Conformational selective binding of metal fluoride complexes to the Plasma Membrane Ca2+-ATPase and structure stabilization.

SAFFIOTI NICOLÁS; RIESCO, ANA; DE SAUTU, MARILINA; BERLIN, JOSHUA; FERREIRA GOMES, MARIELA; ROSSI JPFC; MANGIALAVORI IRENE

BUENOS AIRES

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Biofísica; 2019

Sociedad Argentina de Biofísica

The Plasma Membrane Ca2+ ATPase (PMCA) is a key participant in cytoplasmic Ca 2+regulation. As others P type ATPases, PMCA transports Ca2+ by the Albers and Postmechanism. In this, proteins have two main conformations, E1 and E2, and phosphorylatefrom ATP to transport ions against their electrochemical gradient. Recently, wedemonstrated that fluoride complexes of aluminium (AlFx), beryllium (BeFx) andmagnesium (MgFx) stabilize conformations very similar to the phosphorylatedintermediate of PMCA1.The aim of this work is to understand what conformational changes occur upon thephosphorylation of PMCA. To this end, we measured the fluorescence of the probe eosin,which binds with high affinity to the nucleotide binding site of PMCA. When the proteinbinds fluoride complexes, eosin fluorescence decreases, and the same effect is seenwhen the pump phosphorylates from ATP in presence of Ca2+. However, during transportactivity, part of the fluorescence decrease is due to displacement of eosin by ADP fromits binding site. Our results show that the ADP and ATP affinity for PMCA in E2conformation are 172 ± 28 and 74 ± 17 µM, respectively. The binding of fluoridecomplexes to PMCA depended strictly on Mg2+ concentration. Furthermore, thesefluoride complexes only binds to PMCA-Mg2+. This property allowed us to measure thePMCA affinity for Mg2+ (2,2 ± 0,4 mM) in E2 conformation. Finally, we studied thermalstability of PMCA by measuring the enzyme activity as a function of time. Fluoridecomplexes stabilize PMCA structure and delay activity loss when the protein is incubatedat 44°C compared to the free protein. We can conclude that fluoride complexes areligands of PMCA-Mg2+ complex, they set PMCA structure in a conformation analogous toE2-P, stabilizing it. This property allows to study the PMCA structure in E2 conformations.References1 Saffioti N.A, de Sautu M., Ferreira-Gomes M.S., Rossi R.C., Berlin J., Rossi J.P.,Mangialavori, I.C. (2019) BBA-Biomembranes. 1861, 366-379.AcknowldegmentsThis work was supported by Agencia Nacional de Promoción Científica y Tecnológica PICT 2014 0065,Consejo Nacional de Investigaciones Científicas y Técnicas PIP 11220150100250CO, and Universidad deBuenos Aires Ciencia y Técnica grant 2014-2017: 20020130100254B