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X-Ray Structure Determination Of A Dimeric Hemoglobin From Caudina arenicola
Full text linkThe X-ray structure of a dimeric, cyanomet-liganded hemoglobin D-chain (Hb-D) from Caudina arenicola has been determined by the molecular-replacement method. The search model was a concatenated model of three hemoglobin structures superimposed on the backbone of monomeric, hemichrome hemoglobin C-chain (Hb-C) from the same organism. Hb-D crystallizes in space group P4(1)2(1)2 with cell constants a = b = 77.0 and c = 61.5 Angstrom with one subunit in the asymmetric unit. The dimer twofold axis corresponds to a crystallographic twofold along one of the body diagonals of the unit cell. Rotation and translation searches as well as model refinement were carried out in X-PLOR with the final model having an R value of 0.19 using the data from 5.0 to 2.9 Angstrom resolution (R = 0.26 for 10.0 to 2.9 Angstrom resolution). The homodimeric structure of Caudina Hb-D features close heme-heme contacts with an Fe-Fe distance of 19.0 Angstrom. The subunit-subunit interface involves both the E and F helices from each subunit with the E helices oriented antiparallel at 50 degrees with respect to one another, similar to the quaternary structure observed for the homodimeric hemoglobin from Scapharca inaequivalvis.NIH GM 30105Robert A. Welch FoundationChemistryBiochemistr
3-Dimensional Structure Of A Hemichrome Hemoglobin From Caudina arenicola
No full textThe structure of a monomeric hemichrome form of an invertebrate hemoglobin, Hb-C chain, from Caudina arenicola has been refined to an R value of 0.16 using the data from 5.0 to 2.5 Angstrom resolution (R = 0.21 from 10.0 to 2.5 Angstrom resolution). Hb-C crystallizes in space group P2(1) with cell constants a=45.74, b=45.23 and c=40.92 Angstrom and beta=104.4 degrees with two monomers packed in the unit cell (V-m = 2.34 Angstrom(3) Da(-1)). The phases were determined by the multiple isomorphous replacement method with Hg2+ the major derivative. The structure consists of 157 amino acids with N- and C-terminal regions and eight a-helices forming a heme pocket. The unique feature of this structure is the hemichrome form with the proximal and distal histidines coordinated to the heme Fe atom, which is nearly in the plane of the porphyrin ring. A total of 111 solvent molecules were added to the structure using difference density peaks of at least 3 sigma over background. Interestingly, all the heme groups present in the crystal are nearly coplanar.NIH GM 30105Robert A. Welch FoundationFoundation for ResearchChemistryBiochemistr
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The 2.7 Å resolution structure of the catalytic domain of the dihydrolipoamide succinyltransferase from Escherichia coli in complex with coenzyme A and the 1.45 Å resolution structure of murine macrophage migration inhibitory factor in complex with phenylacetylenepyruvate
No full textThe work presented in this dissertation includes structural analysis of
liganded forms of the truncated catalytic domain of the trimeric form of the
dihydrolipoamide succinyltransferase from E. coli (E2oCD) and the macrophage
migration inhibitory factor (MIF) from mouse. Part one of this dissertation deals with the analysis of the 2.7 Å resolution
structure of E2oCD in complex with coenzyme A. The structure of the native
E2oCD was solved in this laboratory previously. This project resulted in the
crystal structure of the E2oCD·CoA complex obtained by molecular replacement
and the resulting analysis of the chemical nature of the complex. Extensive
comparisons were made with the previously reported structure of E2p from A.
vinelandii, in complex with CoA and DHLA, as well as with the predicted ligand
location based on computer models for E2oCD. Residues in the active site cleft
are implicated in providing substrate-binding specificity. CoA is observed in the
extended “IN” conformation, consistent with the unoccupied DHLA binding site.
