Exploring the role of the linker peptide in Galectin-4 lectin activity

MASSARO, MORA; DI LELLA, SANTIAGO; CAGNONI, ALEJANDRO J.; MANSELLE COCCO, MONTANA; PEREZ SAEZ, JUAN MANUEL; RABINOVICH, GABRIEL A.; MARIÑO, KARINA V.

Buenos Aires

Congreso; Young Initiative on Biophysics (YIB); 2021

Sociedad Argentina de Biofísica

Galectin-4 (Gal-4) is a tandem-repeat carbohydrate-binding protein mainly expressed in the gastrointestinal tract. It presents two carbohydrate recognition domains (CRD) at the N- and C-terminal ends connected by a linker peptide. Even though both domains display lectin activity themselves, the nature and structural significance of the linker regarding the biological activity of the protein is not well understood. While the structure of each domain has already been solved by experimental techniques, no structural data has yet been reported for the full-length Gal-4 protein nor the linker region. In this work, we employed an interdisciplinary approach to gain insight into the relation between structure and function of linking both domains. For this purpose, we recombinantly expressed human Gal-4 and each of its isolated CRDs, and their glycosidic preferences were evaluated. In doing so, we identified different affinities for both full-length Gal-4 and each isolated CRD for specific glycan moieties. To assess key determinants of these differences, we employed different computational approaches to construct three models of the intact Gal-4 using Modeller, Rosseta and AlphaFold2 programs. After careful inspection, molecular dynamic simulations were performed using Amber19 package programs. Simulations were visualized with VMD software, and characterized by structural parameters such as atomic fluctuations, distances and dihedral angles in order to evaluate the orientation and direction of each domain with respect to the linker. Together, these analyses allowed the characterization of the whole protein, mainly its CRD positions and linker peptide conformation. A discussion on the importance and function of the linker region in the protein biological function will be presented.