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      Crystal Structure of the Complex mAb 17.2 and the C-Terminal Region of Trypanosoma cruzi P2β Protein: Implications in Cross-Reactivity

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          Abstract

          Patients with Chronic Chagas' Heart Disease possess high levels of antibodies against the carboxyl-terminal end of the ribosomal P2ß protein of Trypanosoma cruzi (TcP2ß). These antibodies, as well as the murine monoclonal antibody (mAb) 17.2, recognize the last 13 amino acids of TcP2ß (called the R13 epitope: EEEDDDMGFGLFD) and are able to cross-react with, and stimulate, the ß1 adrenergic receptor (ß1-AR). Indeed, the mAb 17.2 was able to specifically detect human β1-AR, stably transfected into HEK cells, by flow cytometry and to induce repolarisation abnormalities and first degree atrioventricular conduction block after passive transfer to naïve mice. To study the structural basis of this cross-reactivity, we determined the crystal structure of the Fab region of the mAb 17.2 alone at 2.31 Å resolution and in complex with the R13 peptide at 1.89 Å resolution. We identified as key contact residues on R13 peptide Glu3, Asp6 and Phe9 as was previously shown by alanine scanning. Additionally, we generated a model of human β1-AR to elucidate the interaction with anti-R13 antibodies. These data provide an understanding of the molecular basis of cross-reactive antibodies induced by chronic infection with Trypanosoma cruzi.

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          Trypanosoma cruzi is a protozoan parasite responsible for Chagas disease. Chronic Chagas' heart disease (cChHD) is not only the most frequent and severe consequence of the chronic infection by T. cruzi, but is also the main cause of cardiomyopathy in South and Central America. Patients with cChHD possess high levels of antibodies against the carboxyl-terminal tail of the ribosomal P proteins of T. cruzi (called the R13 epitope). These antibodies, as well as the murine monoclonal antibody (mAb) 17.2, are able to cross-react with, and stimulate, the ß1 adrenergic receptor (ß1-AR). Indeed, the mAb 17.2 was able to specifically detect human β1-AR and induce some of the classical cardiac symptoms after passive transfer to mice. To study the structural basis of this cross-reactivity, we determined the crystal structure of the Fab region of the mAb 17.2 alone and in complex with R13. Additionally, we generated a model of human β1-AR to elucidate the interaction with anti-R13 antibodies in order to understand the molecular basis of cross-reactive antibodies induced by chronic infection with T. cruzi.

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          Shape complementarity at protein/protein interfaces.

          A new statistic Sc, which has a number of advantages over other measures of packing, is used to examine the shape complementarity of protein/protein interfaces selected from the Brookhaven Protein Data Bank. It is shown using Sc that antibody/antigen interfaces as a whole exhibit poorer shape complementarity than is observed in other systems involving protein/protein interactions. This result can be understood in terms of the fundamentally different evolutionary history of particular antibody/antigen associations compared to other systems considered, and in terms of the differing chemical natures of the interfaces.
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            Pathogenesis of chronic Chagas heart disease.

            Chagas disease remains a significant public health issue and a major cause of morbidity and mortality in Latin America. Despite nearly 1 century of research, the pathogenesis of chronic Chagas cardiomyopathy is incompletely understood, the most intriguing challenge of which is the complex host-parasite interaction. A systematic review of the literature found in MEDLINE, EMBASE, BIREME, LILACS, and SCIELO was performed to search for relevant references on pathogenesis and pathophysiology of Chagas disease. Evidence from studies in animal models and in anima nobile points to 4 main pathogenetic mechanisms to explain the development of chronic Chagas heart disease: autonomic nervous system derangements, microvascular disturbances, parasite-dependent myocardial aggression, and immune-mediated myocardial injury. Despite its prominent peculiarities, the role of autonomic derangements and microcirculatory disturbances is probably ancillary among causes of chronic myocardial damage. The pathogenesis of chronic Chagas heart disease is dependent on a low-grade but incessant systemic infection with documented immune-adverse reaction. Parasite persistence and immunological mechanisms are inextricably related in the myocardial aggression in the chronic phase of Chagas heart disease. Most clinical studies have been performed in very small number of patients. Future research should explore the clinical potential implications and therapeutic opportunities of these 2 fundamental underlying pathogenetic mechanisms.
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              The structural basis for agonist and partial agonist action on a β1-adrenergic receptor

