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1. Identification of the first structurally validated covalent ligands of the small GTPase RAB27A.

Author

Tersa, Montse, De Vita, Elena, Brustur, Delia, Pérez-Dorado, Inmaculada, Hassan, Sarah, Petracca, Rita, Morgan, Rhodri, Sanz-Hernández, Máximo, Norman, Jim, Armstrong, Alan, Mann, David, Cota, Ernesto, Tate, Edward, Jamshidiha, Mostafa, Lanyon-Hogg, Thomas, Sutherell, Charlotte, and Craven, Gregory

Abstract

Rab27A is a small GTPase, which mediates transport and docking of secretory vesicles at the plasma membrane via protein-protein interactions (PPIs) with effector proteins. Rab27A promotes the growth and invasion of multiple cancer types such as breast, lung and pancreatic, by enhancing secretion of chemokines, metalloproteases and exosomes. The significant role of Rab27A in multiple cancer types and the minor role in adults suggest that Rab27A may be a suitable target to disrupt cancer metastasis. Similar to many GTPases, the flat topology of the Rab27A-effector PPI interface and the high affinity for GTP make it a challenging target for inhibition by small molecules. Reported co-crystal structures show that several effectors of Rab27A interact with the Rab27A SF4 pocket (WF-binding pocket) via a conserved tryptophan-phenylalanine (WF) dipeptide motif. To obtain structural insight into the ligandability of this pocket, a novel construct was designed fusing Rab27A to part of an effector protein (fRab27A), allowing crystallisation of Rab27A in high throughput. The paradigm of KRas covalent inhibitor development highlights the challenge presented by GTPase proteins as targets. However, taking advantage of two cysteine residues, C123 and C188, that flank the WF pocket and are unique to Rab27A and Rab27B among the >60 Rab family proteins, we used the quantitative Irreversible Tethering (qIT) assay to identify the first covalent ligands for native Rab27A. The binding modes of two hits were elucidated by co-crystallisation with fRab27A, exemplifying a platform for identifying suitable lead fragments for future development of competitive inhibitors of the Rab27A-effector interaction interface, corroborating the use of covalent libraries to tackle challenging targets.

Published
2022

3. Spin-orbit signatures in the dynamics of singlet-triplet qubits in double quantum dots

Author

Rolon, Juan E., Cota, Ernesto, and Ulloa, Sergio E.

Subjects
Condensed Matter - Mesoscale and Nanoscale Physics
Abstract

We characterize numerically and analytically the signatures of the spin-orbit interaction in a two-electron GaAs double quantum dot in the presence of an external magnetic field. In particular, we obtain the return probability of the singlet state by simulating Landau-Zener voltage detuning sweeps which traverse the singlet-triplet ($S-T_+$) resonance. Our results indicate that non-spin-conserving interdot tunneling processes arising from the spin-orbit interaction have well defined signatures. These allow direct access to the spin-orbit interaction scales and are characterized by a frequency shift and Fourier amplitude modulation of the Rabi flopping dynamics of the singlet-triplet qubits $S-T_0$ and $S-T_+$. By applying the Bloch-Feshbach projection formalism, we demonstrate analytically that the aforementioned effects originate from the interplay between spin-orbit interaction and processes driven by the hyperfine interaction between the electron spins and those of the GaAs nuclei.

Published
2016
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5. Spin filtering through excited states in double quantum dot pumps

Author

Sánchez, Rafael, Cota, Ernesto, Aguado, Ramón, and Platero, Gloria

Subjects
Condensed Matter - Mesoscale and Nanoscale Physics, Condensed Matter - Other Condensed Matter
Abstract

Recently it has been shown that ac-driven double quantum dots can act as spin pumps and spin filters. By calculating the current through the system for each spin polarization, by means of the time evolution of the reduced density matrix in the sequential tunneling regime (Born-Markov approximation), we demonstrate that the spin polarization of the current can be controlled by tuning the parameters (amplitude and frequency) of the ac field. Importantly, the pumped current as a function of the applied frequency presents a series of peaks which are uniquely associated with a definite spin polarization. We discuss how excited states participating in the current allow the system to behave as a bipolar spin filter by tuning the ac frequency and intensity. We also discuss spin relaxation and decoherence effects in the pumped current and show that measuring the width of the current vs frequency peaks allows to determine the spin decoherence time $T_{2}$., Comment: 10 pages. 5 figures

