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Publications NMR Proteins Research Lines

Design and structure-function relationships in peptides.

The determination of the structures of biologically active peptides contributes to our understanding of their biological function and paves the way towards the rational design of peptides with pharmaceutical applications. Among our recent studies, we highlight our results on peptides derived from the glycoprotein gp41 from the virus HIV, which are involved in membrane fusion and have inmunogenic capacity against HIV, and on bactericide and antitumoral peptides, such as crotalicidin. An interesting result is that a peptide encompassing the helical N-terminal region of crotalicidin is inactive, whereas that corresponding to the disordered C-terminal region of crotalicidin is active, but less toxic than the full-length peptide.
Apellániz B, Rujas E, Serrano S, Morante K, Tsumoto K, Caaveiro JM, Jiménez MA, Nieva JL ( 2015 ). The atomic structure of the HIV-1 gp41 transmembrane domain and its connection to the inmmunogenic membrane-proximal external region. J Biol Chem 290, 12999-13015
Falcao CB, Pérez-Peinado C, de la Torre BG, Mayol X, Zamora-Carreras H, Jiménez MA, Radis-Baptista G, Andreu D (2015). Structural dissection of crotalicidin, a rattlesnake venom cathelicidin, retrieves a fragment with antimicrobial and antitumor activity. J Med Chem 58, 8553-8563
Rujas E, Caaveiro JMM, Partida-Hanon A, Gulzar N, Morante K, Apellániz B, García-Porras M, Bruix M, Tsumoto K, Scott JK, Jiménez MA, Nieva JL (2016). Molecular recognition of the broadly neutralizing HIV-1 10E8 epitope at the membrane interface. Sci Rep 6, 38177

Implementation and optimization of NMR methods.

The need to determine rapidly and efficiently protein structures has become an urgent objective as a consequence of the advances accomplished in Proteomics. Towards this goal we are developing and implementing a number of very promising NMR experimental techniques for the structural study of complexes and intermolecular interactions, such as pulse sequences with 13C detection. Among these, strategies aimed at identifying amino acid type that will complement the previously proposed method based on 1H detection are of particular relevance.
Pantoja-Uceda D and Santoro J (2013). A suite of amino acid residue type classification pulse sequences for 13C-detected NMR of proteins. J Magn Reson. 234, 190-6
Pantoja-Uceda D and Santoro J (2014). New 13C-detected experiments for the assignment of intrinsically disordered proteins. J Biomol NMR 59,43-50

Structural transitions in peptides and proteins mediated by interactions.

Autolysin LytA is a protein involved in the virulence of pneumococcus, a pathogenic microorganism in humans. Its C-terminal domain (CLytA) consists of six choline-binding repeats (CBR), arranged in the α-solenoid structure characteristic of choline-binding modules. We have characterized by NMR and CD a 14-residue peptide encompassing the sequence of the core β-hairpin from the third CBR repeat of CLytA. It has been found that this peptide conserves its native β-hairpin fold in aqueous solution, but forms a stable, amphipathic α-helix (i.e. with two faces, one hydrophobic and the other polar) in detergent micelles (with a hydrophilic surface and a hydrophobic core). These β-hairpin and α-helix structures differ greatly in the distribution of polar hydrophobic side chains. To our knowledge, this "chameleonic" behavior of a micelle-induced structural transition between two ordered peptide structures has not been reported before, and shows the dramatic effect of hydrophobic-hydrophilic interactions. These results could not only be of relevance in the field of peptide design and biosensors, but may also help to understand the molecular basis for the peculiar mechanism of LytA translocation from the cytoplasm to the bacterial surface.
Zamora-Carreras H, Maestro B, Strandberg E, Ulrich AS, Sanz JM, Jiménez MA (2015). Micelle-triggered β-hairpin to α-helix transition in a 14-residue peptide from a choline-binding repeat of the pneumococcal autolysin LytA. Chem Eur J 21, 8076-89.

