The T cell immunoglobulin and mucin domain 3 (Tim-3) is a plasma membrane-associated receptor whi... more The T cell immunoglobulin and mucin domain 3 (Tim-3) is a plasma membrane-associated receptor which is involved in a variety of biological responses in human immune cells. It is highly expressed in most acute myeloid leukaemia (AML) cells and therefore may serve as a possible target for AML therapy. However, its biochemical activities in primary human AML cells remain unclear. We therefore analysed the total expression and surface presence of the Tim-3 receptor in primary human AML blasts and healthy primary human leukocytes isolated from human blood. We found that Tim-3 expression was significantly higher in primary AML cells compared to primary healthy leukocytes. Tim-3 receptor molecules were distributed largely on the surface of primary AML cells, whereas in healthy leukocytes Tim-3 protein was mainly expressed intracellularly. In primary human AML blasts, both Tim-3 agonistic antibody and galectin-9 (a Tim-3 natural ligand) significantly upregulated mTOR pathway activity. This ...
Bovine [3-LG ([3-1actoglobulin) has been studied under a variety of solution conditions by one-an... more Bovine [3-LG ([3-1actoglobulin) has been studied under a variety of solution conditions by one-and two-dimensional NMR spectroscopy. At highly acidic pH (pH = 2) and low ionic strength the protein is present in a monomeric form, exhibiting a highly structured [~-sheet core and less ordered regions as evidenced by both CD data and the NOESY spectra. Marginal protection was observed for most of the amide protons as a result of high conformational mobility. This structural state of [3-LG may be considered as an attractive model for a partially folded structure occurring late in the folding process of the protein.
Proceedings of the National Academy of Sciences, 2015
Middle East Respiratory Syndrome (MERS) is a highly lethal pulmonary infection caused by a previo... more Middle East Respiratory Syndrome (MERS) is a highly lethal pulmonary infection caused by a previously unidentified coronavirus (CoV), likely transmitted to humans by infected camels. There is no licensed vaccine or antiviral for MERS, therefore new prophylactic and therapeutic strategies to combat human infections are needed. In this study, we describe, for the first time, to our knowledge, the isolation of a potent MERS-CoV-neutralizing antibody from memory B cells of an infected individual. The antibody, named LCA60, binds to a novel site on the spike protein and potently neutralizes infection of multiple MERS-CoV isolates by interfering with the binding to the cellular receptor CD26. Importantly, using mice transduced with adenovirus expressing human CD26 and infected with MERS-CoV, we show that LCA60 can effectively protect in both prophylactic and postexposure settings. This antibody can be used for prophylaxis, for postexposure prophylaxis of individuals at risk, or for the treatment of human cases of MERS-CoV infection. The fact that it took only 4 mo from the initial screening of B cells derived from a convalescent patient for the development of a stable chinese hamster ovary (CHO) cell line producing neutralizing antibodies at more than 5 g/L provides an example of a rapid pathway toward the generation of effective antiviral therapies against emerging viruses.
The diversity of RNA functions ranges from storage and propagation of genetic information to enzy... more The diversity of RNA functions ranges from storage and propagation of genetic information to enzymatic activity during RNA processing and protein synthesis. This diversity of functions requires an equally diverse arrays of structures, and, very often, the formation of functional RNA-protein complexes. Recognition of specific RNA signals by RNA-binding proteins is central to all aspects of post-transcriptional regulation of gene expression. We will describe how NMR is being used to understand at the atomic level how these important biological processes occur.