              β-Adrenergic receptors (βARs) are G protein-coupled receptors (GPCRs) that activate intracellular G proteins upon binding catecholamine agonist ligands such as adrenaline and noradrenaline1,2. Synthetic ligands have been developed that either activate or inhibit βARs for the treatment of asthma, hypertension or cardiac dysfunction. These ligands are classified as either full agonists, partial agonists or antagonists, depending on whether the cellular response is similar to that of the native ligand, reduced or inhibited, respectively. However, the structural basis for these different ligand efficacies is unknown. Here we present four crystal structures of the thermostabilised turkey (Meleagris gallopavo) β1-adrenergic receptor (β1AR-m23) bound to the full agonists carmoterol and isoprenaline and the partial agonists salbutamol and dobutamine. In each case, agonist binding induces a 1 Å contraction of the catecholamine binding pocket relative to the antagonist bound receptor. Full agonists can form hydrogen bonds with two conserved serine residues in transmembrane helix 5 (Ser5.42 and Ser5.46), but partial agonists only interact with Ser5.42 (superscripts refer to Ballesteros-Weinstein numbering3). The structures provide an understanding of the pharmacological differences between different ligand classes, illuminating how GPCRs function and providing a solid foundation for the structure-based design of novel ligands with predictable efficacies. Determining how agonists and antagonists bind to the β receptors has been the goal of research for more than 20 years4-11. Although the structures of the homologous β1 and β2 receptors12-15 show how some antagonists bind to receptors in the inactive state16, structures with agonists bound are required to understand subsequent structural transitions involved in activation. GPCRs exist in an equilibrium between an inactive state (R) and an activated state (R*) that can couple and activate G proteins17. The binding of a full agonist, such as adrenaline or noradrenaline, is thought to increase the probability of the receptor converting to R*, with a conformation similar to that of opsin18,19. In the absence of any ligand, the βARs exhibit a low level of constitutive activity, indicating that there is always a small proportion of the receptor in the activated state, with the β2AR showing a 5-fold higher level of basal activity than the β1AR20. Basal activity of β2AR is important physiologically, as shown by the T164I4.56 human polymorphism that reduces the basal activity of β2AR to levels similar to β1AR21 and whose expression has been associated with heart disease22. As a first step towards understanding how agonists activate receptors, we have determined the structures of β1AR bound to 4 different agonists. Native turkey β1AR is unstable in detergent23, so crystallization and structure determination relied on using a thermostabilised construct (β1AR-m23) that contained six point mutations, which dramatically improved its thermostability24. In addition, the thermostabilising mutations altered the equilibrium between R and R*, so that the receptor was preferentially in the R state24. However, it could still couple to G proteins after activation by agonists13 (Supplementary Fig. 1, Supplementary Tables 1-3), although the activation energy barrier is predicted to be considerably higher than for the wild-type receptor25. Here we report structures of β1AR-m23 (see Methods) bound to r-isoprenaline (2.85 Å resolution), r,r-carmoterol (2.6 Å resolution), r-salbutamol (3.05 Å resolution) and r-dobutamine (two independent structures at 2.6 Å and 2.5 Å resolution) (Supplementary Table 5). The overall structures of β1AR-m23 bound to the agonists are very similar to the structure with the bound antagonist cyanopindolol13, as expected for a receptor mutant stabilised preferentially in the R state. None of the structures show the outward movement of the cytoplasmic end of transmembrane helix H6 by 5-6 Å that is observed during light activation of rhodopsin18,19,26. This suggests that the structures represent an inactive, non-signaling state of the receptor formed on initial agonist binding. All four agonists bind in the catecholamine pocket in a virtually identical fashion (Fig. 1). The secondary amine and β-hydroxyl groups shared by all the agonists (except for dobutamine, which lacks the β-hydroxyl; see Supplementary Figure 4) form potential hydrogen bonds with Asp1213.32 and Asn3297.39, while the hydrogen bond donor/acceptor group equivalent to the catecholamine meta-hydroxyl (m-OH) generally forms a hydrogen bond with Asn3106.55. In addition, all the agonists can form a hydrogen bond with