Generation of Nanobodies as a tool for structural biology

VANINA A. ALZOGARAY; FERNANDO A. GOLDBAUM

Bruselas

Workshop; Nanobodies for Structural Biology and beyond; 2018

INSTRUCT

Generation of Nanobodies as a tool for structural biologyVanina Alzogaray and Fernando GoldbaumFundación Instituto Leloir, Buenos Aires, Argentina.Our group studies complex macromolecules, being in most cases membrane proteins of relevance for drug design in human diseases. Our project addresses the structural study of the sodium-iodine transport protein NIS (Na+/I- symporter), a glycoprotein that has 13 transmembrane passages. NIS has a crucial role in the transport and accumulation of iodine in the thyroid gland. It is also present in the cells of other tissues such as stomach, small intestine, salivary glands and breast. A series of mutations in NIS cause defects in the transport of iodine leading to disorders in the functioning of the gland (Nicola JP et al., 2015, Ferrandino G et al 2016, Ravera S et al, 2017). Therefore, the structural characterization of the wild type protein and certain point mutants will provide a significant insight into its molecular mechanism. This protein is highly characterized but its three-dimensional structure is unknown. To increase the likelihood of crystal formation of NIS we will use nanobodies (Nb). We aim to obtain Nb against NIS and use them to crystallize and solve its structure by X-ray diffraction. NIS was used in clinical medicine to diagnose and treat thyroid disease for five decades before it was identified at the molecular level in 1996. Since its sequence was determined, significant progress has been made in understanding its tissue distribution and regulation, characterizing its structure/function relations, and using it as a reporter molecule. Fully elucidating the molecular mechanism by which NIS translocates its substrates will ultimately require determining the structure of NIS at atomic resolution—in different conformations and with its various substrates bound and not bound to it. We have previous experience in the generation and characterization of Nb (Koch-Nolte et al., 2007, Wesolowski et al., 2009, Doña et al., 2010, Alzogaray et al., 2011, Unger et al., 2015), and we also have started to work with membrane proteins as targets. In fact, we have already obtained a first library against NIS and other membrane proteins. We also have an in-house X-ray generator, a crystallization robot and expertise in crystal structure determination (Aran et al., 2014, Klinke et al., 2014, Fernandez et al., 2015, Rinaldi et al., 2016, Otero et al., 2016, Cerutti et al., 2017, Soldano et al., 2017).We see this training course as an ideal opportunity to learn in detail the crucial experimental tips in order to increase the likelihood of getting Nb-membrane protein useful crystals.