To date, different kinds of biosensing elements have been used effectively for environmental moni... more To date, different kinds of biosensing elements have been used effectively for environmental monitoring. Microbial cells seem to be well-suited for this task: they are cheap, adaptable to variable field conditions and give a measurable response to a broad number of chemicals. Among different pollutants, heavy metals are still a major problem for the environment. A reasonable starting point for the selection of a biorecognition element to develop a biosensor for metals could be that of a microorganism that exhibits good mechanisms to cope with metals. Pseudomonads are characterized by the secretion of siderophores (e.g., pyoverdine), low-molecular weight compounds that chelate Fe 3+ during iron starvation. Pyoverdine is easily detected by colorimetric assay, and it is suitable for simple online measurements. In this work, in order to evaluate pyoverdine as a biorecognition element for metal detection, the influence of metal ions (Fe 3+ , Cu 2+ , Zn 2+ ), but also of temperature, pH and nutrients, on microbial growth and pyoverdine regulation has been studied in P. fluorescens. Each of these variables has been shown to influence the synthesis of siderophore: for instance, the lower the temperature, the higher the production of pyoverdine. Moreover, the concentration of pyoverdine produced in the presence of metals has been compared with the maximum allowable concentrations indicated in international regulations (e.g., 98/83/EC), and a correlation that could be useful to build a colorimetric biosensor has been observed. OPEN ACCESS Biosensors 2013, 3 386
The International Journal of Biochemistry & Cell Biology, 2015
The T-cell immunoglobulin and mucin domain 3 (Tim-3) is a plasma membrane-associated protein that... more The T-cell immunoglobulin and mucin domain 3 (Tim-3) is a plasma membrane-associated protein that is highly expressed in human acute myeloid leukaemia cells. As an acute myeloid leukaemia antigen, it could therefore be considered as a potential target for immune therapy and highly-specific drug delivery. However, a conceptual understanding of its biological role is required before consideration of this protein for therapeutic settings. Here, we reveal the detailed mechanism of action underlying the biological responses mediated by the Tim-3 receptor in myeloid cells. Our studies demonstrate that Tim-3 triggers growth factor type responses in acute myeloid leukaemia cells by activating a phosphatidylinositol-3 kinase (PI-3K)/mammalian target of rapamycin (mTOR) pathway. In addition, the receptor activates hypoxic signalling pathways upregulating glycolysis and pro-angiogenic responses. These findings suggest that Tim-3 could be used as a potential therapeutic target for immune therapy and drug delivery in human acute myeloid leukaemia cells.
Potential crosslinks between inflammation and leukaemia have been discussed for some time, but ex... more Potential crosslinks between inflammation and leukaemia have been discussed for some time, but experimental evidence to support this dogma is scarce. In particular, it is important to understand the mechanisms responsible for potential upregulation of proto-oncogenic growth factor expressions by inflammatory mediators. Here, we investigated the ability of the highly inflammatory cytokine interleukin-1 beta (IL-1β) to induce the production of stem cell factor (SCF), which is a major hematopoietic growth factor that controls the progression of acute myeloid leukaemia upon malignant transformation of haematopoietic myeloid cells. We found that human IL-1β induced the expression/secretion of SCF in MCF-7 human epithelial breast cancer cells and that this process depended on the hypoxia-inducible factor 1 (HIF-1) transcription complex. We also demonstrated a crucial role of the phosphatidylinositol-3 kinase (PI-3K)/mammalian target of rapamycin (mTOR) pathway in IL-1β-induced HIF-1α accumulation in MCF-7 cells. Importantly, mTOR was also found to play a role in IL-1β-induced SCF production. Furthermore, a tendency for a positive correlation of IL-1β and SCF levels in the plasma of healthy human donors was observed. Altogether, our results demonstrate that IL-1β, which normally bridges innate and adaptive immunity, induces the production of the major haematopoietic/proleukaemic growth factor SCF through the PI-3K/mTOR pathway and the HIF-1 transcription complex. These findings strongly support a cross-talk between inflammation and acute myeloid leukaemia.Cellular & Molecular Immunology advance online publication, 24 November 2014; doi:10.1038/cmi.2014.113.
In this manuscript, we modulate the binding properties of estrogen receptor protein by rationally... more In this manuscript, we modulate the binding properties of estrogen receptor protein by rationally modifying the amino acid composition of its ligand binding domain. By combining sequence alignment and structural analysis of known estrogen receptor-ligand complexes with computational analysis, we were able to predict estrogen receptor mutants with altered binding properties. These predictions were experimentally confirmed by producing single point variants with up to an order of magnitude increased binding affinity towards some estrogen disrupting chemicals and reaching an half maximal inhibitory concentration (IC 50 ) value of 2 nM for the 17a-ethinylestradiol ligand. Due to increased affinity and stability, utilizing such mutated estrogen receptor instead of the wild type as bio-recognition element would be beneficial in an assay or biosensor. Citation: Ferrero VEV, Pedotti M, Chiadò A, Simonelli L, Calzolai L, et al. (2014) Rational Modification of Estrogen Receptor by Combination of Computational and Experimental Analysis. PLoS ONE 9(7): e102658.
