BOLLO Mariana Ines
congresos y reuniones científicas
.. Crystal structure of protein factor required for mg.atp stimulation of the squid nerve Na+/Ca2+ exchanger.
ELSO-BERBERIAN G, BOLLO M, COUSIDO A, MITSCHLER A, DIPOLO R, PODJARNY A AND BEAUGE L..
Congreso; Workshop CeBEM- Reunion de la Sociedad de America Latina de Proteína; 2010
Sociedad Argentina de Biofísica (SAB)
CRYSTAL STRUCTURE OF THE PROTEIN FACTOR REQUIRED FOR MgATP STIMULATION OF THE SQUID NERVE Na+/Ca2+ EXCHANGER. Berberián, G. a, Bollo, M. a, Pogdjarny, A.b, Cousido, A. b, DiPolo, R.c, Beaugé, L. a The Na+/Ca2+ antiporter is a ubiquitous structural plasma membrane protein in charge of exchanging Na+ and Ca2+ ions between the intra- and extra-cellular environments. As such, it is the major protein complex responsible for Ca2+ extrusion from most cells in a variety of organisms. An important characteristic of this exchanger is that it is highly regulated through a large intracellular loop within the protein. These regulations are related to the existence, on this regulatory loop, of non-transporting Ca2+ regulatory sites that must be occupied, in order for any transport mode to take place. MgATP up-regulates the exchanger by protecting the Ca2+ regulatory site from H+i and H+i+Na+i inhibition. In squid nerve Na+/Ca2+ exchanger, that effect requires the phosphorylation of a lipid binding protein (ReP1-NCXSQ) (Berberian et. al 2009, BBA, 1788, 1255-1262).The present work shows the solved crystal structure of ReP1-NCXSQ. The structure folds in a b-sandwich, with 11 b-sheets, typical of lipid-binding proteins. The structural features coincide with those expected for a member of the lipocalin supergene family. The structural results and the mass spectrometry indicate the presence of palmitic acid bound mainly to Tyr 128 and Arg 126. The activity results show that the mutant Y128F is not active, while it can be phosphorylated. Thus, the mutant of Y128F fails to stimulate the exchanger; in addition looses, if not completely at least in a large extent, the capability to retain a fatty acid inside the barrel when purified from the expression system. Moreover, this occurs without any detectable change in its crystal structure. The single mutant R126V remains active while, as expected, the double mutant Y128F-R126V does not. These results suggest that fatty acid transport by REP1-NCXSQ is a necessary feature for its participation in the regulatory process, and that the fatty acid involved in the regulation is bound to Y128. a Laboratorio de Biofísica, Instituto de Investigación Médica Mercedes y Martín Ferreyra (INIMEC-CONICET), Córdoba, Argentina, b Department of Structural Biology and Genomics, IGBMC, CNRS, Illkirch, France c Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela Supported by FONCYT-05-38073 and CONICET- PIP2010-2012/ 11220090100063.