotonase superfamily but includes an additional uncharacterized region of approximately 115 residues that is predicted to mediate ring cleavage. Processing of X-ray diffraction data to 1.5 A resolution revealed that BoxC(C) crystallized with two molecules in the asymmetric unit of the P2(1)2(1)2(1) space group, with a solvent content of 47% and a Matthews coefficient of 2.32 A(3) Da(-1). Selenomethionine BoxC(C) has been purified and crystals are currently being refined for anomalous dispersion studies.
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- 2008
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134. Insect-cell expression, crystallization and X-ray data collection of the bradyzoite-specific antigen BSR4 from Toxoplasma gondii.
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Grujic O, Grigg ME, and Boulanger MJ
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- Animals, Antigens, Protozoan genetics, Antigens, Protozoan metabolism, Cloning, Molecular, Crystallization methods, Gene Expression, Membrane Proteins genetics, Membrane Proteins metabolism, Protozoan Proteins genetics, Protozoan Proteins metabolism, X-Ray Diffraction, Antigens, Protozoan chemistry, Baculoviridae physiology, Membrane Proteins chemistry, Protozoan Proteins chemistry, Toxoplasma metabolism
- Abstract
Toxoplasma gondii is an important global pathogen that infects nearly one third of the world's adult population. A family of developmentally expressed structurally related surface-glycoprotein adhesins (SRSs) mediate attachment to and are utilized for entry into host cells. The latent bradyzoite form of T. gondii persists for the life of the host and expresses a distinct family of SRS proteins, of which the bradyzoite-specific antigen BSR4 is a prototypical member. Structural studies of BSR4 were initiated by first recombinantly expressing BSR4 in insect cells, which was followed by crystallization and preliminary X-ray data collection to 1.95 A resolution. Data processing showed that BSR4 crystallized with one molecule in the asymmetric unit of the P4(1)2(1)2 or P4(3)2(1)2 space group, with a solvent content of 60% and a corresponding Matthews coefficient of 2.98 A(3) Da(-1).
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- 2008
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135. Structural elucidation of the m157 mouse cytomegalovirus ligand for Ly49 natural killer cell receptors.
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Adams EJ, Juo ZS, Venook RT, Boulanger MJ, Arase H, Lanier LL, and Garcia KC
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- Animals, Baculoviridae genetics, Binding Sites, Cell Line, Tumor, Crystallography, X-Ray, Disulfides chemistry, Histocompatibility Antigens Class I immunology, Hydrogen Bonding, Ligands, Lymphoma, T-Cell pathology, Mice, Models, Molecular, NK Cell Lectin-Like Receptor Subfamily A, Protein Binding, Protein Conformation, Protein Structure, Secondary, Protein Structure, Tertiary, Receptors, Immunologic genetics, Receptors, NK Cell Lectin-Like, Antigens, Ly chemistry, Killer Cells, Natural immunology, Lectins, C-Type chemistry, Muromegalovirus immunology, Receptors, Immunologic chemistry, Receptors, Immunologic immunology
- Abstract
Natural killer (NK) cells express activating and inhibitory receptors that, in concert, survey cells for proper expression of cell surface major histocompatibility complex (MHC) class I molecules. The mouse cytomegalovirus encodes an MHC-like protein, m157, which is the only known viral antigen to date capable of engaging both activating (Ly49H) and inhibitory (Ly49I) NK cell receptors. We have determined the 3D structure of m157 and studied its biochemical and cellular interactions with the Ly49H and Ly49I receptors. m157 has a characteristic MHC-fold, yet possesses several unique structural features not found in other MHC class I-like molecules. m157 does not bind peptides or other small ligands, nor does it associate with beta(2)-microglobulin. Instead, m157 engages in extensive intra- and intermolecular interactions within and between its domains to generate a compact minimal MHC-like molecule. m157's binding affinity for Ly49I (K(d) approximately 0.2 microM) is significantly higher than that of classical inhibitory Ly49-MHC interactions. Analysis of viral escape mutations on m157 that render it resistant to NK killing reveals that it is likely to be recognized by Ly49H in a binding mode that differs from Ly49/MHC-I. In addition, Ly49H+ NK cells can efficiently lyse RMA cells expressing m157, despite the presence of native MHC class I. Collectively, our results show that m157 represents a structurally divergent form of MHC class I-like proteins that directly engage Ly49 receptors with appreciable affinity in a noncanonical fashion.
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- 2007
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136. A caged lanthanide complex as a paramagnetic shift agent for protein NMR.
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Prudêncio M, Rohovec J, Peters JA, Tocheva E, Boulanger MJ, Murphy ME, Hupkes HJ, Kosters W, Impagliazzo A, and Ubbink M
- Subjects
- Azurin chemistry, Crystallography, X-Ray, Lanthanoid Series Elements chemical synthesis, Mass Spectrometry, Models, Molecular, Azurin analogs & derivatives, Lanthanoid Series Elements chemistry, Nuclear Magnetic Resonance, Biomolecular methods, Pentetic Acid chemistry
- Abstract
A lanthanide complex, named CLaNP (caged lanthanide NMR probe) has been developed for the characterisation of proteins by paramagnetic NMR spectroscopy. The probe consists of a lanthanide chelated by a derivative of DTPA (diethylenetriaminepentaacetic acid) with two thiol reactive functional groups. The CLaNP molecule is attached to a protein by two engineered, surface-exposed, Cys residues in a bidentate manner. This drastically limits the dynamics of the metal relative to the protein and enables measurements of pseudocontact shifts. NMR spectroscopy experiments on a diamagnetic control and the crystal structure of the probe-protein complex demonstrate that the protein structure is not affected by probe attachment. The probe is able to induce pseudocontact shifts to at least 40 A from the metal and causes residual dipolar couplings due to alignment at a high magnetic field. The molecule exists in several isomeric forms with different paramagnetic tensors; this provides a fast way to obtain long-range distance restraints.