Published
2006
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6. AC-driven double quantum dots as spin pumps and spin filters

Author

Cota, Ernesto, Aguado, Ramon, and Platero, Gloria

Subjects
Condensed Matter - Mesoscale and Nanoscale Physics
Abstract

We propose and analyze a new scheme of realizing both spin filtering and spin pumping by using ac-driven double quantum dots in the Coulomb blockade regime. By calculating the current through the system we demonstrate that the spin polarization of the current can be controlled by tuning the parameters (amplitude and frequency) of the ac field. We also discuss spin relaxation and decoherence effects in the pumped current., Comment: 5 pages, 4 figures

Published
2005
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10. Candidalysin is a fungal peptide toxin critical for mucosal infection

Author

Moyes, David L., Wilson, Duncan, Richardson, Jonathan P., Mogavero, Selene, Tang, Shirley X., Wernecke, Julia, Hofs, Sarah, Gratacap, Remi L., Robbins, Jon, Runglall, Manohursingh, Murciano, Celia, Blagojevic, Mariana, Thavaraj, Selvam, Fbrster, Toni M., Hebecker, Betty, Kasper, Lydia, Vizcay, Gema, Iancu, Simona I., Kichik, Nessim, Hader, Antje, Kurzai, Oliver, Luo, Ting, Kruger, Thomas, Kniemeyer, Olaf, Cota, Ernesto, Bader, Oliver, Wheeler, Robert T., Gutsmann, Thomas, Hube, Bernhard, and Naglik, Julian R.

Subjects
Physiological aspects, Fungi -- Physiological aspects, Mucous membrane -- Physiological aspects, Candida albicans -- Physiological aspects
Abstract

The ability of mucosal surfaces to discriminate between commensal and pathogenic microbes is essential to human health. The fungus Candida albicans is normally a benign member of the human microbiota, [...], Cytolytic proteins and peptide toxins are classical virulence factors of several bacterial pathogens which disrupt epithelial barrier function, damage cells and activate or modulate host immune responses. Such toxins have not been identified previously in human pathogenic fungi. Here we identify the first, to our knowledge, fungal cytolytic peptide toxin in the opportunistic pathogen Candida albicans. This secreted toxin directly damages epithelial membranes, triggers a danger response signalling pathway and activates epithelial immunity. Membrane permeabilization is enhanced by a positive charge at the carboxy terminus of the peptide, which triggers an inward current concomitant with calcium influx. C. albicans strains lacking this toxin do not activate or damage epithelial cells and are avirulent in animal models of mucosal infection. We propose the name 'Candidalysin' for this cytolytic peptide toxin; a newly identified, critical molecular determinant of epithelial damage and host recognition of the clinically important fungus, C. albicans.

Published
2016
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16. Structural Insights into Serine-rich Fimbriae from Gram-positive Bacteria

Author

Ramboarina, Stéphanie, Garnett, James A., Zhou, Meixian, Li, Yuebin, Peng, Zhixiang, Taylor, Jonathan D., Lee, Wei-chao, Bodey, Andrew, Murray, James W., Alguel, Yilmaz, Bergeron, Julien, Bardiaux, Benjamin, Sawyer, Elizabeth, Isaacson, Rivka, Tagliaferri, Camille, Cota, Ernesto, Nilges, Michael, Simpson, Peter, Ruiz, Teresa, Wu, Hui, and Matthews, Stephen

Published
2010
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17. Identification of the first structurally validated covalent ligands of the small GTPase RAB27A

Author

Jamshidiha, Mostafa, primary, Lanyon-Hogg, Thomas, additional, Sutherell, Charlotte L., additional, Craven, Gregory B., additional, Tersa, Montse, additional, De Vita, Elena, additional, Brustur, Delia, additional, Pérez-Dorado, Inmaculada, additional, Hassan, Sarah, additional, Petracca, Rita, additional, Morgan, Rhodri M., additional, Sanz-Hernández, Máximo, additional, Norman, Jim C., additional, Armstrong, Alan, additional, Mann, David J., additional, Cota, Ernesto, additional, and Tate, Edward W., additional

Published
2022
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23. Structural and functional characterization of a frataxin from a thermophilic organism

