Na+/Ca2+ exchangers: Relationship between structure and function

Author(s)

Phelipe Augusto Mariano Vitale

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2022-08-08

Examining board members

Roberto Kopke Salinas; Fabio Ceneviva Lacerda de Almeida; Iolanda Midea Cuccovia

• Advisor: Roberto Kopke Salinas; Henning Ulrich

Abstract

Approximately 30% of the genes from eukaryotic and prokaryotic organisms encode membrane proteins, and approximately 60% of the targets for new drug development are membrane proteins. Despite this, membrane proteins account for only about 3% of all structures deposited in the PDB. Na+/Ca2+ exchangers (NCX) are absolutely essential membrane proteins. They play a key role in the maintenance of Ca2+ homeostasis in different types of cells. The NCX are formed by 10 transmembrane helices and a large cytoplasmic loop. The transmembrane domain is responsible for the transport of Na+ and Ca2+ through the lipid bilayer, while the intracellular loop is responsible for the allosteric regulation of the exchanger by intracellular Ca2+: binding of Ca2+ in a two-domain sensor (CBD12, formed by subdomains CBD1 and CBD2) activates the NCX. The NCX combine a transmembrane domain with globular domains connected by flexible linkers. This multifaceted character makes the NCX structure characterization a difficult task. Despite their recognized physiological importance, little is known about the three-dimensional structure of the eukaryotic NCX, and the mechanism of allosteric regulation remains poorly understood. In this thesis we adopted the exchanger from Drosophila, CALX, as a model system to study the structure and the allosteric regulation mechanism of the eukaryotic NCX. CALX displays anomalous regulation because it is inhibited instead of being activated by Ca2+. In contrast, while Ca2+-binding to CBD2 plays a role in the NCX Ca2+-regulation mechanism, CALX Ca2+ regulation depends only on Ca2+ binding to CBD1. To investigate the NCX Ca2+-regulation mechanism, we designed and investigated the dynamics of a mutant CBD12, containing a flexible linker between CBD1 and CBD2. NMR data indicated that the linker decoupled the dynamics of the two subdomains, while calorimetry data indicated that Ca2+ affinity was not significantly altered by the presence of the linker. The functionality of the NCX and the CALX in eukaryotic cells was examined by Ca2+-imaging. We also developed a protocol for 15N isotopic enrichment of the NCX from Methanococcus janaschii (NCX-Mj). Overall these results open the way to carry out structure-function studies of the CALX, and to investigate the NCX-Mj structure and dynamics using solution NMR spectroscopy. (AU)

• FAPESP's process: 17/05614-8 - Na +/Ca2+ exchangers: relationship between structure and function
• Grantee: Phelipe Augusto Mariano Vitale
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)