Probing the Kinetic and Thermodynamic Fingerprints of Anti-EGF Nanobodies by Surface Plasmon Resonance

Despite the widespread use of antibodies in clinical applications, the precise molecular mechanisms underlying antibody-antigen (Ab-Ag) interactions are often poorly understood. In this study, we exploit the technical features of a typical surface plasmon resonance (SPR) biosensor to dissect the kinetic and thermodynamic components that govern the binding of single-domain Ab or nanobodies to their target antigen, epidermal growth factor (EGF), a key oncogenic protein that is involved in tumour progression. By carefully tuning the experimental conditions and transforming the kinetic data into equilibrium constants, we reveal the complete picture of binding thermodynamics, including the energetics of the complex-formation transition state. This approach, performed using an experimentally simple and high-throughput setup, is expected to facilitate mechanistic studies of Ab-based therapies and, importantly, promote the rational development of new biological drugs with suitable properties

Quantification of interacting cognate odorants with olfactory receptors in nanovesicles

This study aims to improve our understanding of the interaction between olfactory receptors and odorants to develop highly selective biosensing devices. Natural nanovesicles (NVs) from Saccharomyces cerevisiae, ~100 nm in diameter, carrying either the human OR17-40 or the chimpanzee OR7D4 olfactory receptor (OR) tagged with the c-myc epitope at their N-terminus, are presented as model systems to quantify the interaction between odorant and olfactory receptors. The level of expression of olfactory receptors was determined at individual NVs using a novel competitive ELISA immunoassay comparing the values obtained against those from techniques involving the solubilization of cell membrane proteins and the identification of c-myc-carrying receptors. Surface Plasmon Resonance (SPR) measurements on L1 Biacore chips indicate that cognate odorants bind to their Ors, thereby quantifying the approximate number of odorants that interact with a given olfactory receptor. The selectivity of OR17-40-carrying NVs towards helional and OR7D4-carrying NVs towards androstenone has been proven in cross-check experiments with non-specific odorant molecules (heptanal and pentadecalactone, respectively) and in control receptors

Structure of the Homodimeric androgen receptor ligand-binding domain

The androgen receptor (AR) plays a crucial role in normal physiology, development and metabolism as well as in the aetiology and treatment of diverse pathologies such as androgen insensitivity syndromes (AIS), male infertility and prostate cancer (PCa). Here we show that dimerization of AR ligand-binding domain (LBD) is induced by receptor agonists but not by antagonists. The 2.15-Å crystal structure of homodimeric, agonist- and coactivator peptide-bound AR-LBD unveils a 1,000-Å2 large dimerization surface, which harbours over 40 previously unexplained AIS- and PCa-associated point mutations. An AIS mutation in the self-association interface (P767A) disrupts dimer formation in vivo, and has a detrimental effect on the transactivating properties of full-length AR, despite retained hormone-binding capacity. The conservation of essential residues suggests that the unveiled dimerization mechanism might be shared by other nuclear receptors. Our work defines AR-LBD homodimerization as an essential step in the proper functioning of this important transcription factor

An integrative structural biology analysis of von Willebrand factor binding and processing by ADAMTS-13 in solution

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An Integrative Structural Biology Analysis of Von Willebrand Factor Binding and Processing by ADAMTS-13 in Solution