Ser2115.42, as seen for cyanopindolol13, and they also induce the rotamer conformation change of Ser2125.43 so that it makes a hydrogen bond with Asn3106.55. The major difference between the binding of full agonists compared to the partial agonists is that only full agonists make a hydrogen bond to the side chain of Ser2155.46 as a result of a change in side chain rotamer. All of these amino acid residues involved in the binding of the catecholamine headgroups to β1AR are fully conserved in both β1 and β2 receptors (Fig. 2). Furthermore, the role of many of these amino acid residues in ligand binding is supported by extensive mutagenesis studies on β2AR that were performed before the first β2AR structure was determined27. Thus it was predicted that Asp1133.32, Ser2035.42, Ser2075.46, Asn2936.55 and Asn3127.39 in β2AR were all involved in agonist binding4,5,7-9 (Fig. 3). Inspection of the region outside the catecholamine binding pocket in the structures with bound dobutamine and carmoterol allows the identification of non-conserved residues that interact with these ligands (Fig. 2 and Supplementary Figure 7), which may contribute to the subtype specificity of these ligands10,28. There are three significant differences in the β1AR catecholamine binding pocket when full agonists are bound compared to when an antagonist is bound, namely the rotamer conformation changes of side chains Ser2125.43 and Ser2155.46 (Fig.3) and the contraction of the catecholamine binding pocket by ~1 Å, as measured between the Cα atoms of Asn3297.39 and Ser2115.42 (Fig. 4). So why should these small changes increase the probability of R* formation? Agonist binding has not changed the conformation of transmembrane helix H5 below Ser2155.46, although significant changes in this region are predicted once the receptor has reached the fully activated state18,19. The only effect that the agonist-induced rotamer conformation change of Ser2155.46 appears to have is to break the van der Waals interaction between Val1724.56 and Ser2155.46, thus reducing the number of interactions between H4 and H5. As there is only a minimal interface between transmembrane helices H4 and H5 in this region (Supplementary Table 8 and Supplementary Fig. 8), this loss of interaction may be significant in the activation process. In this regard, it is noteworthy that the naturally occurring polymorphism in β2AR at the H4-H5 interface, T164I4.56, converts a polar residue to a hydrophobic residue as seen in β1AR (Val1724.56), which results in both reduced basal activity and reduced agonist stimulation21. This supports the hypothesis that the extent of interaction between H4 and H5 could affect the probability of a receptor transition into the activated state. In contrast to the apparent weakening of helix-helix interactions by the agonist-induced rotamer conformation change of Ser2155.46, the agonist-induced rotamer conformation change of Ser2125.43 probably results in the strengthening of interactions between H5 and H6. Upon agonist binding, Ser2125.43 forms a hydrogen bond with Asn3106.55 (Fig. 3) and, in addition, hydrogen bond interactions to Ser2115.43 and Asn3106.55mediated by the ligand serve to bridge H5 and H6. The combined effects of strengthening the H5-H6 interface and weakening the H4-H5 interface could facilitate the subsequent movements of H5 and H6, as observed in the activation of rhodopsin. Stabilisation of the contracted catecholamine binding pocket is probably the most important role of bound agonists in the activation process (Fig. 4). This probably requires strong hydrogen bonding interactions between the catechol (or equivalent) moiety and both H5 and H6, and strong interactions between the secondary amine and β-hydroxyl groups in the agonist and the amino acid side chains in helices H3 and H7. Reduction in the strength of these interactions is likely to reduce the efficacy of a ligand29. Both salbutamol and dobutamine are partial agonists of β1AR-m23 (Supplementary Table 3) and human β1AR. In the case of salbutamol, there are only two predicted hydrogen bonds between the headgroup and H5/H6, compared to 3-4 potential hydrogen bonds for isoprenaline and carmoterol. Dobutamine lacks the β-hydroxyl group, which similarly reduces the number of potential hydrogen bonds to H3/H7 from 3-4 seen in the other agonists to only 2. We propose that this weakening of agonist interactions with H5/H6 for salbutamol and H3/H7 for dobutamine is a major contributing factor in making these ligands partial agonists rather than full agonists. The agonist-bound structures of β1AR suggest there are three major determinants that dictate the efficacy of any ligand; ligand-induced rotamer