Author

Rasheed, Masooma, Jamshidiha, Mostafa, Puglisi, Rita, Yan, Robert, Cota, Ernesto, Pastore, Annalisa, Rasheed, Masooma, Jamshidiha, Mostafa, Puglisi, Rita, Yan, Robert, Cota, Ernesto, and Pastore, Annalisa

Subjects
Biochemistry & Molecular Biology, METAL-BINDING, Science & Technology, STABILITY, 0304 Medicinal and Biomolecular Chemistry, BIOGENESIS, CYAY, Friedreich's ataxia, iron–sulfur cluster, NFS1, Cell Biology, iron–sulfur clusters, 0601 Biochemistry and Cell Biology, Biochemistry, iron-sulfur clusters, ACTIVATION, 1101 Medical Biochemistry and Metabolomics, BACKBONE DYNAMICS, structural biology, Life Sciences & Biomedicine, INTERACTING PROTEIN, Molecular Biology, BACTERIAL FRATAXIN
Abstract

Frataxins form an interesting family of iron‐binding proteins with an almost unique fold and are highly conserved from bacteria to primates. They have a pivotal role in iron‐sulfur cluster biogenesis as regulators of the rates of cluster formation, as it is testified by the fact that frataxin absence is incompatible with life and reduced levels of the protein lead to the recessive neurodegenerative disease Friedreich's ataxia. Despite its importance, the structure of frataxin has been solved only from relatively few species. Here, we discuss the X‐ray structure of frataxin from the thermophilic fungus Chaetomium thermophilum, and the characterization of its interactions and dynamics in solution. We show that this eukaryotic frataxin has an unusual variation of the classical frataxin fold: the last helix is shorter than in other frataxins which results in a less symmetrical and compact structure. The stability of this protein is comparable to that of human frataxin, currently the most stable amongst the frataxin orthologues. We also characterized the iron‐binding mode of C. thermophilum frataxin and demonstrated that it binds it through a semi‐conserved negatively charged ridge on the first helix and beta‐strand. Moreover, this frataxin is also able to bind the bacterial ortholog of the desulfurase, which is central in iron‐sulfur cluster synthesis, and act as its inhibitor.

Published
2019
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34. The fungal ribonuclease-like effector protein CSEP0064/BEC1054 represses plant immunity and interferes with degradation of host ribosomal RNA

Author

Pennington, Helen G., primary, Jones, Rhian, additional, Kwon, Seomun, additional, Bonciani, Giulia, additional, Thieron, Hannah, additional, Chandler, Thomas, additional, Luong, Peggy, additional, Morgan, Sian Natasha, additional, Przydacz, Michal, additional, Bozkurt, Tolga, additional, Bowden, Sarah, additional, Craze, Melanie, additional, Wallington, Emma J., additional, Garnett, James, additional, Kwaaitaal, Mark, additional, Panstruga, Ralph, additional, Cota, Ernesto, additional, and Spanu, Pietro D., additional

Published
2019
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35. Identification of the first structurally validated covalent ligands of the small GTPase RAB27AElectronic supplementary information (ESI) available. See DOI: 10.1039/d1md00225b

Author

Jamshidiha, Mostafa, Lanyon-Hogg, Thomas, Sutherell, Charlotte L., Craven, Gregory B., Tersa, Montse, De Vita, Elena, Brustur, Delia, Pérez-Dorado, Inmaculada, Hassan, Sarah, Petracca, Rita, Morgan, Rhodri M., Sanz-Hernández, Máximo, Norman, Jim C., Armstrong, Alan, Mann, David J., Cota, Ernesto, and Tate, Edward W.