Von Willebrand Factor (vWF), a 300-kDa plasma protein key to homeostasis, is cleaved at a single site by multi-domain metallopeptidase ADAMTS-13. vWF is the only known substrate of this peptidase, which circulates in a latent form and becomes allosterically activated by substrate binding. Herein, we characterised the complex formed by a competent peptidase construct (AD13-MDTCS) comprising metallopeptidase (M), disintegrin-like (D), thrombospondin (T), cysteine-rich (C), and spacer (S) domains, with a 73-residue functionally relevant vWF-peptide, using nine complementary techniques. Pull-down assays, gel electrophoresis, and surface plasmon resonance revealed tight binding with sub-micromolar affinity. Cross-linking mass spectrometry with four reagents showed that, within the peptidase, domain D approaches M, C, and S. S is positioned close to M and C, and the peptide contacts all domains. Hydrogen/deuterium exchange mass spectrometry revealed strong and weak protection for C/D and M/S, respectively. Structural analysis by multi-angle laser light scattering and small-angle X-ray scattering in solution revealed that the enzyme adopted highly flexible unbound, latent structures and peptide-bound, active structures that differed from the AD13-MDTCS crystal structure. Moreover, the peptide behaved like a self-avoiding random chain. We integrated the results with computational approaches, derived an ensemble of structures that collectively satisfied all experimental restraints, and discussed the functional implications. The interaction conforms to a ‘fuzzy complex’ that follows a ‘dynamic zipper’ mechanism involving numerous reversible, weak but additive interactions that result in strong binding and cleavage. Our findings contribute to illuminating the biochemistry of the vWF:ADAMTS-13 axis.This study was supported in part by grants from Spanish, French, Danish and Catalan public and private bodies (grant/fellowship references PID2019-107725RG-I00, BES-2015-074583, ANR-10-LABX-12-01, 6108-00031B, 8022-00385B, LF18039, NNF18OC0032724, Novo Nordisk Foundation “Bio-MS”, 2017SGR3 and Fundació “La Marató de TV3” 201815). This work was also supported by EPICS-XS, project 823839, funded by the Horizon 2020 programme of the European Union. The CBS is a member of France-BioImaging (FBI) and the French Infrastructure for Integrated Structural Biology (FRISBI), which are national infrastructures supported by the French National Research Agency (grants ANR-10-INBS-04-01 and ANR-10-INBS-05, respectively). Finally, we acknowledge the Structural Mass Spectrometry Unit of CIISB, an Instruct-CZ Centre, which was supported by MEYS CR (LM2018127)

Overview of molecular interactions using Biacore

Podeu consultar el llibre complet a: http://hdl.handle.net/2445/32166Surface Plasmon Resonance (SPR) technology is a powerful tool for studying a wide range of different putative interactions. This kind of optical biosensors allow to obtain (in real time and without labelling) quantitative and qualitative information about the kinetics of the surfacebinding process. The most critical points to keep in mind when using the technique are presented, as well as practical examples of applications

Implicació de la calmodulina en la progressió a través de la fase G1 del cicle cel·lular

Se ha estudiado el papel de la calmodulina (CaM) sobre la regulación de la actividad de la cdk4, cdk2, expresión y localización intracelular de las proteínas que intervienen en la progresión a través de la etapa H1 del ciclo celular. La adición de drogas anti-CaM a un cultivo de células NRK ("normal rat kidney") durante el principio del G1 provoca la inhibición de la actividad cdk4 y cdk2 y también se encuentra la proteína del retinoblastoma hipofosforilada. Los niveles totales de cdk4, ciclina D1, ciclina D2, cicE, p21 y p27 no están afectados por el tratamiento, pero sí que se observó una disminución en la cantidad de cicA y cdc2. La disminución en la actividad de la cdk4 no se debe a un cambio en las proteínas que se le asocian, ni a un cambio en la cantidad de proteínas inhibidoras unidas al complejo ckd4/cicD1. La inhibición de la CaM provoca una translocación del núcleo al citoplasma para la cdk4/cicD1, evitando de este modo que pRb se fosforile bajo el efecto de la droga anti-CaM. Mediante técnicas de inmunoprecipitación, columnas de afinidad y "pull down" se ha demostrado que la cdk4 y la cicD1 se asocian tanto in vivo como in vitro a través de una proteína aceptora de CaM. Como la Hsp90 interactúa con la cdk4 in vivo, y que dicha interacción puede facilitar la translocación de la cdk4 y la cicD1 al núcleo, la Hsp90 es una buena candidata para desempeñar el papel de CaMBP en la transcolocación de la cdk4/ckcD1 al núcleo. Por otro lado se encontró que la p21 coinmunoprecipitaba con la CaM y también en sentido contrario, esta interacción se comprobó que era directa utilizando "pull down". Además se comprobó que la CaM era esencial para acumulación de la p21 en el núcleo

Implicació de la calmodulina en la progressió a través de la fase G1 del cicle cel·lular