The main limitation in NMR-determined structures of nucleic acids and their complexes with protei... more The main limitation in NMR-determined structures of nucleic acids and their complexes with proteins derives from the elongated, non-globular nature of physiologically important DNA and RNA molecules. Since it is generally not possible to obtain long-range distance constraints between distinct regions of the structure, long-range properties such as bending or kinking at sites of protein recognition cannot be determined accurately nor precisely. Here we show that use of residual dipolar couplings in the refinement of the structure of a protein-RNA complex improves the definition of the long-range properties of the RNA. These features are often an important aspect of molecular recognition and biological function; therefore, their improved definition is of significant value in RNA structural biology. * Present address: Forschungsstelle für Enzymologie der Proteinfaltung,
Antibodies play an increasingly important role in both basic research and the pharmaceutical indu... more Antibodies play an increasingly important role in both basic research and the pharmaceutical industry. Since their efficiency depends, in ultimate analysis, on their atomic interactions with an antigen, studying such interactions is important to understand how they function and, in the long run, to design new molecules with desired properties. Computational docking, the process of predicting the conformation of a complex from its separated components, is emerging as a fast and affordable technique for the structural characterization of antibody-antigen complexes. In this manuscript, we first describe the different computational strategies for the modeling of antibodies and docking of their complexes, and then predict the binding of two antibodies to the stalk region of influenza hemagglutinin, an important pharmaceutical target. The purpose is two-fold: on a general note, we want to illustrate the advantages and pitfalls of computational docking with a practical example, using different approaches and comparing the results to known experimental structures. On a more specific note, we want to assess if docking can be successful in characterizing the binding to the same influenza epitope of other antibodies with unknown structure, which has practical relevance for pharmaceutical and biological research. The paper clearly shows that some of the computational docking predictions can be very accurate, but the algorithm often fails to discriminate them from inaccurate OPEN ACCESS Int. J. Mol. Sci. 2011, 12 227 solutions. It is of paramount importance, therefore, to use rapidly obtained experimental data to validate the computational results.
Proceedings of the National Academy of Sciences, 2007
T cell receptor (TCR) recognition of peptide-MHC (pMHC) is central to the cellular immune respons... more T cell receptor (TCR) recognition of peptide-MHC (pMHC) is central to the cellular immune response. A large database of TCR-pMHC structures is needed to reveal general structural principles, such as whether the repertoire of TCR/MHC docking modes is dictated by a ''recognition code'' between conserved elements of the TCR and MHC genes. Although Ϸ17 cocrystal structures of unique TCR-pMHC complexes have been determined, cocrystallization of soluble TCR and pMHC remains a major technical obstacle in the field. Here we demonstrate a strategy, based on NMR chemical shift mapping, that permits rapid and reliable analysis of the solution footprint made by a TCR when binding onto the pMHC surface. We mapped the 2C TCR binding interaction with its allogeneic ligand H-2L d -QL9 and identified a group of NMR-shifted residues that delineated a clear surface of the MHC that we defined as the TCR footprint. We subsequently found that the docking footprint described by NMR shifts was highly accurate compared with a recently determined high-resolution crystal structure of the same complex. The same NMR footprint analysis was done on a highaffinity mutant of the TCR. The current work serves as a foundation to explore the molecular dynamics of pMHC complexes and to rapidly determine the footprints of many L d -specific TCRs. chemical shift mapping ͉ dynamics ͉ NMR ͉ cellular immunity ͉ protein-protein interaction
Proceedings of the National Academy of Sciences, 1999
Coding region and intronic mutations in the tau gene cause frontotemporal dementia and parkinsoni... more Coding region and intronic mutations in the tau gene cause frontotemporal dementia and parkinsonism linked to chromosome 17. Intronic mutations and some missense mutations increase splicing in of exon 10, leading to an increased ratio of four-repeat to three-repeat tau isoforms. Secondary structure predictions have led to the proposal that intronic mutations and one missense mutation destabilize a putative RNA stem-loop structure located close to the splicedonor site of the intron after exon 10. We have determined the three-dimensional structure of this tau exon 10 splicing regulatory element RNA by NMR spectroscopy. We show that it forms a stable, folded stem-loop structure whose thermodynamic stability is reduced by frontotemporal dementia and parkinsonism linked to chromosome 17 mutations and increased by compensatory mutations. By exon trapping, the reduction in thermodynamic stability is correlated with increased splicing in of exon 10.