- Published
- 2004
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137. Compensatory energetic mechanisms mediating the assembly of signaling complexes between interleukin-2 and its alpha, beta, and gamma(c) receptors.
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Rickert M, Boulanger MJ, Goriatcheva N, and Garcia KC
- Subjects
- Calorimetry, Interleukin-2 chemistry, Macromolecular Substances, Models, Molecular, Protein Binding, Protein Isoforms chemistry, Protein Structure, Quaternary, Receptors, Interleukin-2 chemistry, Thermodynamics, Interleukin-2 metabolism, Protein Isoforms metabolism, Receptors, Interleukin-2 metabolism, Signal Transduction physiology
- Abstract
Interleukin-2 is a key immuno-regulatory cytokine whose actions are mediated by three different cell surface receptors: the alpha, beta and the "common gamma" (gamma(c)) chains. We have undertaken a complete thermodynamic characterization of the stepwise assembly cycle for multiple possible combinations of the receptor-ligand, and receptor-receptor interactions that are necessary for formation of the high-affinity IL-2/alphabetagamma(c) signaling complex. We find an entropically favorable high affinity interaction between IL-2 and its alpha receptor, a moderately entropically favorable low affinity interaction between IL-2 and its beta receptor, and no interaction between IL-2 and the shared receptor, gamma(c). Formation of the stable intermediate trimolecular complexes of IL-2 with alpha and beta receptors, as well as IL-2 with beta and gamma(c) receptors proceeds through enthalpy-entropy compensation mechanisms. Surprisingly, we see a moderate affinity interaction between the unliganded receptor alpha and beta chains, suggesting that a preformed alphabeta complex may serve as the initial interaction complex for IL-2. Reconstitution of the IL-2/Ralphabetagamma(c) high-affinity quaternary signaling complex shows it to be assembled through cooperative energetics to form a 1:1:1:1 assembly. Collectively, the favorable entropy of the bimolecular interactions appears to be offset by the loss in rigid body entropy of the receptor components in the higher-order complexes, but overcome by the formation of increasingly enthalpically favorable composite interfaces. This enthalpic mechanism utilized by gamma(c) contrasts with the favorable entropic mechanism utilized by gp130 for degenerate cytokine interaction. In conclusion, we find that several energetically redundant pathways exist for formation of IL-2 receptor signaling complexes, suggesting a more complex equilibrium on the cell surface than has been previously appreciated.
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- 2004
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138. Directing the mode of nitrite binding to a copper-containing nitrite reductase from Alcaligenes faecalis S-6: characterization of an active site isoleucine.
- Author
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Boulanger MJ and Murphy ME
- Subjects
- Alcaligenes genetics, Amino Acid Sequence, Binding Sites, Copper chemistry, Copper metabolism, Crystallography, X-Ray, Isoleucine genetics, Ligands, Models, Molecular, Molecular Sequence Data, Mutagenesis, Nitrite Reductases genetics, Nitrites chemistry, Protein Conformation, Structure-Activity Relationship, Alcaligenes enzymology, Isoleucine metabolism, Nitrite Reductases chemistry, Nitrite Reductases metabolism, Nitrites metabolism
- Abstract
Unlike the heme cd(1)-based nitrite reductase enzymes, the molecular mechanism of copper-containing nitrite reductases remains controversial. A key source of controversy is the productive binding mode of nitrite in the active site. To identify and characterize the molecular determinants associated with nitrite binding, we applied a combinatorial mutagenesis approach to generate a small library of six variants at position 257 in nitrite reductase from Alcaligenes faecalis S-6. The activities of these six variants span nearly two orders of magnitude with one variant, I257V, the only observed natural substitution for Ile257, showing greater activity than the native enzyme. High-resolution (> 1.8 A) nitrite-soaked crystal structures of these variants display different modes of nitrite binding that correlate well with the altered activities. These studies identify for the first time that the highly conserved Ile257 in the native enzyme is a key molecular determinant in directing a catalytically competent mode of nitrite binding in the active site. The O-coordinate bidentate binding mode of nitrite observed in native and mutant forms with high activity supports a catalytic model distinct from the heme cd(1) NiRs. (The atomic coordinates for I257V[NO(2)(-)], I257L[NO(2)(-)], I257A[NO(2)(-)], I257T[NO(2)(-)], I257M[NO(2)(-)] and I257G[NO(2)(-)] AfNiR have been deposited in the Protein Data Bank [PDB identification codes are listed in Table 2].)
- Published
- 2003
- Full Text
- View/download PDF
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