Evidence of direct binding of G-actin and Calmodulin to PMCA by Surface Plasmon Resonance.

JP ROSSI MARIANELA G. DALGHIA, MARISA M. FERNÁNDEZB, EMILIO L. MALCHIODIB , EMANUEL STREHLERC AND JUAN PABLO F.C. ROSSIA.

SAN DIEGO, CALIFORNIA

Congreso; 56th Annual Meeting of Biophysical Society. San Diego; 2012

BIOPHYSICAL SOCIETY

Evidence of direct binding of G-actin and Calmodulin to PMCA by Surface Plasmon Resonance. Marianela G. Dalghia, Marisa M. Fernándezb, Emilio L. Malchiodib , Emanuel Strehlerc and Juan Pablo F.C. Rossia. aIQUIFIB-Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, UBA., bIDEHU-Cátedra de Inmunología, Facultad de Farmacia y Bioquímica, UBA. Junín 956, 1113, Buenos Aires, Argentina and cDepartment of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905   Previous studies of our laboratory suggest that the main isoform of the plasma membrane calcium ATPase (PMCA) expressed in erythrocytes, PMCAh4b, associates with the actin cytoskeleton and its catalytic activity can be regulated through this interaction. Apparently, G-actin and/or short oligomers enhance the catalytic activity of the pump, while F-actin seems to inhibit it. To further explore the interaction between PMCA and G-actin, we performed binding experiments based on Surface Plasmon Resonance technology (SPR) using purified PMCAh4b from human red blood cells and purified rabbit muscle G-actin on a BIAcore T100 system. Our strategy consisted in immobilizing G-actin to the sensor chip surface while PMCA constituted the analyte (range of concentrations from 125 nM to 2 µM). The sensorgrams obtained showed an increase in the response as a function of PMCA concentration with a saturable binding. The unspecific binding values were subtracted from the response obtained in the control flow cell (without immobilized G-actin). The apparent KD (1.25 µM) was determined by kinetic analysis with parameters kon (1.33 ± 0.07) 104 (Ms)-1 and koff (1.66 ± 0.03) 10-2 s-1. Conversely, we immobilized PMCAh4b to the sensor chip surface. The analytes were (i) G-actin (0.6 to 5 µM) or (ii) Calmodulin, a known activator of PMCAh4,b ( 1.8  to 15 nM). Results show that G-actin binds to the activated sensor with a KD of 3 µM, kon (2.98 ± 0.45) 104 (Ms)-1 and koff(8.98 ± 0.67) 10-2 s-1 and calmodulin binds with an apparent KD of (1.14 ± 0.6) 10-8 M. This last value agrees with the known value of calmodulin binding affinity determined by other methods. These results show for the first time a specific and high apparent affinity binding between PMCA and G-actin and reveal the usefulness of SPR technology to determine binding constants between small molecules and a large membrane protein like the plasmatic calcium pump.