The second project reported here involves the study of MIF, a cytokine
with unusual tautomerase activity. MIF is an important immunoregulatory protein
with a potential to mediate inflammatory diseases. The details of the tautomerase
activity MIF and its importance in MIF's biological functions remain elusive. This
project resulted in a high-resolution X-ray structure of mouse MIF at 1.45 Å
resolution in complex with an inhibitor phenylacetylenepyruvate (PAP). Well-defined electron density for Glu-16’ from a neighboring trimer was observed in
one of the three active sites, with density for an inhibitor covalently bound to Pro-1 observed in the other two active sites. An analysis of the apparent covalent bond
between the protein and the inhibitor and the potential ramifications for the understanding of the chemistry of formation of the adduct are discussed. The
observed electron density was not consistent with the predicted Michael addition
product.Chemistry and BiochemistryBiochemistr
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Structural studies of Gαq signaling and regulation
Full text linktextGαq signaling is implicated in a number of physiological processes that include platelet activation, cardiovascular development and smooth muscle function. Historically, Gαq is known to function by activating its effector, phospholipase Cβ. Desensitization of Gαq signaling is mediated by G-protein coupled receptor kinases (GRK) such as GRK2 that phosphorylates the activated receptor and also sequesters activated Gαq and Gβγ subunits. Our crystal structure of Gαq-GRK2-Gβγ complex shows that Gαq forms effector-like interactions with the regulator of G-protein signaling (RGS) homology domain of GRK2 involving the classic effector-binding site of Gα subunits, raising the question if GRK2 can itself be a Gáq effector and initiate its own signaling cascade. In the structure, Gα and Gβγ subunits are completely dissociated from one another and the orientation of activated Gαq with respect to the predicted cell membrane is drastically different from its position in the inactive Gαβγ heterotrimer. Recent studies have identified a novel Gαq effector, p63RhoGEF that activates RhoA. Our crystal structure of the Gαq-p63RhoGEF-RhoA complex reveals that Gαq interacts with both the Dbl homology (DH) and pleckstrin homology (PH) domains of p63RhoGEF with its C-terminal helix and its effector-binding site, respectively. The structure predicts that Gαq relieves auto-inhibition of the catalytic DH domain by the PH domain. We show that Gαq activates p63RhoGEF-related family members, Trio and Kalirin, revealing several conduits by which RhoA is activated in response to Gq-coupled receptors. The Gαq effector-site interaction with p63RhoGEF/GRK2 does not overlap with the Gαq-binding site of RGS2/RGS4 that function as GTPase activating proteins (GAPs). This suggests that activated G proteins, effectors, RGS proteins, and activated receptors can form high-order complexes at the cell membrane. We confirmed the formation of RGS-Gαq-effector complexes and our results suggest that signaling pathways initiated by GRK2 and p63RhoGEF are regulated by RGS proteins via both allosteric and GAP mechanisms. Our structural studies of Gαq signaling provide insight into protein-protein interactions that induce profound physiological changes. Understanding such protein interfaces is a key step towards structure-based drug design that can be targeted to treat diseases concerned with impaired Gαq signaling.Cellular and Molecular Biolog
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Different nucleotide-binding states and implications for mechanism of activation of a G protein coupled receptor kinase
Full text linkG protein coupled receptor (GPCR) kinases (GRKs) phosphorylate activated heptahelical receptors, leading to their uncoupling from G proteins and downregulation. The desensitization of GPCRs is critical to render cells responsive to further stimuli and if not regulated can result in many pathophysiological processes including heart abnormalities and hypertension. How GRKs recognize and are activated by GPCRs are not known, in part because the critical N-terminus and the kinase C-terminal extension were not resolved in GRK2 and GRK6 structures. The long-term goal of this project was to address this question by structural analysis of rhodopsin kinase (also known as GRK1), which represents a model system for studying phosphorylation-dependent desensitization of activated GPCRs. Herein we report structures of GRK1 from six crystal forms that represent three distinct nucleotide-ligand binding states. One of the (Mg²⁺)₂·ADP·GRK1 structures is the most high-resolution structure (1.85 Å) of a GRK to date. In one (Mg²⁺)₂·ATP·GRK1 structure, almost the entire N-terminal region (residues 5-30) is observed. In addition, different segments of the kinase C-terminal extension are ordered in the various nucleotide-bound structures. Together, these two elements form a putative receptor-docking site adjacent to the hinge of the kinase domain. Based on these structures, a model is proposed for how GRK1 interacts with activated rhodopsin and how rhodopsin binding in turn could activate the kinase. Two novel phosphorylation sites were also identified at the N-terminus. The physiological role of phosphorylation sites and the extensive dimerization interface mediated by the regulator of G protein signaling (RGS) homology domain of GRK1 was assessed using site-directed mutagenesis. In addition to mediating interaction with activated GPCRs, the N-terminus of GRKs also forms a binding site for calcium sensing proteins. Although its physiological significance is debated, the structures of these complexes could lend further insights into the conformation of the N-terminus of GRKs. The second chapter deals with attempts to isolate Ca²⁺·recoverin·GRK1 and Ca²⁺·calmodulin·GRK6 complexes. Finally, the RH domain of GRK2 binds G[alpha subscript q], G[alpha]₁₁, and G[alpha]₁₄ subunits thereby blocking their interactions with the downstream effectors. The third chapter involves attempts to isolate a complex of GRK6 and G[alpha]₁₆, a member of G[alpha subscript q] family.Institute for Cellular and Molecular Biolog
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Structure based design of a ricin antidote
Full text linktextRicin is a potent cytotoxin easily purified in large quantities. It presents a significant public health concern due to its potential use as a bioterrorism agent. For this reason, extensive efforts have been underway to develop antidotes against this deadly poison. The catalytic A subunit of the heterodimeric toxin has been biochemically and structurally well characterized, and is an attractive target for structure-based drug design. Aided by computer docking simulations, several ricin toxin A chain (RTA) inhibitors have been identified; the most promising leads belonging to the pterin family. To date, the most potent RTA inhibitors developed using this approach are only modest inhibitors with apparent IC50 values in the 10-4 M range, leaving significant room for improvement. This thesis discusses the development of a subset of inhibitors belonging to the pterin family in which amino acids have been utilized as building blocks. Inhibitors in this family have achieved a significant increase in potency, and have provided valuable structural information for further development.Cellular and Molecular Biolog
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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