conformational changes of (i) Ser2125.43 and (ii) Ser2155.46 and (iii) stabilization of the contracted ligand binding pocket. The full agonists studied here achieve all three. The partial agonists studied here do not alter the conformation of Ser2155.46 and may be less successful than isoprenaline or carmoterol at stabilizing the contracted catecholamine binding pocket due to reduced numbers of hydrogen bonds between the ligand and the receptor. The antagonist cyanopindolol acts as a very weak partial agonist and none of the three agonist-induced changes are observed. In contrast to partial agonists, neutral antagonists or very weak partial agonists such as cyanopindolol may also have a reduced ability to contract the binding pocket due to the greater distance between the secondary amine and the catechol moiety (or equivalent). For example, the number of atoms in the linker between the secondary amine and the headgroup of cyanopindolol is 4 whereas the agonists in this study only contain 2 (Fig. 1 and Supplementary Fig. 4). A ligand with a sufficiently bulky headgroup that binds with high-affinity and which actively prevents any spontaneous contraction of the binding pocket and/or Ser5.46 rotamer change, would be predicted to act as a full inverse agonist. This is indeed what is observed in the recently determined structure15 of β2AR bound to the inverse agonist ICI 118,551. The significant structural similarities amongst GPCRs suggests that similar agonist-induced conformational changes to those we have observed here may also be applicable to many other members of the GPCR superfamily, though undoubtedly there will be many subtle variations on this theme. METHODS SUMMARY Expression, purification and crystallization The β44-m23 construct was expressed in insect cells, purified in the detergent Hega-10 and crystallized in the presence of cholesterol hemisuccinate (CHS), following previously established protocols30. Crystals were grown by vapour diffusion, with the conditions shown in Supplementary Table 4. Data collection, structure solution and refinement Diffraction data were collected from a single cryo-cooled crystal (100 K) of each complex in multiple wedges at beamline ID23-2 at ESRF, Grenoble. The structures were solved by molecular replacement using the β1AR structure13 (PDB code 2VT4) as a model (see Online Methods). Data collection and refinement statistics are presented in Supplementary Table 5. Supplementary Material 1 2
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                plos
                plosntds
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, USA )
                1935-2727
                1935-2735
                November 2011
                1 November 2011
                : 5
                : 11
                : e1375
                Affiliations
                [1 ]Unité d'Immunologie Structurale, Institut Pasteur, Paris, France
                [2 ]Centre National de la Recherche Scientifique, Unité de Recherche Associée 2185, Paris, France
                [3 ]Laboratorio de Biología Molecular de la Enfermedad de Chagas, INGEBI-CONICET, Buenos Aires, Argentina
                [4 ]UPR9021 du CNRS, Strasbourg, France
                [5 ]Institut Pasteur, Paris, France
                National Institutes of Health, United States of America
                Author notes

                ¤: Current address: Structural Genomic Consortium (SGC), University of Toronto, Toronto, Canada

                Conceived and designed the experiments: GAB MJL MH CRS. Performed the experiments: JCP GB GAB KAG MH CRS. Analyzed the data: JCP GAB KAG JH CRS. Contributed reagents/materials/analysis tools: KAG MJL MH GAB CRS. Wrote the paper: JCP GAB MH CRS.

                Article
                PNTD-D-11-00600
                10.1371/journal.pntd.0001375
                3206007
                22069505
                78967d20-f0ac-46b6-9d9a-18a0a57714bc
                Pizarro et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 18 June 2011
                : 11 September 2011
                Page count
                Pages: 10
                Categories
                Research Article
                Biology
                Computational Biology
                Macromolecular Structure Analysis
                Protein Structure
                Immunology
                Immunity
                Immunity to Infections
                Immunoglobulins
                Microbiology
                Parasitology
                Medicine
                Cardiovascular
                Cardiomyopathies

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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