Structural and interaction studies of RNA-binding proteins

Structural and interaction studies of RNA-binding proteins that build up ribonucleoprotein complexes (RNPs) with biological activity in: RNA processing, mRNA export and translational control by stress granules. Most of the target proteins contain RRM and CCCH-type zinc finger domains, which are characterized by their RNA binding abilities. Moreover, we are interested in the analysis of protein-protein interactions mediated by these types of domains, which, during recent years, have been revealed as key for RNP formation. In this field, our principal goal is the structural determination of biologically relevant protein-protein and protein-RNA complexes by NMR.
Martínez-Lumbreras S, Taverniti V, Zorrilla S, Séraphin B, Pérez-Canadillas JM (2016). Gbp2 interacts with THO/TREX through a novel type of RRM domain. Nucleic Acids Res 44, 437-448

Molecular mechanisms of light sensing and response in bacteria.

Light is a crucial environmental factor that directly or indirectly signals diverse biological processes but can also cause photooxidative cellular damage. We study molecular mechanisms involved in light sensing and response in bacteria (Myxococcus xanthus, Thermus thermophilus etc) in collaboration with the Genetics group at the University of Murcia. Our studies have identified a number of novel signal transducers and regulators and we focus on the molecular basis (structures, interactions,..) of their functions. Highlights of our work include, among others, the discovery and molecular characterization of a new and widespread class of RNA polymerase-binding transcriptional regulators, and a novel family of vitamin B12-dependent photoreceptors.
Padmanabhan S, Jost M, Drennan CL, Elías-Arnanz M (2017). A new facet of vitamin B12: gene regulation by cobalamin-based photoreceptors. Ann Rev Biochem 86 (in press)
Jost M, Fernéndez-Zapata J, Polanco MC, Ortiz-Guerrero JM, Chen PY, Kang G, Padmanabhan S, Elías-Arnanz M, Drennan CL (2015). Structural basis for gene regulation by a B12-dependent photoreceptor. Nature 526, 536-41
Kutta RJ, Hardman SJ, Johannissen LO, Bellina B, Messiha HL, Ortiz-Guerrero JM, Elías-Arnanz M, Padmanabhan S, Barran P, Scrutton NS, Jones AR (2015). The photochemical mechanism of a B12-dependent photoreceptor protein. Nat Commun 6, 7907

Recognition events in the dynein microtubule motor.

We have studied by NMR some complexes of DLC8 and DYNLT1 with different peptides in order to understand why these proteins interact with multiple functionally unrelated cell proteins with low sequence homology. Also, we have used NMR spectroscopy to obtain the solution structure of human DYNLT1 forming a complex with DIC1 (dynein intermediate chain), the first mammalian structure described so far.
Merino-Gracia J, Zamora-Carreras H, Bruix M, Rodríguez-Crespo I (2016). Molecular basis for the protein recognition specificity of the dynein light chain DYNLT1/Tctex1: characterization of the interaction with activin receptor IIB. J Biol Chem 291, 20962-20975
Merino-Gracia J, García-Mayoral MF, Rapali P, Valero RA, Bruix M, Rodríguez-Crespo I (2015). DYNLT (Tctex1) forms a tripartite complex with dynein intermediate chain and RagA hence linking this small GTPase to the dynein motor. FEBS J 282, 3945-3958

Structural aspects of the protein-lipid recognition by sticholysins and ribonucleases.

We have characterised at atomic level the interaction of some proteins with membrane mimetic systems or membrane components. In the case of sticholysins, we have obtained important clues about the initial steps of pore formation by these proteins. Also, we have described the interaction between ECP (from the ribonuclease family) with micelles, heparin mimetics and lipopolysaccharides. In all these studies we have described the molecular bases of the interactions that are responsible of the protein potent cytotoxicity.
García-Mayoral MF, Canales A, Díaz D, López-Prados J, Moussaoui M, de Paz JL, Angulo J, Nieto PM, Jiménez-Barbero J, Boix E, Bruix M (2013). Insights into the glycosaminoglycan-mediated cytotoxic mechanism of eosinophil cationic protein revealed by NMR. ACS Chem Biol 8, 144-151
Pulido D, García-Mayoral MF, Moussaoui M, Torrent M, Bruix M, Boix E (2016). Structural basis for endotoxin neutralization by the Eosinophil Cationic Protein. FEBS J 283, 4176-4191

Molecular bases of allergenicity.