Drug-like molecules that bind RNA with sequence selectivity would provide valuable tools to eluci... more Drug-like molecules that bind RNA with sequence selectivity would provide valuable tools to elucidate gene expression pathways and new avenues to the treatment of degenerative and chronic conditions. Efforts at discovering such agents have been hampered, until recently, by the limited knowledge of RNA recognition principles. Several recent structures of aminoglycoside-RNA complexes have begun to reveal the structural basis for RNA-drug recognition. However, the absence of suitable chemical scaffolds known to bind the RNA major groove, where specificity could be provided by the diversity of functional groups exposed on the RNA bases, has represented a major obstacle. Here we report an investigation of the structural basis for recognition of an RNA stem-loop by neomycin, a naturally occurring aminoglycoside antibiotic. We found that neomycin binds the RNA stem-loop that regulates alternative splicing of exon 10 within the gene coding for human tau protein.
In our recent publication`Changes in side-chains and backbone dynamics identify determinants of s... more In our recent publication`Changes in side-chains and backbone dynamics identify determinants of speci®city in RNA recognition by human U1A protein' (Mittermaier et al., J. Mol. Biol. (1999), 294, p. 967), we provided experimental evidence that ms-ms dynamic processes within loop 3, the most critical determinant of speci®city, are quenched upon RNA binding and therefore important for RNA recognition. The importance of dynamics within loop 3 on RNA recognition was ®rst suggested in another study (Kranz and Hall,
If we understand the structural rules governing antibody (Ab)-antigen (Ag) interactions in a give... more If we understand the structural rules governing antibody (Ab)-antigen (Ag) interactions in a given virus, then we have the molecular basis to attempt to design and synthesize new epitopes to be used as vaccines or optimize the antibodies themselves for passive immunization. Comparing the binding of several different antibodies to related Ags should also further our understanding of general principles of recognition.
Bovine [3-LG ([3-1actoglobulin) has been studied under a variety of solution conditions by one-an... more Bovine [3-LG ([3-1actoglobulin) has been studied under a variety of solution conditions by one-and two-dimensional NMR spectroscopy. At highly acidic pH (pH = 2) and low ionic strength the protein is present in a monomeric form, exhibiting a highly structured [~-sheet core and less ordered regions as evidenced by both CD data and the NOESY spectra. Marginal protection was observed for most of the amide protons as a result of high conformational mobility. This structural state of [3-LG may be considered as an attractive model for a partially folded structure occurring late in the folding process of the protein.
Antibodies protect against homologous Dengue virus (DENV) infection but can precipitate severe de... more Antibodies protect against homologous Dengue virus (DENV) infection but can precipitate severe dengue by promoting heterotypic virus entry via Fcg receptors (FcgR). We immortalized memory B cells from individuals after primary or secondary infection and analyzed anti-DENV monoclonal antibodies (mAbs) thus generated. MAbs to envelope (E) protein domain III (DIII) were either serotype specific or cross-reactive and potently neutralized DENV infection. DI/DII-or viral membrane protein prM-reactive mAbs neutralized poorly and showed broad cross-reactivity with the four DENV serotypes. All mAbs enhanced infection at subneutralizing concentrations. Three mAbs targeting distinct epitopes on the four DENV serotypes and engineered to prevent FcgR binding did not enhance infection and neutralized DENV in vitro and in vivo as postexposure therapy in a mouse model of lethal DENV infection. Our findings reveal an unexpected degree of cross-reactivity in human antibodies against DENV and illustrate the potential for an antibodybased therapy to control severe dengue.
