The effect of mutations at Glu99 in the autoinhibition of the Plasma Membrane Ca2+ Pump

MAZZITELLI L.R.; ADAMO HP

Congreso; Latin American Protein Society Meeting; 2010

The PMCA belongs to the P2B family of P-ATPases, which are characterized by their autoinhibitory mechanism. The precise molecular mechanism responsible for the autoinhibition of the pump is not known. It has been proposed that the PMCA’s autoinhibition involves the blockage of the active site of the enzyme by the rearrangement of the N- and C-terminal segments of the molecule (1). The conformational change leading to activation is triggered by the binding of Ca2+-calmodulin to its site in the C-terminal segment. Glu99 is located in the connecting stretch between the PMCA’s “A” domain and transmembrane segment M1. Mutations at the equivalent residue have been shown to activate the autoinhibited plant Ca2+ pump ACA2 (2). We have investigated the effect of substituting Glu99 either by Gln or Lys. We found that both mutants retained about 70% of the maximal activity of the wild type enzyme. In the absence of calmodulin the activity of the wild type enzyme increased with the concentration of  Ca2+ with K1/2 of 1.3 uM Ca2+ , and at saturating Ca2+ attained 65% of its maximal activity in the presence of calmodulin. In the same experimental conditions mutant Glu99Lys exhibited a slightly increased apparent affinity for Ca2+ affinity K1/2 (1 uM Ca2+) but at saturating Ca2+  attained 93% of its  maximal activity in the presence of calmodulin. In contrast the behavior of mutant Glu99Gln was near wild type. These results show that substitution of Glu99 for a positively charged residue partially disrupts the autoihbitition mainly by increasing the maximal activity of the autoinhibited enzyme. With grants from UBA, CONICET PIP 2022 and ANPCyT 15-25965.