Diseño y caracterización estructural de sistemas minimizados para el estudio de interacciones biomoleculares mediante RMN

Tesis de la Universidad Complutense de Madrid, Facultad de Ciencias Químicas, Departamento de Bioquímica y Biología Molecular, leída el 26/10/2018The molecular processes that take place in living organisms are regulated by an intricate network of large complexes interacting among them. In this context, the biomolecular recognition processes involving each component are essential to produce the fully biologically active complex. Understanding the fine details on how these processes occur, which are the important players in each step of the recognition, which forces are involved, how they are balanced and drive the process, etc., is the current challenge in Biology. One approximation to solve these open questions is the characterization of these systems at atomic resolution. However, the study of the native biomolecular associations in the form of multiple interacting networks, at this resolution level, is extremely difficult. Given these premises, the best approach is to perform an experimental analysis of the biological systems of interest by using simplified versions with the help of model systems...Los procesos moleculares que tienen lugar en los organismos vivos se regulan a través de una enmarañada red de grandes complejos biológicos que interaccionan entre sí. En este contexto, los mecanismos de reconocimiento biomolecular que se producen en cada uno de ellos son esenciales para dar lugar al complejo activo y biológicamente funcional. Por su importancia, uno de los retos actuales en Biología es comprender a fondo los detalles específicos que definen estos procesos. Por ejemplo, necesitamos conocer cómo y cuándo ocurren, cuáles son los factores esenciales en cada tipo de reconocimiento, qué fuerzas están en juego, cómo se equilibran y dirigen estos mecanismos, etc. Una de las estrategias más apropiadas para esclarecer estas incógnitas es la caracterización de estos sistemas a escala atómica. Sin embargo, el estudio de estas asociaciones biomoleculares que forman grandes redes de interacciones, a este nivel de detalle, es extremadamente complicado. En este escenario, una aproximación realista para el estudio de un sistema biológico complejo consiste en llevar a cabo la caracterización experimental de versiones simplificadas del mismo en sistemas modelo...Depto. de Bioquímica y Biología MolecularFac. de Ciencias QuímicasTRUEunpu

Involvement of loops 2 and 3 of alpha-sarcin on its ribotoxic activity

Ribotoxins are a family of fungal ribosome-inactivating proteins displaying highly specific ribonucleolytic activity against the sarcin/ricin loop (SRL) of the larger rRNA, with a-sarcin as its best-characterized member. Their toxicity arises from the combination of this activity with their ability to cross cell membranes. The involvement of a-sarcin's loops 2 and 3 in SRL and ribosomal proteins recognition, as well as in the ribotoxin-lipid interactions involving cell penetration, has been suggested some time ago. In the work presented now different mutants have been prepared in order to study the role of these loops in their ribonucleolytic and lipid-interacting properties. The results obtained confirm that loop 3 residues Lys 111, 112, and 114 are key actors of the specific recognition of the SRL. In addition, it is also shown that Lys 114 and Tyr 48 conform a network of interactions which is essential for the catalysis. Lipid-interaction studies show that this Lys-rich region is indeed involved in the phospholipids recognition needed to cross cell membranes. Loop 2 is shown to be responsible for the conformational change which exposes the region establishing hydrophobic interactions with the membrane inner leaflets and eases penetration of ribotoxins target cells

Molecular recognition of a membrane-anchored HIV-1 pan-neutralizing epitope.

Antibodies against the carboxy-terminal section of the membrane-proximal external region (C-MPER) of the HIV-1 envelope glycoprotein (Env) are considered as nearly pan-neutralizing. Development of vaccines capable of producing analogous broadly neutralizing antibodies requires deep understanding of the mechanism that underlies C-MPER recognition in membranes. Here, we use the archetypic 10E8 antibody and a variety of biophysical techniques including single-molecule approaches to study the molecular recognition of C-MPER in membrane mimetics. In contrast to the assumption that an interfacial MPER helix embodies the entire C-MPER epitope recognized by 10E8, our data indicate that transmembrane domain (TMD) residues contribute to binding affinity and specificity. Moreover, anchoring to membrane the helical C-MPER epitope through the TMD augments antibody binding affinity and relieves the effects exerted by the interfacial MPER helix on the mechanical stability of the lipid bilayer. These observations support that addition of TMD residues may result in more efficient and stable anti-MPER vaccines.This study was supported by MCIN/AEI/10.13039/501100011033 - “ERDF A way of making Europe” (Grant PID2021-126014OB-I00 to J.L.N. and B.A.), MCIN/AEI/10.13039/501100011033 (Grant PID2020-112821GB-I00 to M.A.J.), Basque Government (Grant: IT1449-22 to J.L.N. and B.A.) and Kiban-B grant 20H03228 from JSPS to J.M.M.C. L.R.-M. acknowledges funding from the Agence National de la Recherche (ANR), as part of the ‘Investments d′Avenir’ Program (I-SITE ULNE/ANR-16-IDEX-0004 ULNE). This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 895819 (to C.V.). Work at Pompeu Fabra University was supported by the María de Maeztu network of Units of Excellence of the Spanish Ministry of Science and Innovation. Technical assistance from Miguel García-Porras is greatly acknowledged. The NMR experiments were performed in the “Manuel Rico” NMR laboratory, LMR, CSIC, a node of the Spanish Large-Scale National Facility ICTS R-LRB