The T cell immunoglobulin and mucin domain 3 (Tim-3) is a plasma membrane-associated receptor whi... more The T cell immunoglobulin and mucin domain 3 (Tim-3) is a plasma membrane-associated receptor which is involved in a variety of biological responses in human immune cells. It is highly expressed in most acute myeloid leukaemia (AML) cells and therefore may serve as a possible target for AML therapy. However, its biochemical activities in primary human AML cells remain unclear. We therefore analysed the total expression and surface presence of the Tim-3 receptor in primary human AML blasts and healthy primary human leukocytes isolated from human blood. We found that Tim-3 expression was significantly higher in primary AML cells compared to primary healthy leukocytes. Tim-3 receptor molecules were distributed largely on the surface of primary AML cells, whereas in healthy leukocytes Tim-3 protein was mainly expressed intracellularly. In primary human AML blasts, both Tim-3 agonistic antibody and galectin-9 (a Tim-3 natural ligand) significantly upregulated mTOR pathway activity. This ...
Bovine [3-LG ([3-1actoglobulin) has been studied under a variety of solution conditions by one-an... more Bovine [3-LG ([3-1actoglobulin) has been studied under a variety of solution conditions by one-and two-dimensional NMR spectroscopy. At highly acidic pH (pH = 2) and low ionic strength the protein is present in a monomeric form, exhibiting a highly structured [~-sheet core and less ordered regions as evidenced by both CD data and the NOESY spectra. Marginal protection was observed for most of the amide protons as a result of high conformational mobility. This structural state of [3-LG may be considered as an attractive model for a partially folded structure occurring late in the folding process of the protein.
Proceedings of the National Academy of Sciences, 2015
Middle East Respiratory Syndrome (MERS) is a highly lethal pulmonary infection caused by a previo... more Middle East Respiratory Syndrome (MERS) is a highly lethal pulmonary infection caused by a previously unidentified coronavirus (CoV), likely transmitted to humans by infected camels. There is no licensed vaccine or antiviral for MERS, therefore new prophylactic and therapeutic strategies to combat human infections are needed. In this study, we describe, for the first time, to our knowledge, the isolation of a potent MERS-CoV-neutralizing antibody from memory B cells of an infected individual. The antibody, named LCA60, binds to a novel site on the spike protein and potently neutralizes infection of multiple MERS-CoV isolates by interfering with the binding to the cellular receptor CD26. Importantly, using mice transduced with adenovirus expressing human CD26 and infected with MERS-CoV, we show that LCA60 can effectively protect in both prophylactic and postexposure settings. This antibody can be used for prophylaxis, for postexposure prophylaxis of individuals at risk, or for the treatment of human cases of MERS-CoV infection. The fact that it took only 4 mo from the initial screening of B cells derived from a convalescent patient for the development of a stable chinese hamster ovary (CHO) cell line producing neutralizing antibodies at more than 5 g/L provides an example of a rapid pathway toward the generation of effective antiviral therapies against emerging viruses.
The diversity of RNA functions ranges from storage and propagation of genetic information to enzy... more The diversity of RNA functions ranges from storage and propagation of genetic information to enzymatic activity during RNA processing and protein synthesis. This diversity of functions requires an equally diverse arrays of structures, and, very often, the formation of functional RNA-protein complexes. Recognition of specific RNA signals by RNA-binding proteins is central to all aspects of post-transcriptional regulation of gene expression. We will describe how NMR is being used to understand at the atomic level how these important biological processes occur.