Abstract

Rab27A is a small GTPase, which mediates transport and docking of secretory vesicles at the plasma membrane viaprotein–protein interactions (PPIs) with effector proteins. Rab27A promotes the growth and invasion of multiple cancer types such as breast, lung and pancreatic, by enhancing secretion of chemokines, metalloproteases and exosomes. The significant role of Rab27A in multiple cancer types and the minor role in adults suggest that Rab27A may be a suitable target to disrupt cancer metastasis. Similar to many GTPases, the flat topology of the Rab27A-effector PPI interface and the high affinity for GTP make it a challenging target for inhibition by small molecules. Reported co-crystal structures show that several effectors of Rab27A interact with the Rab27A SF4 pocket (‘WF-binding pocket’) viaa conserved tryptophan–phenylalanine (WF) dipeptide motif. To obtain structural insight into the ligandability of this pocket, a novel construct was designed fusing Rab27A to part of an effector protein (fRab27A), allowing crystallisation of Rab27A in high throughput. The paradigm of KRas covalent inhibitor development highlights the challenge presented by GTPase proteins as targets. However, taking advantage of two cysteine residues, C123 and C188, that flank the WF pocket and are unique to Rab27A and Rab27B among the >60 Rab family proteins, we used the quantitative Irreversible Tethering (qIT) assay to identify the first covalent ligands for native Rab27A. The binding modes of two hits were elucidated by co-crystallisation with fRab27A, exemplifying a platform for identifying suitable lead fragments for future development of competitive inhibitors of the Rab27A-effector interaction interface, corroborating the use of covalent libraries to tackle challenging targets.

Published
2022
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44. A fungal ribonuclease-like effector protein inhibits plant host ribosomal RNA degradation

Author

Pennington, Helen G., primary, Jones, Rhian, additional, Kwon, Seomun, additional, Bonciani, Giulia, additional, Thieron, Hannah, additional, Chandler, Thomas, additional, Luong, Peggy, additional, Morgan, Sian, additional, Przydacz, Michal, additional, Bozkurt, Tolga, additional, Bowden, Sarah, additional, Craze, Melanie, additional, Wallington, Emma, additional, Garnett, James, additional, Kwaaitaal, Mark, additional, Panstruga, Ralph, additional, Cota, Ernesto, additional, and Spanu, Pietro D., additional

Published
2018
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46. Automated assignment in selectively methyl-labeled proteins

Author

Yingqi Xu, Minhao Liu, Simpson, Peter J., Isaacson, Rivka, Cota, Ernesto, Marchant, Jan, Daiwen Yang, Xiaodong Zhang, Freemont, Paul, and Matthews, Stephen

Subjects
Methyl groups -- Chemical properties, Methyl groups -- Structure, Mutagenesis -- Analysis, Nuclear magnetic resonance spectroscopy -- Usage, Proteins -- Structure, Proteins -- Analysis, Chemistry
Abstract

A new automated procedure is developed that is able to rapidly assign the majority of methyl groups in very large proteins, without recourse to mutagenesis or truncated fragments. The effectiveness of this approach is demonstrated on the 300 kDa, ILV-labeled proteasome.

Published
2009

47. A new labeling method for methyl transverse relaxation-optimized spectroscopy NMR spectra of alanine residues

Author

Isaacson, Rivka L., Simpson, Peter J., Minhao Liu, Cota, Ernesto, Xiaodong Zhang, Freemont, Paul, and Matthews, Stephen

Subjects
Nuclear magnetic resonance spectroscopy -- Usage, Alanine -- Research, Alanine -- Chemical properties, Chemistry
Abstract

An efficient method is described for the synthesis and incorporation of L-alanine-3-[super 13]C,2,-super 2]H into protein sequences. The use of specific protonation of alanine residues is shown along with methyl TROSY experiments.

Published
2007

49. Peptide Probes for Plasmodium falciparumMyoA Tail Interacting Protein (MTIP): Exploring the Druggability of the Malaria Parasite Motor Complex

Author

Saunders, Charlie N., Cota, Ernesto, Baum, Jake, and Tate, Edward W.

Abstract

Malaria remains an endemic tropical disease, and the emergence of Plasmodium falciparumparasites resistant to current front-line medicines means that new therapeutic targets are required. The Plasmodiumglideosome is a multiprotein complex thought to be essential for efficient host red blood cell invasion. At its core is a myosin motor, Myosin A (MyoA), which provides most of the force required for parasite invasion. Here, we report the design and development of improved peptide-based probes for the anchor point of MyoA, the P. falciparumMyoA tail interacting protein (PfMTIP). These probes combine low nanomolar binding affinity with significantly enhanced cell penetration and demonstrable competitive target engagement with native PfMTIP through a combination of Western blot and chemical proteomics. These results provide new insights into the potential druggability of the MTIP/MyoA interaction and a basis for the future design of inhibitors.