[spa] Se ha estudiado el papel de la calmodulina (CaM) sobre la regulación de la actividad de la cdk4, cdk2, expresión y localización intracelular de las proteínas que intervienen en la progresión a través de la etapa H1 del ciclo celular. La adición de drogas anti-CaM a un cultivo de células NRK ("normal rat kidney") durante el principio del G1 provoca la inhibición de la actividad cdk4 y cdk2 y también se encuentra la proteína del retinoblastoma hipofosforilada. Los niveles totales de cdk4, ciclina D1, ciclina D2, cicE, p21 y p27 no están afectados por el tratamiento, pero sí que se observó una disminución en la cantidad de cicA y cdc2. La disminución en la actividad de la cdk4 no se debe a un cambio en las proteínas que se le asocian, ni a un cambio en la cantidad de proteínas inhibidoras unidas al complejo ckd4/cicD1. La inhibición de la CaM provoca una translocación del núcleo al citoplasma para la cdk4/cicD1, evitando de este modo que pRb se fosforile bajo el efecto de la droga anti-CaM. Mediante técnicas de inmunoprecipitación, columnas de afinidad y "pull down" se ha demostrado que la cdk4 y la cicD1 se asocian tanto in vivo como in vitro a través de una proteína aceptora de CaM. Como la Hsp90 interactúa con la cdk4 in vivo, y que dicha interacción puede facilitar la translocación de la cdk4 y la cicD1 al núcleo, la Hsp90 es una buena candidata para desempeñar el papel de CaMBP en la transcolocación de la cdk4/ckcD1 al núcleo. Por otro lado se encontró que la p21 coinmunoprecipitaba con la CaM y también en sentido contrario, esta interacción se comprobó que era directa utilizando "pull down". Además se comprobó que la CaM era esencial para acumulación de la p21 en el núcleo

Quantification of interacting cognate odorants with olfactory receptors in nanovesicles

This study aims to improve our understanding of the interaction between olfactory receptors and odorants to develop highly selective biosensing devices. Natural nanovesicles (NVs) from Saccharomyces cerevisiae, ∼100 nm in diameter, carrying either the human OR17-40 or the chimpanzee OR7D4 olfactory receptor (OR) tagged with the c-myc epitope at their N-terminus, are presented as model systems to quantify the interaction between odorant and olfactory receptors. The level of expression of olfactory receptors was determined at individual NVs using a novel competitive ELISA immunoassay comparing the values obtained against those from techniques involving the solubilization of cell membrane proteins and the identification of c-myc-carrying receptors. Surface Plasmon Resonance (SPR) measurements on L1 Biacore chips indicate that cognate odorants bind to their Ors, thereby quantifying the approximate number of odorants that interact with a given olfactory receptor. The selectivity of OR17-40-carrying NVs towards helional and OR7D4-carrying NVs towards androstenone has been proven in cross-check experiments with non-specific odorant molecules (heptanal and pentadecalactone, respectively) and in control receptors. © 2017 The Author(s).This work was supported by the “Nanoscience, nanotechnologies, materials, and new production technologies” programme under the BOND project (228685-2) from the 7th Research Framework Programme of the European Union (PI and AC), by the FIS Project PI10/01171 funded by the Instituto de Salud Carlos III, by the Botin fundation, and by the predoctoral fellowship of the Instituto de Salud Carlos III-Ministry of Science and Innovation (Spain) (MS). The Nb4D group and Nanobioengineering SIC-BIO group are supported by the Commission for Universities and Research of the Department of Innovation, Universities, and Enterprise of the Generalitat de Catalunya (2014 SGR 1442 and 2014 SGR 1484). This work was partially supported by the MINDS project (TEC2015-70104-P), awarded by the Spanish Ministry of Economy and Competitiveness and the CERCA program from Generalitat de Catalunya. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with support from the European Regional Development Fund. The authors also thank Dr. Marie-Annick Persuy and Dr. Edith Pajot-Augy (NBO, INRA, Université Paris-Saclay) for olfactory receptors expression in yeast cells and yeast membrane material. Dr. José Amable Bernabé (ICMAB/CSIC/CIBER BBN) for his help with Nanosight measurements and Dr. Carmen López (CCiT, UB) for her help with Cryo-EM measurements.Peer reviewe