To date, different kinds of biosensing elements have been used effectively for environmental moni... more To date, different kinds of biosensing elements have been used effectively for environmental monitoring. Microbial cells seem to be well-suited for this task: they are cheap, adaptable to variable field conditions and give a measurable response to a broad number of chemicals. Among different pollutants, heavy metals are still a major problem for the environment. A reasonable starting point for the selection of a biorecognition element to develop a biosensor for metals could be that of a microorganism that exhibits good mechanisms to cope with metals. Pseudomonads are characterized by the secretion of siderophores (e.g., pyoverdine), low-molecular weight compounds that chelate Fe 3+ during iron starvation. Pyoverdine is easily detected by colorimetric assay, and it is suitable for simple online measurements. In this work, in order to evaluate pyoverdine as a biorecognition element for metal detection, the influence of metal ions (Fe 3+ , Cu 2+ , Zn 2+ ), but also of temperature, pH and nutrients, on microbial growth and pyoverdine regulation has been studied in P. fluorescens. Each of these variables has been shown to influence the synthesis of siderophore: for instance, the lower the temperature, the higher the production of pyoverdine. Moreover, the concentration of pyoverdine produced in the presence of metals has been compared with the maximum allowable concentrations indicated in international regulations (e.g., 98/83/EC), and a correlation that could be useful to build a colorimetric biosensor has been observed. OPEN ACCESS Biosensors 2013, 3 386
The International Journal of Biochemistry & Cell Biology, 2015
The T-cell immunoglobulin and mucin domain 3 (Tim-3) is a plasma membrane-associated protein that... more The T-cell immunoglobulin and mucin domain 3 (Tim-3) is a plasma membrane-associated protein that is highly expressed in human acute myeloid leukaemia cells. As an acute myeloid leukaemia antigen, it could therefore be considered as a potential target for immune therapy and highly-specific drug delivery. However, a conceptual understanding of its biological role is required before consideration of this protein for therapeutic settings. Here, we reveal the detailed mechanism of action underlying the biological responses mediated by the Tim-3 receptor in myeloid cells. Our studies demonstrate that Tim-3 triggers growth factor type responses in acute myeloid leukaemia cells by activating a phosphatidylinositol-3 kinase (PI-3K)/mammalian target of rapamycin (mTOR) pathway. In addition, the receptor activates hypoxic signalling pathways upregulating glycolysis and pro-angiogenic responses. These findings suggest that Tim-3 could be used as a potential therapeutic target for immune therapy and drug delivery in human acute myeloid leukaemia cells.
Potential crosslinks between inflammation and leukaemia have been discussed for some time, but ex... more Potential crosslinks between inflammation and leukaemia have been discussed for some time, but experimental evidence to support this dogma is scarce. In particular, it is important to understand the mechanisms responsible for potential upregulation of proto-oncogenic growth factor expressions by inflammatory mediators. Here, we investigated the ability of the highly inflammatory cytokine interleukin-1 beta (IL-1β) to induce the production of stem cell factor (SCF), which is a major hematopoietic growth factor that controls the progression of acute myeloid leukaemia upon malignant transformation of haematopoietic myeloid cells. We found that human IL-1β induced the expression/secretion of SCF in MCF-7 human epithelial breast cancer cells and that this process depended on the hypoxia-inducible factor 1 (HIF-1) transcription complex. We also demonstrated a crucial role of the phosphatidylinositol-3 kinase (PI-3K)/mammalian target of rapamycin (mTOR) pathway in IL-1β-induced HIF-1α accumulation in MCF-7 cells. Importantly, mTOR was also found to play a role in IL-1β-induced SCF production. Furthermore, a tendency for a positive correlation of IL-1β and SCF levels in the plasma of healthy human donors was observed. Altogether, our results demonstrate that IL-1β, which normally bridges innate and adaptive immunity, induces the production of the major haematopoietic/proleukaemic growth factor SCF through the PI-3K/mTOR pathway and the HIF-1 transcription complex. These findings strongly support a cross-talk between inflammation and acute myeloid leukaemia.Cellular & Molecular Immunology advance online publication, 24 November 2014; doi:10.1038/cmi.2014.113.
In this manuscript, we modulate the binding properties of estrogen receptor protein by rationally... more In this manuscript, we modulate the binding properties of estrogen receptor protein by rationally modifying the amino acid composition of its ligand binding domain. By combining sequence alignment and structural analysis of known estrogen receptor-ligand complexes with computational analysis, we were able to predict estrogen receptor mutants with altered binding properties. These predictions were experimentally confirmed by producing single point variants with up to an order of magnitude increased binding affinity towards some estrogen disrupting chemicals and reaching an half maximal inhibitory concentration (IC 50 ) value of 2 nM for the 17a-ethinylestradiol ligand. Due to increased affinity and stability, utilizing such mutated estrogen receptor instead of the wild type as bio-recognition element would be beneficial in an assay or biosensor. Citation: Ferrero VEV, Pedotti M, Chiadò A, Simonelli L, Calzolai L, et al. (2014) Rational Modification of Estrogen Receptor by Combination of Computational and Experimental Analysis. PLoS ONE 9(7): e102658.