We have determined the 3D structure and characterised the molecular interactions of several protein allergens and immunogenic peptides to understand the antigen/antibody recognition events. We are covering a wide range of protein systems from plants, parasites, to human proteins involved in important diseases.
García-Mayoral MF, Trevino M, Pérez-Pinar T, Caballero ML, Knaute T, Umpierrez A, Bruix M, Rodríguez R (2013). Solution structure and identification of IgE and IgG4 sequential epitiopes of Ani s 5, an Anisakis simplex allergen. Allergy 68, 126
García-Mayoral MF, Trevino M, Pérez-Pinar T, Caballero ML, Knaute T, Umpierrez A, Bruix M, Rodríguez R (2014). The relationship between IgE and IgG4 epitopes, structure and function in Anisakis simplex Ani s 5, a member of the SXP/RAL-2 protein family. PLoS Negl Trop Dis 8, e2735
Zamora-Carreras H, Torres M, Bustamante N, Macedo A, Rodríguez R, Villalba M, Bruix M (2015). The C-terminal domains of two homologous Oleaceae β-1,3-glucanases recognize carbohydrates differently: laminarin binding by NMR. Arch Biochem Biophys 580, 93-101

Structural study of proteins of biomedical relevance: applications in cancer and Alzheimer’s disease.

Apoptin is a promising anti cancer lead from a chicken anemia virus that induces apoptosis in 80 different cancer cell lines, while it does not harm normal cells. We have characterized the conformation of a soluble, monomeric construct of Apoptin that conserves most of the wild type protein's exquisitely selectively biological activity and find that it is an intrinsically disordered protein. Our studies of the effects of phosphorylation, divalent cations and reduction on Apoptin's conformational ensemble reveal insight into its mechanism of action and pave the way for the rational design of improved Apoptin variants. With over 50 million patients worldwide, Alzheimer's disease and other dementia are daunting public health and social challenges. Improving Alzheimer's treatments will require safer and less expensive diagnostic probes and low cost inhibitors of amyloid formation, and we are collaborating with other labs to address these needs.
Martins AF, Dias DM, Morfin JF, Lacerda S, Laurents DV, Tóth É, Geraldes CF (2015). Interaction of PiB-derivative metal complexes with beta-amyloid peptides: selective recognition of the aggregated forms. Chem Eur J 21, 5413-542
Ruiz-Martínez S, Pantoja-Uceda D, Castro J, Vilanova M, Rib? M, Bruix M, Benito A, Laurents DV (2017). Insights into the mechanism of Apoptin's exquisitely selective anti-tumor action from atomic level characterization of its conformation and dynamics. Arch Biochem Biophys 614, 53-64

Comparison of the structure and formation of pathological versus functional prion-like domains in proteins implicated in and Amyotrophic Lateral Sclerosis (ALS) and memory consolidation.

Whereas amyloids and prions are infamous as protagonists in neurodegenerative diseases, over the last decade, it has become clear that amyloids and prion-like domains also play crucial functional roles in regulating gene expression. TDP-43 is an essential protein whose prion-like domain plays physiological roles in RNA maturation, transport and regulation, but rarely forms pathological aggregates which appear to drive ALS and Frontotemporal Lobar Degeneration, a closely related dementia. Like TDP-43, CPEB3 contains RNA binding modules and a prion-like domain. Amyloid formation by the latter is key to the fascinating process of long term memory consolidation. By characterizing and contrasting the prion-like domains of TDP-43 and CPEB3 and their stabilizing interactions, we aim to understand the differences between functional versus pathological amyloids. These studies could aid the design of selective inhibitors which block the latter without interfering with the labor of the former.
Mompeán M, Hervás R, Xu Y, Tran TH, Guarnaccia C, Buratti E, Baralle F, Tong L, Carrión-Vázquez M, McDermott AE, Laurents DV (2015). Structural evidence of amyloid fibril formation in the putative aggregation domain of TDP-43. J Phys Chem Lett 6, 2608-2615
Hervás R, Li L, Majumdar A, Fernéndez-Ramírez MC, Unruh JR, Slaughter BD, Galera-Prat A, Santana E, Suzuki M, Nagai Y, Bruix M, Casas-Tintó S, Menéndez M, Laurents DV, Si K, Carrión-Vázquez M (2016). Molecular basis of Orb2 amyloidogenesis and blockade of memory consolidation. PLoS Biol 14, e1002361
Mompeán M, Romano V, Pantoja-Uceda D, Stuani C, Baralle FE, Buratti E, Laurents DV (2016). The TDP-43 N-terminal domain structure at high resolution. FEBS J 283,1242-1260
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