Published
2020
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50. Structural insight into host recognition by aggregative adherence fimbriae of enteroaggregative Escherichia coli

Author

Berry, Andrea A., Yang, Yi, Pakharukova, Natalia, Garnett, James A., Lee, Wei-chao, Cota, Ernesto, Marchant, Jan, Roy, Saumendra, Tuittila, Minna, Liu, Bing, Inman, Keith G., Ruiz-Perez, Fernando, Mandomando, Inacio, Nataro, James P., Zavialov, Anton V., Matthews, Steve, and Derrick, JP

Subjects
Bacterial Diseases, Models, Molecular, Travel-Associated Diseases, Magnetic Resonance Spectroscopy, Protein Conformation, Mutagenesis and Gene Deletion Techniques, Spectrum analysis techniques, Crystallography, X-Ray, Biochemistry, Bacterial Adhesion, Macromolecular Structure Analysis, Medicine and Health Sciences, polycyclic compounds, Intestinal Mucosa, Biology (General), Macromolecular Complex Analysis, Escherichia coli Infections, Escherichia Coli, Adhesins, Escherichia coli, Ecology, Escherichia coli Proteins, Bacterial Biochemistry, Bacterial Pathogens, Infectious Diseases, Medical Microbiology, Host-Pathogen Interactions, Protein Structure Determination, Research Article, Protein Structure, QH301-705.5, Immunoblotting, Molecular Sequence Data, Microbiology, Microbial Ecology, NMR spectroscopy, Protein Domains, Enterobacteriaceae, Humans, Amino Acid Sequence, Molecular Biology Techniques, Gram Negative Bacteria, Microbial Pathogens, Molecular Biology, Bacteria, Sequence Homology, Amino Acid, Organisms, Biology and Life Sciences, Proteins, Bacteriology, RC581-607, Fibronectins, Research and analysis methods, Emerging Infectious Diseases, Microscopy, Fluorescence, Biofilms, Fimbriae, Bacterial, Mutation, Mutagenesis, Site-Directed, Molecular Complexes, Immunologic diseases. Allergy, Bacterial Biofilms
Abstract

Enteroaggregative Escherichia coli (EAEC) is a leading cause of acute and persistent diarrhea worldwide. A recently emerged Shiga-toxin-producing strain of EAEC resulted in significant mortality and morbidity due to progressive development of hemolytic-uremic syndrome. The attachment of EAEC to the human intestinal mucosa is mediated by aggregative adherence fimbria (AAF). Using X-ray crystallography and NMR structures, we present new atomic resolution insight into the structure of AAF variant I from the strain that caused the deadly outbreak in Germany in 2011, and AAF variant II from archetype strain 042, and propose a mechanism for AAF-mediated adhesion and biofilm formation. Our work shows that major subunits of AAF assemble into linear polymers by donor strand complementation where a single minor subunit is inserted at the tip of the polymer by accepting the donor strand from the terminal major subunit. Whereas the minor subunits of AAF have a distinct conserved structure, AAF major subunits display large structural differences, affecting the overall pilus architecture. These structures suggest a mechanism for AAF-mediated adhesion and biofilm formation. Binding experiments using wild type and mutant subunits (NMR and SPR) and bacteria (ELISA) revealed that despite the structural differences AAF recognize a common receptor, fibronectin, by employing clusters of basic residues at the junction between subunits in the pilus. We show that AAF-fibronectin attachment is based primarily on electrostatic interactions, a mechanism not reported previously for bacterial adhesion to biotic surfaces., Author Summary Enteroaggregative Escherichia coli (EAEC) is a major cause of diarrhea worldwide and is commonly present as an infection in symptomatic travelers returning from developing countries. The attachment of EAEC to the human intestine is mediated protein filaments extending from the bacterial surface known as aggregative adherence fimbria (AAF). Here we use X-ray crystallography and nuclear magnetic resonance (NMR) structures to provide an atomic structure of the protein fibers made by the two major variants, AAF/I and AAF/II. The structures of the major subunit proteins show that the AAFs assemble into flexible, linear polymers that are capped by a single minor protein subunit at the tip. Biochemical assays reveal that the AAFs recognize a common receptor, the extracellular matrix protein fibronectin, via clusters of positively-charged amino acid residues running along the length of the fimbriae. Our structures suggest a unique mechanism based on ionic interactions for AAF-mediated receptor binding and biofilm formation.

Published
2014

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