The main limitation in NMR-determined structures of nucleic acids and their complexes with protei... more The main limitation in NMR-determined structures of nucleic acids and their complexes with proteins derives from the elongated, non-globular nature of physiologically important DNA and RNA molecules. Since it is generally not possible to obtain long-range distance constraints between distinct regions of the structure, long-range properties such as bending or kinking at sites of protein recognition cannot be determined accurately nor precisely. Here we show that use of residual dipolar couplings in the refinement of the structure of a protein-RNA complex improves the definition of the long-range properties of the RNA. These features are often an important aspect of molecular recognition and biological function; therefore, their improved definition is of significant value in RNA structural biology. * Present address: Forschungsstelle für Enzymologie der Proteinfaltung,
Antibodies play an increasingly important role in both basic research and the pharmaceutical indu... more Antibodies play an increasingly important role in both basic research and the pharmaceutical industry. Since their efficiency depends, in ultimate analysis, on their atomic interactions with an antigen, studying such interactions is important to understand how they function and, in the long run, to design new molecules with desired properties. Computational docking, the process of predicting the conformation of a complex from its separated components, is emerging as a fast and affordable technique for the structural characterization of antibody-antigen complexes. In this manuscript, we first describe the different computational strategies for the modeling of antibodies and docking of their complexes, and then predict the binding of two antibodies to the stalk region of influenza hemagglutinin, an important pharmaceutical target. The purpose is two-fold: on a general note, we want to illustrate the advantages and pitfalls of computational docking with a practical example, using different approaches and comparing the results to known experimental structures. On a more specific note, we want to assess if docking can be successful in characterizing the binding to the same influenza epitope of other antibodies with unknown structure, which has practical relevance for pharmaceutical and biological research. The paper clearly shows that some of the computational docking predictions can be very accurate, but the algorithm often fails to discriminate them from inaccurate OPEN ACCESS Int. J. Mol. Sci. 2011, 12 227 solutions. It is of paramount importance, therefore, to use rapidly obtained experimental data to validate the computational results.
Proceedings of the National Academy of Sciences, 2007
T cell receptor (TCR) recognition of peptide-MHC (pMHC) is central to the cellular immune respons... more T cell receptor (TCR) recognition of peptide-MHC (pMHC) is central to the cellular immune response. A large database of TCR-pMHC structures is needed to reveal general structural principles, such as whether the repertoire of TCR/MHC docking modes is dictated by a ''recognition code'' between conserved elements of the TCR and MHC genes. Although Ϸ17 cocrystal structures of unique TCR-pMHC complexes have been determined, cocrystallization of soluble TCR and pMHC remains a major technical obstacle in the field. Here we demonstrate a strategy, based on NMR chemical shift mapping, that permits rapid and reliable analysis of the solution footprint made by a TCR when binding onto the pMHC surface. We mapped the 2C TCR binding interaction with its allogeneic ligand H-2L d -QL9 and identified a group of NMR-shifted residues that delineated a clear surface of the MHC that we defined as the TCR footprint. We subsequently found that the docking footprint described by NMR shifts was highly accurate compared with a recently determined high-resolution crystal structure of the same complex. The same NMR footprint analysis was done on a highaffinity mutant of the TCR. The current work serves as a foundation to explore the molecular dynamics of pMHC complexes and to rapidly determine the footprints of many L d -specific TCRs. chemical shift mapping ͉ dynamics ͉ NMR ͉ cellular immunity ͉ protein-protein interaction
Proceedings of the National Academy of Sciences, 1999
Coding region and intronic mutations in the tau gene cause frontotemporal dementia and parkinsoni... more Coding region and intronic mutations in the tau gene cause frontotemporal dementia and parkinsonism linked to chromosome 17. Intronic mutations and some missense mutations increase splicing in of exon 10, leading to an increased ratio of four-repeat to three-repeat tau isoforms. Secondary structure predictions have led to the proposal that intronic mutations and one missense mutation destabilize a putative RNA stem-loop structure located close to the splicedonor site of the intron after exon 10. We have determined the three-dimensional structure of this tau exon 10 splicing regulatory element RNA by NMR spectroscopy. We show that it forms a stable, folded stem-loop structure whose thermodynamic stability is reduced by frontotemporal dementia and parkinsonism linked to chromosome 17 mutations and increased by compensatory mutations. By exon trapping, the reduction in thermodynamic stability is correlated with increased splicing in of exon 10.
