Crystal structure of the protein factor required for MgATP stimulation of the squid Na/Ca exchanger.

G. ELSO DE BERBERIAN, M. BOLLO, A. COUSIDO, A. MITSCHLER, R. DIPOLO, A. PODJARNY AND L. BEAUGÉ.

Salta

Congreso; Latin American Protein Society Meeting; 2010

Latin American Protein Society and Sociedad Argentina de Biofisica

CRYSTAL STRUCTURE OF THE PROTEIN FACTOR REQUIRED FOR MgATP STIMULATION OF THE SQUID NERVE Na+/Ca2+ EXCHANGER.                      G. Elso-   Berberián, a,M.  Bolloa, A. Cousido b, A.  Mitschler b ,R. DiPoloc, A. Podjarnyb , L. Beaugé. a 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, INSERM, U. de Strasbourg, Illkirch, Francec  Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela e-mail: gelso@immf.uncor.edu The Na+/Ca2+ antiporter is a structural plasma membrane protein in charge of exchanging Na+ and Ca2+ between the intra- and extra-cellular environments. It is the major protein complex  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)1.The present work shows the solved crystal structure of the wild type  ReP1-NCXSQ and of its mutant Y128F. 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; and binds a much shorter fatty acid (heptanoic acid) in a more disordered way. Furthermore, the mutant shows a conformational change at the level of the portal region (loop of Phe58) which is open. In the case of the this loop is closed against the tail of the palmitic acid .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. (1) (Berberián et. al , BBA, 1788, 1255-1262, 2009) Supported by FONCYT-05-38073 and CONICET- PIP2010-2012/ 11220090100063,  the CNRS, ENSERM and the Hopital de Strasbourg.