Drug-like molecules that bind RNA with sequence selectivity would provide valuable tools to eluci... more Drug-like molecules that bind RNA with sequence selectivity would provide valuable tools to elucidate gene expression pathways and new avenues to the treatment of degenerative and chronic conditions. Efforts at discovering such agents have been hampered, until recently, by the limited knowledge of RNA recognition principles. Several recent structures of aminoglycoside-RNA complexes have begun to reveal the structural basis for RNA-drug recognition. However, the absence of suitable chemical scaffolds known to bind the RNA major groove, where specificity could be provided by the diversity of functional groups exposed on the RNA bases, has represented a major obstacle. Here we report an investigation of the structural basis for recognition of an RNA stem-loop by neomycin, a naturally occurring aminoglycoside antibiotic. We found that neomycin binds the RNA stem-loop that regulates alternative splicing of exon 10 within the gene coding for human tau protein.
In our recent publication`Changes in side-chains and backbone dynamics identify determinants of s... more In our recent publication`Changes in side-chains and backbone dynamics identify determinants of speci®city in RNA recognition by human U1A protein' (Mittermaier et al., J. Mol. Biol. (1999), 294, p. 967), we provided experimental evidence that ms-ms dynamic processes within loop 3, the most critical determinant of speci®city, are quenched upon RNA binding and therefore important for RNA recognition. The importance of dynamics within loop 3 on RNA recognition was ®rst suggested in another study (Kranz and Hall,
If we understand the structural rules governing antibody (Ab)-antigen (Ag) interactions in a give... more If we understand the structural rules governing antibody (Ab)-antigen (Ag) interactions in a given virus, then we have the molecular basis to attempt to design and synthesize new epitopes to be used as vaccines or optimize the antibodies themselves for passive immunization. Comparing the binding of several different antibodies to related Ags should also further our understanding of general principles of recognition.
Bovine [3-LG ([3-1actoglobulin) has been studied under a variety of solution conditions by one-an... more Bovine [3-LG ([3-1actoglobulin) has been studied under a variety of solution conditions by one-and two-dimensional NMR spectroscopy. At highly acidic pH (pH = 2) and low ionic strength the protein is present in a monomeric form, exhibiting a highly structured [~-sheet core and less ordered regions as evidenced by both CD data and the NOESY spectra. Marginal protection was observed for most of the amide protons as a result of high conformational mobility. This structural state of [3-LG may be considered as an attractive model for a partially folded structure occurring late in the folding process of the protein.
Antibodies protect against homologous Dengue virus (DENV) infection but can precipitate severe de... more Antibodies protect against homologous Dengue virus (DENV) infection but can precipitate severe dengue by promoting heterotypic virus entry via Fcg receptors (FcgR). We immortalized memory B cells from individuals after primary or secondary infection and analyzed anti-DENV monoclonal antibodies (mAbs) thus generated. MAbs to envelope (E) protein domain III (DIII) were either serotype specific or cross-reactive and potently neutralized DENV infection. DI/DII-or viral membrane protein prM-reactive mAbs neutralized poorly and showed broad cross-reactivity with the four DENV serotypes. All mAbs enhanced infection at subneutralizing concentrations. Three mAbs targeting distinct epitopes on the four DENV serotypes and engineered to prevent FcgR binding did not enhance infection and neutralized DENV in vitro and in vivo as postexposure therapy in a mouse model of lethal DENV infection. Our findings reveal an unexpected degree of cross-reactivity in human antibodies against DENV and illustrate the potential for an antibodybased therapy to control severe dengue.
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Papers by Luca Varani